THE COMPARISON OF DIAGNOSTIC TESTING WITH PHENOTYPIC EXPRESSION OF PARENT AND HYBRID BRASSICA VARIETIES

Transcription

1 THE COMPARISON OF DIAGNOSTIC TESTING WITH PHENOTYPIC EXPRESSION OF PARENT AND HYBRID BRASSICA VARIETIES by Sandra Sawall A Master s Thesis Submitted to the Graduate Faculty in Partial Fulfillment of The requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURAL EDUCATION Major Advisor Director, Graduate Studies UNIVERSITY OF WISCONSIN-RIVER FALLS 2011

2 ABSTRACT Many seed companies and producers use diagnostic testing to determine the genetic purity of their seed. Diagnostic labs, such as isoelectric focusing and isozyme electrophoresis, are able to verify a variety s genetic purity within a few days of extracting the proteins from the seed. Growout testing can also be used to determine genetic purity of a seed variety; however, it can be months before a seed company can obtain results. The purpose of this experiment was to collectively use isoelectric focusing and isozyme electrophoresis technologies with a growout method to analyze the genetic purity of parent and hybrid varieties for three Brassica oleracea species: broccoli, cabbage, and cauliflower. Two varieties were used with three variables consisting of five female, five male, and twenty hybrid seeds for each crop. A total of 180 pots were placed in a controlled environment; and benches were blocked by crop and sub-blocked by variety. Plants were cared for daily and visually inspected for phenotypic differences. Data measurements of height and leaf spread were collected weekly and final mass and weights of the yield were assessed at the end of the experiment. Tissue samples were also collected from each plant and sampled by both isoelectric focusing and isozyme electrophoresis. The comparison of the growout with lab results verified the accuracy of 98.89% for Brassica oleracea genetic purity testing. Results indicated not only accuracy but also identified useful polymorphic stains and created a nomenclature for Brassica oleracea species which can be used for future research.

3 ACKNOWLEDGEMENTS A special thanks to those who made this research project possible: Dr. Tim Buttles Faculty Advisor Dr. James Graham Faculty Advisor Dr. Veronica Justen Research Project Advisor BioDiagnostics Inc. Appreciated their financial support and the use of their Isoelectric Focusing and Isozyme Electrophoresis Departments. An extended thanks to Craig Nelson, Wendy Zillgitt, Carol Betzel, Joyce Connolly, Erin Murphy-Untz, Cindy Robey, and Kathy Deiss for all of their support and assistance that made the completion of this study possible University of River-Falls Greenhouse Committee & Greenhouse manager Dan Waletzko - Glad to have them host and provide greenhouse resources for the growout portion of the trial. My family Dad, Mom and my sisters; for all they have done to help me accomplish my educational goals. Zachary Sawall Thankful for my wonderful husband who took time to help me prepare and collect data throughout the project and for his constant encouragement and support. Thank You!!! iii

4 TABLE OF CONTENTS Page Chapter I. Introduction... 1 Background... 1 Growouts... 1 Isoelectric Focusing... 3 Isozyme Electrophoresis... 4 Statement of Problem... 5 Purpose of the Study... 5 Objectives... 6 Hypotheses... 6 Delimitations... 7 Limitations... 7 Definitions... 8 Chapter II. Review of Literature... 9 Brassica oleracea L. and Diagnostic Technologies... 9 Importance of Pure Seed Lots... 9 Polymorphic stains Nomenclature for Isozyme Electrophoresis Accuracy of Laboratory versus Actual Field Growout Tests Benefits Challenges in Isoelectric Focusing and Isozyme Electrophoresis The Future of Isoelectric Focusing and Isozyme Electrophoresis with DNA Chapter III. Research Methodology Introduction Description of Variables Planting Lab Testing Sample Preparation Gel Preparation Loading Gels Staining Data Collection Procedures Data Analysis Procedures Summary iv

5 Chapter IV. Results Growout SAS Nomenclature Isoelectric Focusing Isozyme Electrophoresis Percent Purity and Percent Error Diagnostic Testing Accuracy Chapter V. Conclusions Overview Analysis of Results Potential Impact Future Research Summary References Appendices A SAS code B Design of Isozyme Electrophoresis Gel Layout C Design of Isoelectric Focusing Gel Layout D Pictures of Plants Outlining Visual Differences E Pictures of Isoelectric Focusing Gel F Isozyme Electrophoresis Genotypes for Variety A, B, C, D, E and F G Greenhouse Growout Study Measurements H Pictures of Isozyme Electrophoresis Gels V

6 CHAPTER I INTRODUCTION Background The maintenance of genotypically uniform inbred lines and commercial hybrids that are true to their original type is an essential requirement in the production and marketing of hybrid crops. Uniform hybrid varieties of Brassica oleracea species are becoming increasingly more important especially for mechanical harvesting and optimized yield. The self-incompatibility system, which is generally used as the method of pollination control, does not always provide a complete protection against self or foreign pollination between plants of the female inbred resulting in unacceptable numbers of non-hybrid seeds. For this reason, seed companies test for contaminated seeds in an operation regularly. For it is only through strict quality control procedures that the full genetic potential can be gained through breeding and delivered to the farmer. Nonuniform seed lots can cause seed lots to deviate unpredictably from their expected performance. The presence of female or male selfs can also reduce yield and economic value through the loss of hybrid individuals. Growouts Growout testing is one method used for effective quality control and spans throughout the entire timeline of the seed production process. A growout is simply an analysis of genetic purity based on visual detection. Traditionally, morphological appearance in crops during growouts alone has been used to assess genotypic purity. 1

7 When using morphological traits, a comprehensive quality control procedure is based on field appearance of plants during seed increase and hybrid seed production followed by growouts in winter nurseries of F1 hybrid seed lots. However, morphological attributes are often unreliable as indicators of genotypes because results can also be subjective and dependent on environmental conditions which may not be consistent with the conditions where the seed product will be planted. In addition, winter nurseries require significant planning, expense, and travel. Most importantly, data is not available from winter nurseries until about three months after seed lots need to be identified for shipment to seed distributors. Therefore, the use of isoelectric focusing and isozyme electrophoresis is a very powerful tool for genotyping inbred lines and hybrids as well as for performing genotypic tests of purity. In contrast with morphological trait data, isozyme data from electrophoresis can give results within several days. This compared to an entire growth cycle makes a big difference. Isoelectric focusing and isozyme electrophoresis testing can also give results that are not subjective since they are based on well-defined patterns of protein-based markers and are not influenced by the environment like growouts. So results are more indicative of the crops genotype which is not always displayed visually. A few other advantages are that the data can be collected prior to harvest, analyses can be performed locally, and the seed producers can save time and money because of these advantages. Thus, comprehensive quality control for commercial seed operations can now utilize both field observations made during F1 seed production and laboratory test made immediately following harvest. 2

8 Isoelectric focusing (IEF) Isoelectric focusing (IEF) is a technique for separating different molecules by their electric charge differences (Westermeier, 1997). It is performed on an immobilized ph gradient gel composed of polyacrylamide, starch, or agarose where a ph gradient has been established (Garfin, 1990). Typically 100 individuals are tested. Male and female lines are run to determine the hybrid pattern. For hybrids, female selfs, male selfs, offtypes, and total purity percentages are determined. For inbred varieties, off-types and total purity percentages are determined. When the proteins are first introduced to the gel, the gel takes advantage of the fact that a molecule's charge changes with the ph of its surroundings. A protein that is in a ph region below the ph at which a particular molecule carries no net electrical charge is known as the isoelectric point (IP) and will become positively charged and migrate towards the cathode or electrode through which an electric current flows out of a polarized electrical device (Garfin, 1995). An electric current is passed through the medium, creating a positive anode and negative cathode end (Allen, 1994). Negatively charged molecules migrate through the ph gradient in the medium toward the positive end while positively charged molecules move toward the negative end. As the extraction migrates through a gradient of increasing ph, the protein's overall charge will decrease until the protein reaches the ph region that corresponds to its IP (Micklos, n.d.). At this point the molecule no longer has a net electric charge and will not proceed any further within the gel. As a result, the proteins become focused into stationary bands with each protein positioned at a point in the ph gradient corresponding to its IP (Dunn, 1993). 3

9 The technique is capable of extremely high resolution with proteins differing by only a single charge being fractioned into separate bands. Through the method of isoelectric focusing, proteins can be resolved when they differ in an IP value as little as (Micklos, n.d.). The method is applied particularly in the study of proteins, which separate based on their relative content of acidic and basic residues, whose value is represented by the IP (Garfin, 1990). Hence this method uses ph and molecular charges to identify proteins in crop varieties. After the IEF process is complete, the molecules in the gel can be stained to make them visible. This data can then be analyzed for the crop s genetic purity as the proteins are detected according to their position in the gel. Genetic purity results in identical banding patterns. Different molecular components of the protein represent an offtype or parent seed mixed in the hybrid. Isozyme Electrophoresis Isozyme electrophoresis is a technique used for separating multiple forms of an enzyme or more specifically isozymes from each other using an electric field applied to a gel matrix (Reghetti, 1998). Isozymes are multiple molecular forms of specific enzymes and it is single genes that determine each molecular form of an isozyme (Dunn, 1993). Electrophoresis refers to the electromotive force that is used to move the molecules through the gel matrix (Andrews, 1986). By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined mainly by their mass. When the charge to mass ratio is uniform (toward the anode if negatively charged or toward the cathode if positively 4

10 charged) it stops (Andrews, 1986). The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule (Andrews, 1986). Like IEF, when the electrophoresis is complete, the molecules in the gel can be stained with specific substrate-containing solutions to make them visible. These different forms can then be ascribed to specific genetic alleles, thus, establishing the genotype of an individual by the locus coding for the enzyme variant (Dunn, 1993). Gel electrophoresis is used in testing plants/crops amongst other things. The results are analyzed to determine genetic purity. The benefit of isozyme electrophoresis is that it gives strong data genotypically for crops. Statement of Problem Research indicates very little work in the development of Brassica oleracea diagnostic testing, especially in isozyme electrophoresis. Instead, most research done in these fields focus on primary crops, such as corn and sunflower. Moreover, there was very little recent data on the testing of isoelectric focusing and isozyme electrophoresis, especially in correlation with a growout test that compare genotypic with phenotypic expression in the field. Purpose and Importance of the Study The purpose of this experiment was to conduct results for recent data on the comparison of growout testing and isoelectric focusing and isozyme electrophoresis. This study was also able to provide recent data on the Brassica oleracea species which is a more infrequent crop of study. Subsequently, this data can used in a commercial lab to 5

11 market the test of Brassica oleracea species with their isoelectric focusing and isozyme electrophoresis technologies; or by a seed company s decision on utilizing isoelectric focusing and isozyme electrophoresis processes to determine the genetic purity of their seed and if it is more cost effective than SNPS or DNA testing. Objectives The following objectives served to guide the research for this study: 1.) To determine if isoelectric focusing and isozyme electrophoresis technologies can detect genetic purity in Brassica oleracea species 2.) Define which stains are most polymorphic in isoelectric focusing and in isozyme electrophoresis for tissue testing of Brassica oleracea species. 3.) Create a nomenclature for Brassica oleracea species in isozyme electrophoresis. 4.) To verify the accuracy of laboratory testing to actual field expression in Brassica oleracea species. 5.) Conclude the advantages and disadvantages of diagnostics testing to determine the value laboratory testing has to a seed company. Hypotheses For the purpose of this study, the following hypotheses were tested and stated in the null form: 1. There is no difference in testing Brassica oleracea species in isoelectric focusing and isozyme electrophoresis compared with tissue samples from other crops. 6

12 2. There is no significant difference in the accuracy of using a grow-out method of Brassica oleracea species with the visual detection of phenotypic traits between parent and hybrid seed and in genotypic traits identified with isoelectric focusing and isozyme electrophoresis processes. Delimitations The delimitations for the study included the following: 1. The study was delimited by the amount of available research conducted on diagnostic testing on Brassica oleracea species. 2. The study was delimited by the amount of available research conducted on laboratory testing versus phenotypic expression in actual field studies. 3. The study was delimited on the amount of recent research in isoelectric focusing and isozyme electrophoresis Limitations The following limitations have been identified: 1. This project was limited (by space, time and resources) to testing only three Brassica oleracea species: Broccoli, Cabbage, and Cauliflower with a limited number of parent and hybrid seed varieties. 2. It was limited to a single lab for isoelectric focusing and isozyme electrophoresis testing, BioDiagnostics, Inc. 3. The study was limited to a summer growout which excluded most of the fall harvest. 7

13 Definitions Isozyme Electrophoresis - a technique used on crops for genetic purity testing as a quality assurance test to evaluate true-types, off-types, variants and self-levels. Isoelectric focusing (IEF) - a technique used on crops for genetic purity testing that can be described as isozyme electrophoresis in a ph gradient. Nomenclature - Individual isozymes distinguished and numbered on the basis of electrophoretic mobility. In this study, the number 1 is assigned to a form having the lowest mobility. Replicates - Each individual replicate will be abbreviated by variety, seed type, and number of replicate. Two varieties per species are used in this study: A and B are Broccoli, B and C are Cabbage, and E and F are Cauliflower. The tree types of seed are female, male, and hybrid. The parent seed (female and male plants) will each have five replicates, while the hybrid seed will have 20 replicates. Therefore abbreviations will be short-handed to AF1, AF2, AF3, AF4, AF5, AM1-5, AH1-20, BH1, EF4 etc. The abbreviations mean that, for example, AH1 refers to a hybrid, plant, or replicate number one from variety A. Abbreviations for the stains are: ADH - alcohol dehydrogenase ACO - aconitase ACP - acid phosphatase AMP - aminopeptidase GLU - 2-glucosidase GOT - glutamic-oxaloacetic transaminase PGD - 6-phosphogluconate PGM - phosphoglucomutase LAP - leucine amino peptidase MDH - malate dehydrogenase IDH - isocitrate dehydrogenase PHI - phosoglucomutase 8

14 CHAPTER II REVIEW OF LITERATURE Brassica oleracea and Diagnostic Technologies Isoelectric focusing and isozyme electrophoresis profiles of Brassica oleracea species of seed protein and enzymes were first applied to genetic purity assessment in hybrid varieties in 1971 by Nijenhuis. He studied the banding patterns of various species at a region of the acid phosphatase zymogram and observed four bands in which he later gave a genetic interpretation to the pattern that was produced by single seed extracts. He also proposed the use of isozymes for the determination of purity in F1 hybrid seeds. Most of the further work on isozyme analysis of Brassica oleracea has had the objective to increasing the number of known loci to facilitate the assessment of purity in hybrids (Yadava, et al, 1999). Importance of Pure Seed Lots Ideally, an inbred line is expected to be homozygous at all loci on all plants of a seed lot and fixed for the same isozymic allele at each locus assayed (Woods & Thurman, 2006). Woods and Thurman tested of over 17,000 individual plants representing seed lots of at least 100 seeds each for 67 inbred lines involved in commercial seed production in the United States. Of this, a mean of.75% of plants showed heterozygosity at one or more loci and for 90% of inbred lines, just 0.2% of plants revealed heterozygosity at one or more loci (Woods & Thurman, 2006). Thus, routine breeding and procedures to increase, average seven or eight generations of self-pollination during inbred 9

15 development. So with careful analysis of inbred lines, diagnostic testing can provide isozymic profiles that are sufficiently fixed and stable within inbred lines to allow their usage in purity assessment. Any sign of possible segregation or contamination in inbred lines would then dictate if additional seed lots should be checked for their degree of uniform appearance in the laboratory and possible in a field grow out test. Hybrid isozymic profiles can be constructed from the hybrid seed directly; but if possible, hybrid profiles should be made from assays of the inbred parents. This is because isozyme bands that may be hidden by overlapping and co-migrating bands. Therefore, the hybrid can be more accurately assayed by the inbred parents (Woods & Thurman, 2006). In addition, genotyping of inbreds provides at least some data on levels of segregation if they should exist within in the inbred lines. Even if segregation levels do not appear to warrant further tests of genotypic purity within the inbred line, knowledge of their occurrence greatly aids in the interpretation of data subsequently collected from hybrid seed lots (Woods & Thurman, 2006). In routine evaluations of hybrid purity, testing for female selfs alone should normally involve at least two unlinked loci that are fixed for alternate alleles in the inbred parents (Woods & Thurman, 2006). Their study also concluded that it is advisable to assay for at least 75% of the most polymorphic loci when outcrosses are suspected. Polymorphic stains Isozymic loci that are polymorphic (frequency of most common allele <0.95) in commercial crops are acid phosphatase (ACP), alcohol dehydrogenase (ADH), 2- glucosidase (GLU), phosphoglucomutase (PGM), aminopeptidase (AMP), malate 10

16 dehydrogenase (MDH), isocitrate dehydrogenase (IDH), and 6-phosphogluconate (PGD) (Stuber et al, 1989). For genotypic characterization of lines, it is desirable to collect data from most if not all of these listed loci. However, the variability at eight enzyme loci (PGM1, PGM2, ACO, PHI, AMP, LAP, ADH, and GOT) has been studied in 1993 by Arus, Shields, and Orton in 19 commercial open pollinated cultivars of Brassica oleracea: three cultivars each of cauliflower, broccoli, Brussels sprouts, cabbage, and collards and two of kale and kohlrabi. And they found that most of the 18 alleles described in this study were present in each agronomic group: collards and kohlrabi had all of them, 17 were found in cauliflower and cabbage, and 16 in broccoli, Brussels sprouts and kale. Each allele predominated in at least one of the cultivars analyzed. Predominate alleles were often different in cultivars of the same group, indicating that none of the agronomic groups had an isozymic profile clearly different from the rest (Arus, et al. 1993). The measurement of the extent of genetic variability in the study replicated another study in using two criteria: the average heterozygosity and the proportion of loci polymorphic averaged over all cultivars (Gottleib, 1981). High values were obtained for both parameters. These variation estimates indicate a trend towards a high level of variability that seems to be confirmed in most of the remaining known loci or regions of activity (Arus, et al. 1993). Segregation data in Brassica species yielded similar values. Several studies have been conducted in an effort to evaluate associations of alleles at isozyme loci with agronomically useful traits and to test the possible use of isozyme for more efficiently improving certain agronomics traits such as grain yield (Halim, et al., 2006). Stuber et al. in 1998 reported in his study that alleles at eight maize isozyme loci 11

17 were responsive to directional selection and were associated with changes in grain yield. A subsequent study was conducted to test the hypothesis that manipulation of frequencies of isozyme alleles would produce responses in correlated quantitative traits (Westermeier, 1997). This study showed that selection based solely on seven isozyme loci produced improvement in grain yield equivalent to one and a half to two cycles of full-sib family selection for yield alone. Selection at the isozyme loci also significantly increased yield. The changes in the quantitatively inherited traits are very likely mediated for the most part through linkages of agronomically favorable alleles for other traits (Westermeier, 1997). Studies by researchers have also provided evidence specifically on the genetic basis of phosphoglucomutase (PGM). PGM is active in seeds, leaf tissue, pollen, and even roots. This enzyme is well resolved in starch gels especially when using histidine at ph 7.0 as an extraction buffer (Bradford, 2006). Two loci on the PGM stain have been described as PGM1 and PGM2. PGM1is expressed by two bands in the upper region. The faster migrating band is said to be less active than the slower migrating band (Allen, 1994). PGM2 is known to be a four single-banded protein. Heterozygous individuals for PGM1 and PGM2 exhibit only the bands that are present in the homozygous parents (Burg, et al., 1996). Gotoh in 2002 also did a study that used inbred lines and their F1 and F2 populations in cauliflower. In the study, isozyme loci were examined with eleven enzyme stains. Polymorphism was detected for PGM as well as glutamate-oxalacetate transaminase (GOT) by polyacrylamide gel electrophoresis. In a subsequent study, fifteen commercial hybrid varieties of Brassica species were compared for the isozyme loci. All 12

18 the loci identified in this study showed co-dominance and multiple alleles, and were found to be useful in determination of genetic purity of hybrid seed or in varietal identification (Gotoh et al, 2002). Nomenclature for Isozyme Electrophoresis Extraction techniques, gel and electrode buffers, electrophoresis conditions, and staining methods used for each substrate tend to be variable in research. These differences do not effect the banding patterns of the enzyme, but instead change the relative mobility of migrants (Wills et al, 2009). Thus, comparisons between alleles found by different researchers on the basis of their position are not used in this study. Accuracy of Laboratory versus Actual Field Growout Tests Little recent research was found on isoelectric focusing and isozyme electrophoresis. However, one study done by Zhao in 2005 tested for inbreds among F1 seeds of two-line hybrid rice in the lab using isoelectric focusing of seed proteins. Embryoless half-seeds and whole seeds were used for isoelectric focusing to test inbreds. The corresponding embryo-containing half-seeds were then used to germinate to form seedlings on vermiculite. Seedlings were then transplanted into the field for grow-out tests. The genetic purity determined by isoelectric focusing of proteins from embryoless half-seeds was confirmed by grow-out tests of the corresponding embryo-containing halfseeds (Zhao et al, 2005). This work shows that isoelectric focusing of seed proteins can replace grow-out tests to determine F1 genetic purity of two-line hybrid rice and potentially other crops. The study also found that using half-seeds, the comparison 13

19 between laboratory and field tests was more objective and accurate than using whole seeds. Another study done by Halim and others found that laboratory testing (other than their test which used sodium hydroxide) for hybrid mixtures, was more accurate than field methods. Their study looked at field methods consisting of a modified growout test (20 d) compared to the traditional grow-out-test and found that the modified growout test was superior. They then concluded that the accuracy of laboratory methods is of great significance in varietal identification and genetic purity testing programs for the more economic use of money, time and land (Halim et al, 2006 p. 693). Benefits One of the primary advantages of isoelectric focusing and isozyme electrophoresis is that genetic purity can be determined prior to field tests so that the evaluation of conventional characters can be restricted to groups of varieties with the same isozyme genotype (Van den berg, 1998). Then, the number of entries of each trial may be considerably reduced. The assessment of purity can be done in seeds or seedlings (or even tissue samples), which can save time and costs in comparison with other methods, for example, a growout where identifications must be done in adult plants. A study was done in 1996 by Burg and others that indicated the cost efficiency of the equipment and procedures. They developed two hybrid purity test methods for Brassica species using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using alcohol dehydrogenase (ADH) from imbibed seeds. The study showed that one person can routinely analyze 1,536 individual seeds per day at a cost of 14

20 about US 0.11 per seed for chemicals, materials, and electrophoresis equipment (Burg et al, 1996). In addition, species variable for isozymes and with various alleles per locus, like Brassica oleracea, are especially suitable for isozyme electrophoresis since the number of possible isozyme phenotypes is very large (Hunter & Kannenberg, 2001). In Hunter and Kannenberg s study, 22 of the 32 F1 hybrids could be individually distinguished on the basis of their isozyme phenotype at six loci. This finding then concluded that the use of isozymes for the analysis of distinctions in F1hybrids, or inbred lines of the Brassica oleracea species, would greatly simplify the plant breeding process. Challenges in Isoelectric Focusing and Isozyme Electrophoresis One of the biggest challenges is in scoring and interpreting protein expression. For a few loci, some isozyme bands migrate very closely and it is difficult to accurately identify homozygous versus heterozygous plants (Garfin, 1995). Another component that makes scoring hard is that there can be co-migrating isozyme bands and is therefore difficult to accurately assess whether particular isozyme alleles are present in a homozygous or heterozygous condition (Garfin, 1995). If these alleles are donated by the male parent then their absence in a hybrid (a female self) can easily be seen. But, if these alleles are donated by the female, then a heterozygote (hybrid) cannot readily be differentiated from a homozygote (a female self). Moreover, the presence of the PGD2 allele is associated with a reduction in overall enzymatic activity at both the PGD 1 and 2 loci and frequently causes a reduction in resolution of the isozyme bands associated with these loci (Garfin, 1995). 15

21 Another challenge comes into play when both inbred lines are fixed for the same allozyme at the locus observed (Garfin, 1995). Then, electrophoresis assays cannot be used. Thus, it is important that the gene or genes analyzed be variable in the species. Using a polymorphic isozyme does not guarantee that both inbreds will be fixed for a different allele, however, this is more likely of various loci known to be variable are analyzed (Garfin, 1995). However, the variability observed at the isozyme loci known in Brassica oleracea species is a good indication that most of the hybrid varieties that already exist can be tested with confidence with laboratory techniques. According to a study done by Arus, Shields and Orton, only three out of 32 commercial Brassica F1 hybrids analyzed for six enzyme loci were homozygous for all of them (1993). The Future of Isoelectric Focusing and Isozyme Electrophoresis with DNA Van den berg did a study in 1998 on different types of procedures developed for hybrid purity testing. These were: starch gel electrophoresis (SGE), vertical polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (IEF). For most major vegetables, these methods proved to be much faster than plant grow-outs and relatively inexpensive. A Comparison of SGE and PACE methods to IEF proved to be more efficient for large-scale hybrid purity testing (Van den berg, 1998). Furthermore, there was a comparison of IEF with DNA fingerprinting methods along with the future of both techniques with respect to hybrid purity testing in the vegetable seed industry. When it comes to a choice between IEF or a DNA-based method, efficiency and effectiveness ultimately determines the method which is best suited for hybrid purity testing (Van den 16

22 berg, 1998). It was also concluded that in the future, we will see an increased use of both technologies for hybrid purity testing because expectations of plant breeders and growers has increased. As a result, they will accept fewer inbreds in a hybrid variety, especially when growing in a greenhouse (Van den berg, 1998). 17

23 CHAPTER III RESEARCH METHODOLOGY Introduction and Design of the Study This study incorporates three methods of varietal purity testing: a growout, isoelectric focusing, and isozyme electrophoresis. Initially, the growout was performed on two varieties from each of the Brassica oleracea species (Broccoli, Cabbage, and Cauliflower). The three variables of female, male, and hybrid seeds from the two varieties of three crops were planted on May 13, 2011 into each labeled corresponding pot. The 180 (which were 30.5 cm by 30.5 cm in diameter) pots were placed in a controlled environment, in this case a greenhouse, to avoid major environmental factors that can affect plant appearance, and ultimately the results. Benches were blocked by crop and sub-blocked by variety. Plants were cared for daily and visually inspected for phenotypic differences. Data measurements of height and leaf spread were collected weekly until August 9 th. Final mass and weights of the yield were then assessed at the end of the experiment. During the growout, tissue samples consisting of the cotyledons were collected from each plant after the first and second true leaves were fully developed. The tissue samples were then placed immediately in a cooler to prevent the degradation of proteins and later sampled by both isoelectric focusing and isozyme electrophoresis. After results were tabulated from the growout, a comparison study evaluated the results from diagnostic technologies and appraised results from all three methods. The most polymorphic stains were then identified in both laboratory methods and a new 18

24 nomenclature was created to correspond to the protein expression in isozyme electrophoresis. Description of Variables List of Treatments: 1.) Female Parent Seed - 5 replicates 2.) Male Parent Seed - 5 replicates 3.) Hybrid Seed - 20 replicates 30 replicates x 2 varieties x 3 crops = 180 pots Planting Planting took place on May 13, A basic potting soil was placed into round plastic nursery pots that were 30.5 cm in diameter. Five seeds were randomly selected from the seed packed and planted at the recommended level of cm and watered to ensure at least one plant per pot. Plants were watered and cared for daily. The date of the first plant emergence was May 25, Almost all plants emerged within a day of each other. After emergence, the five plants in each pot were randomly thinned out to one plant per pot. Visual notes and pictures were recorded for physiological or other characteristics of varietal distinctions or differences as plants were growing. Measurement data collection began on May 31 where plant height and leaf spread were recorded. Weekly measurements continued throughout the course of the study and daily visuals were recoded as well. About a tablespoon of Fertilizer was applied when the first true leaves developed and again right before the heads began to form. Final measurements included height, leaf spread and head height, width, and weight (if applicable). 19

25 Lab Testing Sample Preparation Leaf tissue was cut from the growing plant. Cotyledon tissue was selected because it has a high level of enzyme activity and because it is possible to remove the lower leaf tissue without sacrificing the plant as these plants are grown out for subsequent cross evaluation. Leaf tissue was then chilled and cut into 1/2 inch cubed pieces and placed into two polyethylene microcentrifuge (one for Isoelectric Focusing and one for Isozyme electrophoresis). Each section was then homogenized for about 30 seconds with 4 drops of a stock solution containing g Tris,.5 g L-Ascorbic Acid,.5g L-Cystine which had a final ph of 7.0 and was crushed. Filter paper was then used to absorb the protein extraction and frozen in an ultafeeezer at -80º C to preserve enzyme activity. Gel Preparation Starch gels are prepared by using buffer systems for the specific enzyme under investigation. Starch gels are composed of starch and sucrose with buffer. This method entails the use of a hot plate in order to thicken the solution. Three fourths of the prepared buffer required for one gel was heated in a 5000 ml Erlenmeyer flask until boiling (which takes about minutes) while the remaining volume of buffer was mixed with the starch and sucrose in another flask and mixed until it formed an even suspension. The solution was then poured into an acrylic gel mold. The gel was allowed to cool for approximately one hour and then the top was sliced off and the entire gel was covered with polyethylene film to prevent dehydration and left overnight at room temperature. 20

26 Loading Gels Wicks that were frozen overnight in the ultra-freezer at -80 degrees Celsius was then placed into the gel. The gel was then boosted electrically for 30 minutes. Then the wicks were removed. During this process, any air bubbles introduced into the gel during the placement of the wicks were removed by squeezing the top of the gel. The gel was then placed into the electrode buffer trays in a refrigerated cooling chamber at 2 degrees Celsius to prevent the degradation of proteins. An ice pack was placed into the water coolant system for additional cooling. An electrical current of about 500V, 200mA, 100W was applied across the gel from the power supply and a constant power for about five hours. No more than a 2% deviation in constant power was maintained during the electrophoresis separation. After electrophoresis, the gel was removed from the refrigerator and a rectangular slab was cut by trimming away the side boarders and the anodal electrode strip. A diagonal slash was cut in the upper left corner of the gel to identify the top of the gel. The slab of gel was then placed on an acrylic slicing bed. A fine wire is drawn horizontally through the gel to cut a thin 1.25mm slice which was then removed and placed into a stainbox. The procedure is repeated until the desired number of slices was removed. The slicing sequence was as follows: ACO, AMP, PGD, PGM, IDH, and PHI. 21

27 Staining Zones of enzymatic activity are revealed by immersing the gel slice into a stain assay. In most cases a single slice is immersed in approximately 50 ml of stain solution. After a two hour incubation at 36 degrees Celsius, the banding patterns were scored and then photographed. Because different stains fade at different rates, most scoring is preformed immediately after staining. Data Collection Procedures The data for this study was collected on a weekly basis beginning on May 13 th and concluding on August 9 th. Plant treatments were cared for and watered daily (with exceptions of July 4 th plants were watered the following day). Recording of any visual manifestations and signs of phenotypic differences (i.e. color, stem, leaf, and any other differences) were noted during watering. Every week the plant height and width/spread was measured in mm. Photographs were also taken throughout the growout visually verifying observations. After the first and second true leaves were fully developed, leaf samples consisting of the plant s cotyledons were cut off and placed into corresponding labeled zip lock bags and cooled on ice packs in a cooler. These samples were then run as described in the process above in the isoelectric focusing and isozyme electrophoresis labs. Final plant measurements were recorded August 9 th. The height and width/leaf spread of the plant was measurements along with yield weights. Concluding photographs were taken at the time as well. 22

28 Data Analysis Procedures Collected data from the plant s final measurements were entered directly and stored in the HP Pavilion dv6000 PC using Microsoft Excel and Word processor during the growout. An analysis of the data was done using the Statistical Analysis Software (SAS/STAT) with the proc mixed approach. An alpha level of 0.05 was used throughout the analysis to determine if means were considered significantly different. From the notes and pictures during the data collection phase and of final recording, visual manifestations and signs of phenotypic differences (i.e. color, stem, leaf, and any other differences) were correlated. Observations were made and concluding remarks were based on the statistical differences, visual notes, and photographs. A variety s genetic purity profiles was then made of what the growout considered as an offtype, female self, male self, and true self. In the lab, the samples were run and analyzed for genetic purity based on protein expression. Since nomenclature is a means to identify a protein banding pattern, this study identifies proteins having the lowest mobility with the number 1. Letters are used when more than one loci are present and effect mobility. In this case, protein shape is used instead. A number of stains were also run in this experiment. The stains selected were: ACP, MDH, ACO, AMP, PGD, PGM, IDH, and PHI. The most polymorphic stains were identified and noted to be used for future procedures. Lastly, the percent purity of variety was calculated as: Percent Purity = (# of identical hybrid phenotypic expression /total # hybrid variety) for each treatment. Lab results were then compared to the growout and a percent error was calculated as: Percent 23

29 Error = ([Greenhouse Result Lab Results] / Lab Results) x 100. Additionally, each plant served as a repetition and was compared to all of the plant s results from the study. From this, the final accuracy of the lab results was affirmed. Summary A growout was performed on parent and hybrid seed from two varieties from each of the Brassica oleracea species (Broccoli, Cabbage, and Cauliflower). Data was collected from the growout to phenotypically determine the genetic purity of each variety s parent and hybrid seed. The same plant tissue used for the growout was then tested with the isoelectric and isozyme electrophoresis technologies, which in turn determined the genotypic genetic purity. Results compared the growout s findings with the diagnostic technologies and concluded accuracy by percent error. The study also identified the most polymorphic stains after testing all of the stains and determining which stains revealed the most diagnostic banding patterns. And finally, nomenclature was created for the Brassica species to correspond to the protein expression. 24

30 CHAPTER IV RESULTS Growout From the notes and pictures during the data collection phase and of the final recording of visual manifestations and signs of phenotypic differences (i.e. color, stem, leaf, and any other differences) observations concluded the following genetic purity profiles: Profile A (broccoli) Female: Stem and branching is thicker, curves downward and occurs beneath the head, head yields on the top of the plant. Also has a light leaf color with more curvature in the leaves. Also is smaller than the male and hybrid. Male: First to yield, leaves grow through the broccoli head, branching surpasses the head and the plant is slightly darker than the female and male. It is smaller than the hybrid but taller than the female. Hybrid: Branching surpasses the head and leaves grow through the broccoli head. The hybrid is taller than both parents. Notes of Phenotypic differences: H4, H6, H17 appear to exhibit characteristics of female. Therefore these three were concluded to be female selfs. Profile B (broccoli) Female: Smaller than hybrid and male, pronounced axillary buds Male: Smaller than hybrid but slightly taller than the female. Also pronounced axillary buds. 25

31 Hybrid: Taller than both parents with the pronounced axillary buds. Notes of Phenotypic differences: H15 and H16 appear to be offtypes. H15 had the same characteristics of the other hybrids but was significantly smaller. H16 was also smaller and headed out three weeks earlier that the other hybrids. Additionally, H16 was much lighter in color, has a much thicker stem and its branching did not have the pronounced axillary buds as the hybrid. Profile C (cabbage) Female: Smaller and yields later than the hybrid, but bigger and yields earlier than the male. Looser symmetrical leaf pattern. Male: Smaller and behind on yield than both hybrid and female. Leaf structure was a tightly held more symmetrical leaf pattern. Hybrid: Taller, larger, and yields more and before the parent seed. Carries the same loose leaf pattern as female. Notes of Phenotypic differences: None Profile D (cabbage) Female: Smaller and behind on yield compared to the hybrid. Darker color with an unsymmetrical florette. Male: Dead Hybrid: Taller and larger and has more yield than parent seed. It is also a lighter plant in color and has an unsymmetrical florette like the female. 26

32 Notes of Phenotypic differences: H3, H4, H8, H15 appeared to be offtypes. H3 was taller than the hybrid and female plants, had a different color, and the head develops farther down, closer to the stem. H4 and H8 appeared to be the same off type that had a different color and larger head. They also have a flatter leaf pattern that was more symmetrical. H15 was a different color with a smaller head, flatter leaf pattern, and the leaves curved upright and were more symmetrical. Profile E (cauliflower) Female: Lighter color than the male, purplish color on axil and axillary buds, compound branching. Male: Darker green than both the female and hybrid. No purple was around the axil/axillary buds. It had an elongated stem compared to the female and had more erect branching. Hybrid: Same color as the female with a more elongated stem (little shorter than the male) and had erect branching like the male. Notes of Phenotypic differences: H15 appear to exhibit characteristics of female. Therefore it was concluded to be a female self. Profile F (cauliflower) Female: Dead Male: Smaller than the hybrid with erect branching. Hybrid: Larger than the parent seed with erect branching Notes of Phenotypic differences: None 27

33 A weekly measurement of the plant s height and leaf spread was merely used as a tool to assess visual manifestations and signs of phenotypic differences via plant mass between individuals. It also served as a more sound and tangible tool to reference back to in instances of growth-rate and plant development. Therefore, the final measurements of the weekly study were collected on August 9, SAS The SAS code can be found in Appendix B along with the tables for the following data. The model that was used for SAS was class=plant treatment which is one for the females, two for the males, and three for the hybrids. This was run through the proc mixed procedure in SAS comparing plant height, leaf spread width, head height and width, and head weight. The significant figures are shown below: Table 1. P-values for Variety A - Broccoli Plant Head Head Head Width Height Width Weight Female vs. Male 0.02* <.0001* <.0001* <.0001* Female vs. Hybrid 0.01* <.0001* <.0001* <.0001* Male vs. Hybrid * P-value (p = < 0.05) Variety B (broccoli): No significant differences 28

34 Table 2. P-values for Variety C - Cabbage Plant Plant Head Head Head Height Width Height Width Weight Female vs. Male <.0001*.0023* <.0001* <.0001*.001* Female vs. Hybrid <.0001* <.0001* <.0001* <.0001* <.0001* Male vs. Hybrid 0.11 <.0001* <.0001* 0.03* <.0001* * P-value (p = < 0.05) Table 3. P-values for Variety D - Cabbage Plant Plant Head Head Head Height Width Height Width Weight Female vs. Male <.0001* <.0001* <.0001* <.0001*.0003* Female vs. Hybrid 0.24 <.0001* *.0024* Male vs. Hybrid <.0001* <.0001* <.0001* 0.03* <.0001* * P-value (p = < 0.05) Variety E (cauliflower): Plant Width - Males were smaller than hybrids

35 Variety F (cauliflower): Plant Width Males were smaller than hybrids <.0001 The SAS program also identified statistical differences in varieties A, B, D, and E. Females visually identified before were confirmed statistically different in leaf spread and yield. In Variety B, statistical differences for hybrids 15 and 16 were found in plant height and leaf spread and for hybrid 18, yield. For variety D, visual offtypes were confirmed by statistics as significant differences were found in all parameters for hybrids 3, 4, 8, and 15. In variety E, identified offtype (hybrid 15) was significantly different in height. The identified figures validate those significant differences that were visually and statistically found between male, female, and hybrid lines. One can therefore conclude assessments such as selfing or offtypes because the significant figures match the actual differences found in the plant lines. Nomenclature Isozyme electrophoresis has been applied to genetic purity assessment in hybrid varieties. Most inbreds have one allele per locus so a copy of the female allele and a copy of the male allele combine to make the hybrid pattern. The basic scheme employed is that if each parental line used for hybrid production is fixed for a different allele at an enzyme locus, then contaminants can be identified since they will not be heterozygous for both parental alleles. 30

36 Bands observed on a gel after staining represent multiple forms of an enzyme. The banding patterns appear vertically with each lane representing one sample. These patterns are visible forms of alleles present in the seed. In routine evaluations of hybrid purity, testing for selfs should involve at least two unlinked loci that are fixed for alternate alleles in the inbred parents. Offtype detection can be seen by observing physical characteristics different than that of the parents or the hybrid. Appendix H outlines the techniques and scoring procedures used in this study for the starch gel electrophoresis of isozymes from broccoli, cauliflower, and cabbage. Although techniques are adapted from various published sources, an original nomenclature system is identified and developed in this study. The techniques and scoring procedures presented here can serve as a technical reference for future research and other applications. Isoelectric Focusing There are many stains that can be used in isoelectric focusing. Based on previous research on these varieties, certain stains were used to correspond with seed lots. Typically an ADH stain is used exclusively on tissue samples because it is a total protein stain. Unfortunately, because of limited resources, only certain stains were run and not all stains were diagnostic. Only varieties A,C,E, and F could be completed. The results were as follows: 31

37 Variety A: Female Self on H4, H6, and H17. Offtype was also found on H13. Variety C: No differences. Variety E: Female Self on H15. Potential offtype on Female 1. Variety F: Offtype on Male 2. For pictures of these stains see Appendix F Isozyme Electrophoresis Diagnostic results appeared on ACO, AMP, PGD, PGM, IDH, and PHI. MDH and ACP, however, did not show any differences. Nomenclature was established for the Brassica species by their protein banding pattern. The proteins having the lowest mobility were assigned with the number 1. Letters were used when more than one loci were present and effect mobility of the protein. In this case, the shape of the protein banding pattern was used. The isozyme electrophoresis lab indicated the following results: Variety A (broccoli): H4, H6, and H17 are Female Selfs Variety B (broccoli): H5, H8, and H16 are Variants. Variety C (cabbage): No differences Variety D (cabbage): H3, H4, and H8 are Offtypes. H15 is a Male Self. Variety E (cauliflower): H15 is a Female Self. Variety F (cauliflower):h6 is a Variant. For a complete review of pictures and genotype, please see appendices G and I. 32

38 Percent Purity and Percent Error The percent purity of variety was calculated for hybrids only. Due to limitations of recourses and space, there was not enough parent seed to determine the percent purity for the inbreds. As a result, the percent purity for the hybrid varieties are as follows: Table 4. A Comparison of the Percent of Hybrid Genetic Purity between a Growout, Isoelectric Focusing, and Isozyme Electrophoresis Technologies Field Growout Test Isoelectric Focusing Isozyme Electrophoresis Variety A (broccoli): 85% 80% 85% Variety B (broccoli): 90% - 100% * Variety C (cabbage): 100% 100% 100% Variety D (cabbage): 80% - 80% Variety E (cauliflower): 95% 95% 95% Variety F (cauliflower): 100% 100% 100% * Three variants were noted in this hybrid; however a variant is not enough evidence in diagnostic testing to signify an offtype. Only Broccoli (varieties A and B) reveal differences in the percent purity of the hybrids between the three tests. Therefore there was no percent error on cabbage or cauliflower testing. Lab results did indicate a 5.88% error in isoelectric focusing on variety A and an 11.11% error in Isozyme electrophoresis in Variety B. 33

39 Diagnostic Testing Accuracy In the growout, there were a total of ten plants that revealed differences from parent seed and hybrids. When testing for these differences diagnostically, isoelectric focusing identified an additional five offtypes to the ones phenotypically found, but missed two offtypes that were identified in the growout. Isozyme electrophoresis found three variants (one of which was an offtype) and missed one offtype that was phenotypically identified. For both diagnostic tests, two different offtypes were missed. Meaning that two seeds out of 180 (or 1.11%) for each test was unsuccessfully identified as an offtype. This indicates that diagnostic testing for both isoelectric focusing and isozyme electrophoresis according to these growout results provide up to a 98.89% accuracy in genetic purity testing. 34

40 CHAPTER V CONCLUSIONS Overview The purpose of this study was to conduct recent data on the comparison of growout testing, isoelectric focusing, and isozyme electrophoresis for the Brassica oleracea species. Results were examined to determine five main objectives which were all met in this study. The specific objectives were: 1.) To determine if isoelectric focusing and isozyme electrophoresis technologies can detect genetic purity in Brassica oleracea species. 2.) Define which stains are most polymorphic in isoelectric focusing and in isozyme electrophoresis for tissue testing of Brassica oleracea species. 3.) Create a nomenclature for Brassica oleracea species in isozyme electrophoresis. 4.) To verify the accuracy of laboratory testing to actual field expression in Brassica oleracea species. 5.) Conclude the advantages and disadvantages of diagnostics testing to determine the value laboratory testing has to a seed company. This study found that (1) isoelectric focusing and isozyme electrophoresis technologies can detect genetic purity in Brassica oleracea species. (2) The stain most polymorphic for tissue testing in isoelectric focusing is ADH (a total protein stain). The stains most polymorphic in isozyme electrophoresis are: ACO, AMP, PGD, PGM, IDH, and PHI. (3) An original nomenclature was created for Brassica oleracea species in 35

41 isozyme electrophoresis - see Appendix G. (4) The accuracy of laboratory testing to actual field expression in Brassica oleracea species was found to be at 98.89% and was verified by percent error from percent purity as explained below. (5) The advantages and disadvantages of diagnostics testing were determined in this study and are also analyzed below. Analysis of Results Final analysis indicated that variety A had an 85% genetic purity in the hybrid. Three female selfs were identified in the hybrid lot; they were AH4, 6, and 17. In variety B, one known offtype BH16 and one potential offtype BH15 were identified indicating a 90% genetic purity percentage. Variety C reached 100% purity in female, male, and hybrid lines. The male seed on D never grew which made testing a little more challenging. Nonetheless, offtypes were found on DH 3, 4, and 8 and a male self was recognized through genotypic evaluation which concluded an 80% genetic purity. Variety E had one female self on EH15 determining a 95% genetic purity and variety F was a pure line with 100% genetic purity. Industry standards, although not enforced, strive to be around at least 95% purity. According to the lab tests, two varieties would be considered below that mark and impure. Those lines are Hybrid A (85%) and D (80%). In the growout test, those same lines were identified. However, B was also recognized as an unfit hybrid line. Although there is a slight discrepancy, it was noted that three variants were found in this hybrid in isozyme electrophoresis. But since variants are not enough evidence to suggest an offtype, there appears to be error even though testing more loci could prove otherwise. 36

42 Tests on cabbage and cauliflower were compatible throughout all three methods of study with no percent error. Broccoli, however, (varieties A and B) reveal differences in the percent purity of the hybrids between the three tests. Lab results specify a 5.88% error in isoelectric focusing on variety A and an 11.11% error in isozyme electrophoresis in Variety B. An inclination could be granted that cabbage and cauliflower are easier to test for with these technologies than broccoli. Results established that isoelectric focusing and isozyme electrophoresis are still accurate ways to test the Brassica oleracea species. In all six varieties, results from the three methods were congruent. The only exceptions were variety A Hybrid 13 and the variants on the B hybrid variety. However, this does not provide evidence against the diagnostic testing accuracy because, for example in A Hybrid 13, genotypic differences are not always displayed phenotypically. Isoelectric focusing could have caught a genetic difference that a growout could not test for based on phenotypic traits. Likewise, in variety B. Hybrid 15 has all of the same characteristics of the other hybrids; it just had stunted growth which could be an environmental factor. Also, isozyme electrophoresis did find a variant on B hybrid 16, but there may not have been enough loci tested to verify an offtype. Nevertheless, isozyme electrophoresis and isoelectric focusing are accurate tests when the right stains are used. A total protein stain known as ADH worked best in isoelectric focusing and should be used in all future tissue sampling. Other stains, although still diagnostic, were still blurred and harder to assess. ACO, AMP, PGD, PGM, IDH, and PHI are the stains that worked best in isozyme electrophoresis. 37

43 Potential Impact Although new technologies in SNPS and DNA are more accurate, none of the technologies have replaced isoelectric focusing and isozyme electrophoresis. Only a selected group of crops with high seed value such as corn are used because of the cost inefficiencies. They are expensive especially for smaller seed companies that have multiple lines to test and develop and for the plant breeder who works with minority crops. Hence, isoelectric focusing and isozyme electrophoresis are still viable tests for seed producers. Consequently, it is essential to keep up current research on isoelectric focusing and isozyme electrophoresis technologies to continue to enhance, improve, and remain competitive. This study confirms that isoelectric focusing and isozyme electrophoresis will give strong genotypic data and still remains as an accurate way to test Brassica oleracea species. This data also provides a list of stains that can be used to show polymorphism for the Brassica oleracea species in diagnostic testing. Furthermore, nomenclature for Brassica oleracea species in isozyme electrophoresis was created to be used as a resource for future studies. This data can also be used in a commercial lab to market the test of Brassica oleracea species with their isoelectric focusing and isozyme electrophoresis technologies; or by a seed company s decision on utilizing isoelectric focusing and isozyme electrophoresis processes to determine the genetic purity of their seed and if it is more cost effective than SNPS or DNA testing. 38

44 Future Research Although this study has concluded some of the stains that are most polymorphic for isoelectric focusing and isozyme electrophoresis for Brassica oleracea species, there are still a number of other stains that can be tested and evaluated for polymorphism. Suggested future research in isozyme electrophoresis stains could include at least leucine amino peptidase (LAP), alcohol dehydrogenase (ADH), and glutamic-oxaloacetic transaminase (GOT). These stains have been used in previous research and may also prove to be useful in diagnostic testing of the Brassica oleracea species. This study also concludes that a total protein stain, ADH works best on tissue samples in isoelectric focusing; however, additional procedural developments with other stains could be diagnostics as well and therefore could be tested. This study furthermore poses questions as to the significance of identified variants in diagnostic testing. A variant means that there was only a difference found at one locus. Since most labs require differences for at least two loci, identified variants are not considered offtypes. Yet in this study, one variant was unquestionably an offtype while two variants showed no phenotypic difference. Future studies should look into testing variants as potential offtypes. While the stains used in this study tested over 26 known loci, there is still a possibility that there were differences at other non-tested loci that could identify an offtype. Possible future research could include incorporation of more stains per diagnostic test that is still economically viable to commercial diagnostic testing. 39

45 Summary A high level of genetic purity in crop varieties must be reached and maintained for agronomic performance. This ultimately will encourage investment and advancement in plant breeding and ensure that the improvements in productivity and quality will be shared by breeders and delivered to the farmer and, eventually, to consumers. Traditionally, morphological comparisons were done to evaluate genetic purity. However, over time breeders have found that replicated field observations are timeconsuming, expensive, and unreliable. Morphology cannot always provide information on the purity of specific genetic attributes, but isozyme electrophoresis and isoelectric focusing technologies can accurately (as much as 98.89%) and efficiently distinguish varieties within several species including Brassica oleracea. Stains effective in doing so are ACO, AMP, PGD, PGM, IDH, and PHI for isozyme electrophoresis and ADH for isoelectric focusing. Future research will need to be completed to designate comprehensive and cost efficient stains, as well as address variants as potential offtypes. 40

46 REFERENCES Allen R.C. & Budowle, B. (1994). Gel Electrophoreis of proteins and nucleic acids. Retrieved from DeGruyter, Berlin Andrews, A.T (1986). Electrophoresis: theory, techniques, and biochemical and clinical applications Retrieved from (2 nd ed) Oxford University Press. Arus, P. et al. (1993). Inheritence and linkage of isozyme loci in brassica oleracea. Journal of Genetics, 31, Bradford, K.J. (2006). Methods to maintain genetic purity of seed stock. No p Retrieved from UC Davis Publication Burg, C., Henk, J., & Bartel, M. (1996). State of the art of large-scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at Petosluis. Electrophoresis, 17(3), Dunn, M.J (1993). Gel Electrophoresis: Proteins Retrieved from BIO Scientific Publishers Oxford. Garfin, D.E. (1990). Methods in Enzymeology (ed M.P. Deutscher) Vol. 182 p.459 Retrieved from Academic Press, San Diego Garfin, D.E. (1995). Introduction to biophysical methods for protein and nucleic acid research. Retrieved from Academic Press, San Diego. Garfin, D.E. (2000). Handbook of biosepartaions (ed S. Ahuja) p.263 Retrieved from Academic Press, San Diego Gotoh, S. & Ikehasi, H., (2002). Survey of isozyme genes by polyacrylamide gel electrophoresis in cauliflower broccoli and cabbage brassica-oleracea. Japanese 41

47 Journal of Breeding, 42(1), Halim, S., Shaista, & Saxena, (2006). Survey of isozyme genes by polyacrylamide gel electrophoresis in cauliflower broccoli and cabbage brassica-oleracea. Seed Science and Technology, 23(3), Harrington M.G. et al. (1991). Methods: a companion to methods in enzymology Vol. 2 No. 2 p. 98 Retrieved from Academic Press, San Diego Hunter, R.B. & Kannenberg, L.W., (2001). Isozyme characterization of Brassica oleracea L. inbreds and its relationship to single cross hybrid performance. Canadian Journal of Genetic Cytology, 13, Laas, T. (1989). In Protein purification: principals, high resolution methods, and applications (ed. J.-C. Janson and L. Ryden) p. 376 Retrieved from VCH Press Weinheim Micklos, D.A. (n.d.) Gel Electrophoresis What is it and how does it work? Board of Regents And Center for Image Processing in Education. Retrieved from < Nijenhuis, G. (1991). Electrophoretic variability as a tool for determining seed purity And for breeding hybrid varieties in brassica oleracea. Journal of Genetics, 20, Reghetti P.G, Bossi A., & Gelfi C (1998). Gel Electrophoresis: a practical approach Retrieved (3 rd ed.) Oxford University Press Thomson, K., & Taylor, J. (2006). The breakdown of self-incompatibility in cultivars of brassica oleracea. Heredity, 21, Van den berg, M., (1998). Isoelectric focusing in the vegetable seed industry. 42

48 Electrophoresis, 19(10), Vaughan, J., & Waite, A. (2006). Comparitive study of the seed proteins of brassica campestris, brassica oleracea and brassica nigra. Journal of Botany, 17, Westermeier R. (1997). Electrophoresis in practice: a guide to methods and applications of DNA and protein separators (2 nd ed.) Retrieved from VCH Press Weinheim Wills, A., Fyfe, S., & Wiseman, E. (2009). Testing f1 hybrids of brassica oleracea for sibs by seed isozyme analysis. Annals of Applied Biology, 91, Wills, A., & Wiseman, E. (2008). Acid phophatase isoenzyme of brassica oleracea seedlings and their application to sib testing in f1 hybrids. Annals of Applied Biology, 94, Woods, S., & Thurman, D. (2006). The use of seed acid phosphatases in the determination of the purity in f1 hybrids. Journal of Plant Science, 25, Yadava, J.S., Chowdbury, J.B., Kakar, S.N., & Nainawatee, H.S. (1999). Comparitive electrophoretic studies of proteins and enzymes of some brassica species. Theoretical and Applied Genetics, 54, Zhao, T. et al. (2005). Genetic purity testing of two-line hybrid rice seeds by ultrathin Layer isoelectric focusing of proteins. Seed Science and Technology, 33(1),

49 APPENDIX A SAS Code The SAS code is as follows: data planttest; set planttest; proc mixed; class plant trt; model height= trt /ddfm=satterth; random plant; lsmeans trt /pdiff; run; proc mixed; class plant trt; model width= trt /ddfm=satterth; random plant; lsmeans trt /pdiff; run; proc mixed; class plant trt; model heighthead= trt /ddfm=satterth; random plant; lsmeans trt /pdiff; run; proc mixed; class plant trt; model widthhead= trt /ddfm=satterth; random plant; lsmeans trt /pdiff; run; proc mixed; class plant trt; model weighthead= trt /ddfm=satterth; random plant; lsmeans trt /pdiff; run; The model that was used for SAS was class=plant treatment which is one for the females, two for the males and three for the hybrids. This was run through the proc mixed procedure in SAS comparing plant height, leaf spread width, head height and width, and head weight. Here are the results: 44

50 The Mixed Procedure Model Information Data Set Dependent Variable Covariance Structure Estimation Method Residual Variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.PLANTTEST Width Variance Components REML Profile Model-Based Satterthwaite Class Level Information Class Levels Values Plant trt Dimensions Covariance Parameters 2 Columns in X 4 Columns in Z 30 Subjects 1 Max Obs Per Subject 30 Number of Observations Number of Observations Read 30 Number of Observations Used 30 Number of Observations Not Used 0 Iteration History Iteration Evaluations -2 Res Log Like Criterion Convergence criteria met. 45

51 Plant A Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt trt Differences of Least Squares Means head height Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt Differences of Least Squares Means head width Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt Differences of Least Squares Means head weight Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt Plant B Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt trt

52 Plant C Differences of Least Squares Means plant height Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt <.0001 trt <.0001 Differences of Least Squares Means head height Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt <.0001 Differences of Least Squares Means head width Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt <.0001 Differences of Least Squares Means head weight Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt <.0001 trt <

53 Plant D Differences of Least Squares Means plant height Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt trt <.0001 Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt <.0001 Differences of Least Squares Means head height Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt trt <.0001 Differences of Least Squares Means head width Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt trt <.0001 Differences of Least Squares Means head weight Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt trt <

54 Plant E Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt trt <.0001 trt Plant F Differences of Least Squares Means plant width Standard Effect trt trt Estimate Error DF t Value Pr > t trt <.0001 trt <.0001 trt <

55 APPENDIX B Design of Isozyme Electrophoresis Gel Layout The following displays an example of the design of the isozyme electrophoresis test gel layout. The top of the gel is read from the top left and is marked by the notch. The order of the treatments is as follows: AF 1-5, AM 1-5, AH Next is B,C,D,E and F in the same numeric order. There are two protein bands right next to each other for each sample. Each vertical line stains a control used to separate treatments. AF 1-5 AM 1-5 AH 1-10 AH AH B Female BM Control D=Dead Male 50

56 APPENDIX C Design of Isoelectric Focusing Gel Layout Gel is read left to right starting with the first ten hybrids, followed by the female, male and then the remaining the remaining ten hybrids as shown below. Hybrid 1-10 Female 1-5 Male 1-5 Hybrid

57 APPENDIX D Pictures of Plants Outlining Visual Differences Variety A Three female selfs were found in variety A. Pictured below are the three differences initially detected in this study. AH13, AH4 AH13, AH6 AH13, AH17 Each picture begins with a female 2 (far left) and is followed by male 3 and then the found female self. The first picture does show hybrid 13 on the far right to give a comparative visual. The last picture shows a typical female, male and hybrid. Please note the leaf spread from AH17 and AH13 on the bottom two pictures. AF2, AM3, AH4, AH13 AF2, AM3, AH6 52

58 AF2, AM3, AH17 AF2, AM3, AH13 Variety B There were two offtypes visually seen in variety B, Hybrid 15 and Hybrid 16. Pictures displayed show female 3, male 3, and offtype (15 or 16) and hybrid 8 comparatively. Visual differences are shown below. BF3, BM3, BH15, BH 8 53

59 BF3, BM3, BH16, BH 8 Variety D The order of the plants in the picture below (beginning row, the top left) is: D Female 2, D Hybrid 15, D Hybrid 12, and bottom row is D Hybrid 3, D Hybrid 4 and D hybrid 8. Note the male seed was dead on this variety. From visual assessment, D hybrids 3, 4, 8, and 15 are classified as offtypes as they look nothing like the female parent (top left) or the typical hybrid (top right). H3 was taller than the hybrid and female plants, had a different color, and the head develops farther down, closer to the stem. H4 and H8 appeared to be the same off type that had a lighter color and larger head. H15 was a different color with a smaller head, flatter leaf pattern, and the leaves curved upright and were more symmetrical. 54

60 Variety D Pictured below shows a side view of each of the phenotypically found offtypes. DF2, DH3, DH12 DF2, DH4, DH12 55

61 DF2, DH8, DH12 DF2, DH15, DH12 Yield was also part of the visual evaluations. As shown below, head mass varies from each of the offtypes described above. Each picture uses the a female (DF2) which is shown on the far left, the offtype D hybrid 15 (second to the left) and the representative actual hybrid 12 to gauge size relations to the other three offtypes that are shown last. DF2, DH15, DH12, DH3 DF2, DH15, DH12, DH4 DF2, DH15, DH12, DH8 56

62 Variety E Note the branching pattern on variety E identifies a Female Self on Hybrid 15. E Female 2 (EF2) E Male 3 (EM3) E Hybrid 10 (EH10) E Hybrid 15 (EH15) 57

63 IEF Broccoli Variety A APPENDIX E Pictures of Isoelectric Focusing (IEF) Gel 58

64 IEF Broccoli Variety B IEF Cabbage Variety C 59

65 IEF Cabbage Variety D IEF Cauliflower Variety E 60

66 IEF Cauliflower Variety F 61

67 APPENDIX F Isozyme Electrophoresis Genotypes for Variety A, B, C, D, E and F Female, Male and Hybrid Identified Genotypes Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female A 2/2 2/2 A/A,C/C* B/B 2/2 1/1,1/2* 4/4 Male A 2/2 2/2 B/B B/B 1/1,1/2,2/2 1/1 4/4 Hybrid A 2/2 2/2 A/B,B/C B/B 1/2** 1/2** 4/4 * IDH & PGM2 - female appears to segregate, but only saw two types in the five individuals tested ** PGM1 - hybrid does not appear to segregate, different lots in the parent seed was used in the production for the hybrid? Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female B 2/2 2/2 A/A B/B 2/2 2/2 4/4 Male B 3/3 2/2 A/A B/B 2/2 1/1 4/4 Hybrid B 2/3 2/2 A/A B/B 2/2 1/2 4/4 Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female C 1/1 2/2 A/A A/A 2/2 1/1 3/3 Male C 3/3 1/1 D/D C/C 2/2 2/2 1/1 Hybrid C 1/3 1/2 A/D A/C 2/2 1/2 1/3 Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female D 1/1 2/2 B/B D/D 2/2 2/2 1/1 Male D Dead Dead Dead Dead Dead Dead Dead Hybrid D 1/3 1/2 B/D C/D 2/2 2/2 1/1 Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female E 3/3 2/2 E/E B/B 1/1 2/2 1/1 Male E 3/3 2/2 A/A B/B 2/2 2/2 1/1 Hybrid E 3/3 2/2 A/E B/B 1/2 2/2 1/1 Parent Pedigree ACO AMP IDH PGD PGM1 PGM2 PHI Female F Dead Dead Dead Dead Dead Dead Dead Male F 3/3 2/2 A/A B/B 1/1 2/2 1/1 Hybrid F 3/3 2/2 A/A B/B 1/2 2/2 1/1 62

68 Individual Genotypes Experimental Code ACO AMP IDH PGD PGM1 PGM2 PHI A-F-1 2/2 2/2 A/A B/B 2/2 1/2 4/4 A-F-2 2/2 2/2 A/A B/B 2/2 1/2 4/4 A-F-3 2/2 2/2 A/A B/B 2/2 1/2 4/4 A-F-4 2/2 2/2 C/C B/B 2/2 1/1 4/4 A-F-5 2/2 2/2 A/A B/B 2/2 1/2 4/4 A-M-1 2/2 2/2 B/B B/B 1/1 1/1 4/4 A-M-2 2/2 2/2 B/B B/B 1/1 1/1 4/4 A-M-3 2/2 2/2 B/B B/B 1/2 1/1 4/4 A-M-4 2/2 2/2 B/B B/B 1/2 1/1 4/4 A-M-5 2/2 2/2 B/B B/B 2/2 1/1 4/4 A-H-1 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-2 2/2 2/2 B/C B/B 1/2 1/2 4/4 A-H-3 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-4 2/2 2/2 A/A B/B 2/2 2/2 4/4 A-H-5 2/2 2/2 B/C B/B 1/2 1/2 4/4 A-H-6 2/2 2/2 A/A B/B 2/2 2/2 4/4 A-H-7 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-8 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-9 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-10 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-11 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-12 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-13 2/2 2/2 B/C B/B 1/2 1/2 4/4 A-H-14 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-15 2/2 2/2 B/C B/B 1/2 1/2 4/4 A-H-16 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-17 2/2 2/2 A/A B/B 2/2 2/2 4/4 A-H-18 2/2 2/2 A/B B/B 1/2 1/2 4/4 A-H-19 2/2 2/2 B/C B/B 1/2 1/2 4/4 A-H-20 2/2 2/2 A/B B/B 1/2 1/2 4/4 B-F-1 2/2 2/2 A/A B/B 2/2 2/2 4/4 B-F-2 2/2 2/2 A/A B/B 2/2 2/2 4/4 B-F-3 2/2 2/2 A/A B/B 2/2 2/2 4/4 B-F-4 2/2 2/2 A/A B/B 2/2 2/2 4/4 B-F-5 2/2 2/2 A/A B/B 2/2 2/2 4/4 B-M-1 3/3 2/2 A/A B/B 2/2 1/1 4/4 63

69 B-M-2 3/3 2/2 A/A B/B 2/2 1/1 4/4 B-M-3 3/3 2/2 A/A B/B 2/2 1/1 4/4 B-M-4 3/3 2/2 A/A B/B 2/2 1/1 4/4 B-M-5 3/3 2/2 A/A B/B 2/2 1/1 4/4 B-H-1 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-2 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-3 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-4 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-5 2/3 2/2 A/C B/B 2/2 1/2 4/4 B-H-6 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-7 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-8 2/3 2/2 A/C B/B 2/2 1/2 4/4 B-H-9 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-10 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-11 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-12 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-13 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-14 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-15 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-16 2/3 2/2 A/C B/B 2/2 1/2 4/4 B-H-17 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-18 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-19 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-20 2/3 2/2 A/A B/B 2/2 1/2 4/4 C-F-1 1/1 2/2 A/A A/A 2/2 1/1 3/3 C-F-2 1/1 2/2 A/A A/A 2/2 1/1 3/3 C-F-3 1/1 2/2 A/A A/A 2/2 1/1 3/3 C-F-4 1/1 2/2 A/A A/A 2/2 1/1 3/3 C-F-5 1/1 2/2 A/A A/A 2/2 1/1 3/3 C-M-1 3/3 1/1 D/D C/C 2/2 2/2 1/1 C-M-2 3/3 1/1 D/D C/C 2/2 2/2 1/1 C-M-3 3/3 1/1 D/D C/C 2/2 2/2 1/1 C-M-4 3/3 1/1 D/D C/C 2/2 2/2 1/1 C-M-5 3/3 1/1 D/D C/C 2/2 2/2 1/1 C-H-1 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-2 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-3 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-4 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-5 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-6 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-7 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-8 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-9 1/3 1/2 A/D A/C 2/2 1/2 1/3 64

70 C-H-10 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-11 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-12 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-13 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-14 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-15 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-16 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-17 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-18 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-19 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-20 1/3 1/2 A/D A/C 2/2 1/2 1/3 D-F-1 1/1 2/2 B/B D/D 2/2 2/2 1/1 D-F-2 1/1 2/2 B/B D/D 2/2 2/2 1/1 D-F-3 1/1 2/2 B/B D/D 2/2 2/2 1/1 D-F-4 1/1 2/2 B/B D/D 2/2 2/2 1/1 D-F-5 1/1 2/2 B/B D/D 2/2 2/2 1/1 D-M-1 Dead Dead Dead Dead Dead Dead Dead D-M-2 Dead Dead Dead Dead Dead Dead Dead D-M-3 Dead Dead Dead Dead Dead Dead Dead D-M-4 Dead Dead Dead Dead Dead Dead Dead D-M-5 Dead Dead Dead Dead Dead Dead Dead D-H-1 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-2 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-3 3/3 1/2 B/D C/D 2/3 2/2 1/3 D-H-4 3/3 1/2 B/D C/D 2/2 2/2 1/3 D-H-5 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-6 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-7 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-8 3/3 1/2 B/D C/D 2/2 2/2 1/3 D-H-9 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-10 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-11 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-12 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-13 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-14 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-15 3/3 1/1 D/D C/C 2/2 2/2 1/1 D-H-16 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-17 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-18 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-19 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-20 1/3 1/2 B/D C/D 2/2 2/2 1/1 E-F-1 3/3 2/2 E/E B/B 1/1 2/2 1/1 E-F-2 3/3 2/2 E/E B/B 1/1 2/2 1/1 65

71 E-F-3 3/3 2/2 E/E B/B 1/1 2/2 1/1 E-F-4 3/3 2/2 E/E B/B 1/1 2/2 1/1 E-F-5 3/3 2/2 E/E B/B 1/1 2/2 1/1 E-M-1 3/3 2/2 A/A B/B 2/2 2/2 1/1 E-M-2 3/3 2/2 A/A B/B 2/2 2/2 1/1 E-M-3 3/3 2/2 A/A B/B 2/2 2/2 1/1 E-M-4 3/3 2/2 A/A B/B 2/2 2/2 1/1 E-M-5 3/3 2/2 A/A B/B 2/2 2/2 1/1 E-H-1 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-2 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-3 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-4 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-5 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-6 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-7 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-8 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-9 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-10 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-11 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-12 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-13 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-14 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-15 3/3 2/2 E/E B/B 1/1 2/2 1/1 E-H-16 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-17 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-18 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-19 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-20 3/3 2/2 A/E B/B 1/2 2/2 1/1 F-F-1 Dead Dead Dead Dead Dead Dead Dead F-F-2 Dead Dead Dead Dead Dead Dead Dead F-F-3 Dead Dead Dead Dead Dead Dead Dead F-F-4 Dead Dead Dead Dead Dead Dead Dead F-F-5 Dead Dead Dead Dead Dead Dead Dead F-M-1 3/3 2/2 A/A B/B 1/1 2/2 1/1 F-M-2 3/3 2/2 A/A B/B 1/1 2/2 1/1 F-M-3 3/3 2/2 A/A B/B 1/1 2/2 1/1 F-M-4 3/3 2/2 A/A B/B 1/1 2/2 1/1 F-M-5 3/3 2/2 A/A B/B 1/1 2/2 1/1 F-H-1 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-2 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-3 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-4 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-5 3/3 2/2 A/A B/B 1/2 2/2 1/1 66

72 F-H-6 3/3 2/2 A/C B/B 1/2 2/2 1/1 F-H-7 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-8 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-9 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-10 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-11 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-12 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-13 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-14 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-15 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-16 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-17 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-18 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-19 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-20 3/3 2/2 A/A B/B 1/2 2/2 1/1 67

73 Analysis of Genotypes Variety A Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis A-F-1 2/2 2/2 A/A B/B 2/2 1/2 4/4 Stem and Branching is thicker, curves do A-F-2 2/2 2/2 A/A B/B 2/2 1/2 4/4 " A-F-3 2/2 2/2 A/A B/B 2/2 1/2 4/4 " A-F-4 2/2 2/2 C/C B/B 2/2 1/1 4/4 " A-F-5 2/2 2/2 A/A B/B 2/2 1/2 4/4 " A-M-1 2/2 2/2 B/B B/B 1/1 1/1 4/4 First to yield, leaves grow through the br A-M-2 2/2 2/2 B/B B/B 1/1 1/1 4/4 " A-M-3 2/2 2/2 B/B B/B 1/2 1/1 4/4 " A-M-4 2/2 2/2 B/B B/B 1/2 1/1 4/4 " A-M-5 2/2 2/2 B/B B/B 2/2 1/1 4/4 " Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female 2/2 2/2 A/A,C/C* B/B 2/2 1/1,1/2* 4/4 Male 2/2 2/2 B/B B/B 1/1,1/2,2/2 1/1 4/4 Hybrid 2/2 2/2 A/B,B/C B/B 1/2** 1/2** 4/4 * IDH & PGM2 - female appears to segregate, but only saw two types in the five individuls tested ** PGM1 - hybrid does not appear to segregate, different lots in the parent seed was used in the production for the hybrid? A-H-1 2/2 2/2 A/B B/B 1/2 1/2 4/4 Branching surpasses head, leaves grow t A-H-2 2/2 2/2 B/C B/B 1/2 1/2 4/4 " A-H-3 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-4 2/2 2/2 A/A B/B 2/2 2/2 4/4 Female Self Female Self Female self A-H-5 2/2 2/2 B/C B/B 1/2 1/2 4/4 " A-H-6 2/2 2/2 A/A B/B 2/2 2/2 4/4 Female Self Female Self Female self A-H-7 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-8 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-9 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-10 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-11 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-12 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-13 2/2 2/2 B/C B/B 1/2 1/2 4/4 Offtype " A-H-14 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-15 2/2 2/2 B/C B/B 1/2 1/2 4/4 " A-H-16 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-17 2/2 2/2 A/A B/B 2/2 2/2 4/4 Female Self Female Self Female Self A-H-18 2/2 2/2 A/B B/B 1/2 1/2 4/4 " A-H-19 2/2 2/2 B/C B/B 1/2 1/2 4/4 " A-H-20 2/2 2/2 A/B B/B 1/2 1/2 4/4 " 68

74 Variety B Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis B-F-1 2/2 2/2 A/A B/B 2/2 2/2 4/4 Not diagnositc on PGM Smaller than hybrid and male, pronounced axillary B-F-2 2/2 2/2 A/A B/B 2/2 2/2 4/4 " " B-F-3 2/2 2/2 A/A B/B 2/2 2/2 4/4 " " B-F-4 2/2 2/2 A/A B/B 2/2 2/2 4/4 " " B-F-5 2/2 2/2 A/A B/B 2/2 2/2 4/4 " " B-M-1 3/3 2/2 A/A B/B 2/2 1/1 4/4 " Smaller than hybrid, slightly taller than female, pro B-M-2 3/3 2/2 A/A B/B 2/2 1/1 4/4 " " B-M-3 3/3 2/2 A/A B/B 2/2 1/1 4/4 " " B-M-4 3/3 2/2 A/A B/B 2/2 1/1 4/4 " " B-M-5 3/3 2/2 A/A B/B 2/2 1/1 4/4 " " Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female 2/2 2/2 A/A B/B 2/2 2/2 4/4 Male 3/3 2/2 A/A B/B 2/2 1/1 4/4 Hybrid 2/3 2/2 A/A B/B 2/2 1/2 4/4 B-H-1 2/3 2/2 A/A B/B 2/2 1/2 4/4 " Taller than parents, pronounced axillary buds B-H-2 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-3 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-4 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-5 2/3 2/2 A/C B/B 2/2 1/2 4/4 Variant different on only one locus " " B-H-6 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-7 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-8 2/3 2/2 A/C B/B 2/2 1/2 4/4 Variant different on only one locus " " B-H-9 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-10 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-11 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-12 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-13 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-14 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-15 2/3 2/2 A/A B/B 2/2 1/2 4/4 " Offtype, smaller but with similar characteristics as B-H-16 2/3 2/2 A/C B/B 2/2 1/2 4/4 Variant different on only one locus " Offtype, smaller - heads 3 weeks earlier, lighter co B-H-17 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-18 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-19 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " B-H-20 2/3 2/2 A/A B/B 2/2 1/2 4/4 " " 69

75 Variety C Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis C-F-1 1/1 2/2 A/A A/A 2/2 1/1 3/3 Smaller than hybrid - behind on yield, bi C-F-2 1/1 2/2 A/A A/A 2/2 1/1 3/3 " C-F-3 1/1 2/2 A/A A/A 2/2 1/1 3/3 " C-F-4 1/1 2/2 A/A A/A 2/2 1/1 3/3 " C-F-5 1/1 2/2 A/A A/A 2/2 1/1 3/3 " C-M-1 3/3 1/1 D/D C/C 2/2 2/2 1/1 Smaller than hybrid and female - behind C-M-2 3/3 1/1 D/D C/C 2/2 2/2 1/1 " C-M-3 3/3 1/1 D/D C/C 2/2 2/2 1/1 " C-M-4 3/3 1/1 D/D C/C 2/2 2/2 1/1 " C-M-5 3/3 1/1 D/D C/C 2/2 2/2 1/1 " Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female 1/1 2/2 A/A A/A 2/2 1/1 3/3 Male 3/3 1/1 D/D C/C 2/2 2/2 1/1 Hybrid 1/3 1/2 A/D A/C 2/2 1/2 1/3 C-H-1 1/3 1/2 A/D A/C 2/2 1/2 1/3 Taller and larger than parent seed - large C-H-2 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-3 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-4 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-5 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-6 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-7 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-8 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-9 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-10 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-11 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-12 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-13 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-14 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-15 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-16 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-17 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-18 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-19 1/3 1/2 A/D A/C 2/2 1/2 1/3 " C-H-20 1/3 1/2 A/D A/C 2/2 1/2 1/3 " 70

76 Variety D Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis D-F-1 1/1 2/2 B/B D/D 2/2 2/2 1/1 Smaller than hybrid - behind in yield, un D-F-2 1/1 2/2 B/B D/D 2/2 2/2 1/1 " D-F-3 1/1 2/2 B/B D/D 2/2 2/2 1/1 " and infected with Fungus D-F-4 1/1 2/2 B/B D/D 2/2 2/2 1/1 " D-F-5 1/1 2/2 B/B D/D 2/2 2/2 1/1 " D-M-1 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead D-M-2 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead D-M-3 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead D-M-4 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead D-M-5 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female 1/1 2/2 B/B D/D 2/2 2/2 1/1 Male DeadDead Dead Dead Dead Dead Dead Hybrid 1/3 1/2 B/D C/D 2/2 2/2 1/1 D-H-1 1/3 1/2 B/D C/D 2/2 2/2 1/1 Taller and larger than parent seed - large D-H-2 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-3 3/3 1/2 B/D C/D 2/3 2/2 1/3 Offtype Offtype Offtype, taller than hybrid and female, d D-H-4 3/3 1/2 B/D C/D 2/2 2/2 1/3 Offtype Offtype, different color head, larger hea D-H-5 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-6 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-7 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-8 3/3 1/2 B/D C/D 2/2 2/2 1/3 Offtype Offtype, different color head, larger hea D-H-9 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-10 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-11 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-12 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-13 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-14 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-15 3/3 1/1 D/D C/C 2/2 2/2 1/1 Male Self Male Self Offtype/Male? Different color, smaller h D-H-16 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-17 1/3 1/2 B/D C/D 2/2 2/2 1/1 Offtype " D-H-18 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-19 1/3 1/2 B/D C/D 2/2 2/2 1/1 " D-H-20 1/3 1/2 B/D C/D 2/2 2/2 1/1 Offtype " 71

77 Variety E Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis E-F-1 3/3 2/2 E/E B/B 1/1 2/2 1/1 Offtype Lighter color than male, purplish color of E-F-2 3/3 2/2 E/E B/B 1/1 2/2 1/1 " E-F-3 3/3 2/2 E/E B/B 1/1 2/2 1/1 " E-F-4 3/3 2/2 E/E B/B 1/1 2/2 1/1 " E-F-5 3/3 2/2 E/E B/B 1/1 2/2 1/1 " E-M-1 3/3 2/2 A/A B/B 2/2 2/2 1/1 Darker green than female and hybrid, no E-M-2 3/3 2/2 A/A B/B 2/2 2/2 1/1 " E-M-3 3/3 2/2 A/A B/B 2/2 2/2 1/1 " E-M-4 3/3 2/2 A/A B/B 2/2 2/2 1/1 " E-M-5 3/3 2/2 A/A B/B 2/2 2/2 1/1 " Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female 3/3 2/2 E/E B/B 1/1 2/2 1/1 Male 3/3 2/2 A/A B/B 2/2 2/2 1/1 Hybrid 3/3 2/2 A/E B/B 1/2 2/2 1/1 E-H-1 3/3 2/2 A/E B/B 1/2 2/2 1/1 Same color as female, elongated stem co E-H-2 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-3 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-4 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-5 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-6 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-7 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-8 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-9 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-10 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-11 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-12 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-13 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-14 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-15 3/3 2/2 E/E B/B 1/1 2/2 1/1 Female Self Female Self Female Self E-H-16 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-17 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-18 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-19 3/3 2/2 A/E B/B 1/2 2/2 1/1 " E-H-20 3/3 2/2 A/E B/B 1/2 2/2 1/1 " 72

78 Variety F Experimental CodeACO AMP IDH PGD PGM1 PGM2 PHI Electrophoresis Analysis IEF Analysis Phenotypic Analysis F-F-1 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead F-F-2 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead F-F-3 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead F-F-4 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead F-F-5 DeadDead Dead Dead Dead Dead Dead Dead Dead Dead F-M-1 3/3 2/2 A/A B/B 1/1 2/2 1/1 Smaller than the hybrid, erect branching F-M-2 3/3 2/2 A/A B/B 1/1 2/2 1/1 Offtype " with an elongated stem before branchi F-M-3 3/3 2/2 A/A B/B 1/1 2/2 1/1 " F-M-4 3/3 2/2 A/A B/B 1/1 2/2 1/1 " F-M-5 3/3 2/2 A/A B/B 1/1 2/2 1/1 " Parent ACO AMP IDH PGD PGM1 PGM2 PHI Female DeadDead Dead Dead Dead Dead Dead Male 3/3 2/2 A/A B/B 1/1 2/2 1/1 Hybrid 3/3 2/2 A/A B/B 1/2 2/2 1/1 F-H-1 3/3 2/2 A/A B/B 1/2 2/2 1/1 Larger than the parent seed, erect branch F-H-2 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-3 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-4 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-5 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-6 3/3 2/2 A/C B/B 1/2 2/2 1/1 Variant " F-H-7 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-8 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-9 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-10 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-11 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-12 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-13 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-14 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-15 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-16 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-17 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-18 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-19 3/3 2/2 A/A B/B 1/2 2/2 1/1 " F-H-20 3/3 2/2 A/A B/B 1/2 2/2 1/1 " 73

79 APPENDIX G Greenhouse Growout Study Measurements Variety A A 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug head cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height WidthWeight g AF AF AF AF AF AM AM AM AM AM AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH AH

80 Variety B B 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug head cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width height width weight g BF BF BF BF BF BM BM BM BM BM BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH BH

81 Variety C C 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug head cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width height width weight g CF CF CF CF CF CM CM CM CM CM CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH

82 Variety D D 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug head cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width height width weight g DF DF DF DF DF DM1 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DM2 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DM3 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DM4 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DM5 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH

83 Variety E E 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width EF EF EF EF EF EM EM EM EM EM EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH

84 Variety F F 31-May 31-May 7-Jun 7-Jun 14-Jun 14-Jun 21-Jun 21-Jun 28-Jun 28-Jun 5-Jul 5-Jul 12-Jul 12-Jul 19-Jul 19-Jul 26-Jul 26-Jul 2-Aug 2-Aug cm Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width Height Width FF1 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD FF2 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD FF3 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD FF4 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD FF5 DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD DEAD FM FM FM FM FM FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH FH

85 APPENDIX H Pictures of Isozyme Electrophoresis Gels ACO - Aconitase 80

86 Rank Two and Three - 2/2, 3/3, 2/3 B Female 2/2 B Male 3/3 B Hybrid 2/3 81

87 Rank Four and Five 1/1, 3/3, 1/3 D Female 1/1 D Hybrid 1/3 ****D Male Dead (should be 3/3)**** Offtypes 3/3 (D hybrids 3, 4, 8, 15) OT 3/3 OT 3/3 1/3 OT 3/3 OT = Offtype 82

88 AMP - Arginine aminopeptidase 83

89 Rank Five 1/1, 2/2, 1/2 (male dead) D Female 2/2 D Hybrid 1/2 ****D Male Dead (should be 1/1)**** D Male Self 1/1 (hybrid 15) D Hybrid 11 1/2 D Hybrid 15 - Male Self (1/1) 84

90 IDH Isocitric dehydrogenase 85

91 Rank One A/A, B/B, C/C, A/B, B/C A Segregating Female - A/A, C/C A Male - B/B 1,2,3,5 - A/A 4 - C/C A Hybrids 11, 12, 13 - A/B, B/C A/B A/B B/C A Female Self - A/A (hybrid 4 and 6) Female Self (A/A) 86

92 Rank Three - A/A, D/D, A/C B Female, Male and Hybrid - A/A (with A/C Variants on hybrid 8 and 16) A/C 8 A/A 11 A/C 16 C Female - A/A C Male - D/D Rank Four - A/D, B/B C Hybrid A/D D Female B/B 87

93 Rank Five B/B, D/D, B/D D Female B/B D Hybrid B/D ****D Male Dead (should be D/D)**** D Male Self D/D (hybrid 15) Hybrid 11 B/D Hybrid 15 - D/D 88

94 Rank Seven - E/E, A/E E Female E/E E Male - A/A E Hybrid A/E E Female Self E/E (hybrid 15) Hybrid 12 A/E Hybrid 15 E/E (female self) 89

95 Rank Eight A/C, A/A F Male A/A F Hybrid A/C ****F Female Dead (should be C/C)**** E Variant A/A (hybrid 6) Hybrid 6 A/A Hybrid 8 - A/C 90

96 PGD - 6-phosphogluconate dehydrogenase 91

97 Ranks Three and Four - A/A, B/B, C/C, D/D, A/C B Hybrid B/B C Female A/A C Male C/C C Hybrid A/C D Female D/D 92

98 Rank Five - C/C, D/D, C/D D Female D/D D Hybrid C/D ****D Male Dead (should be C/C)**** D Male Self C/C (hybrid 15) Hybrid 11 C/D Hybrid 15 C/C(light, but 3 banded) 93

99 PGM - Phosoglucomutase 94

100 PGM 1 Ranks One and Two 1/1, 2/2, 1/2 A Female 2/2 A Segregating Male 1/1, 2/2, 1/2 1/1 1/1 1/2 1/2 2/2 Male1&2 = 1/1, Male 3&4 = 1/2, Male 5 = 2/2 A Hybrid 1/2 A Female Self 2/2 (hybrids 4, 6 and 17)

101 Rank Five 2/3, 2/2 D Female 2/2 D Hybrid 2/2 ****D Male Dead (should be 2/2)**** Offtype 2/3 (D Hybrid 3) D Hybrid 1 2/2 D Hybrid 3 2/3 Offtype 96

102 Rank Seven 1/1, 2/2, 1/2 E Female 1/1 E Male 2/2 E Hybrid 1/2 E Female Self 1/1 (hybrid 15) E Hybrid 15 (female self) 1/1 E Hybrid 18 1/2 97

103 PGM 2 Rank One 1/1, 1/2 A Segregating Female 1/1, 1/2 A Male 1/1 ***Female also should be 2/2*** 1,2,3,5 1/2 4 1/1 A Hybrid - 1/2 A Female Self 2/2 (hybrids 4, 6 and 17) 4 2/2 6 2/2 8 1/2 17 2/2 98

104 PHI - Phosphohexose isomerase 99

105 Ranks Three and Four - 1/1, 3/3, 4/4, 1/3 B Female, Male and Hybrid 4/4 C Female 3/3 C Male 1/1 B Hybrid - 4/4 C Female - 3/3 C Male 1/1 C Hybrid 1/3 100

106 Rank Five - 1/1, 1/3 D Female 1/1 D Hybrid 1/1 ****D Male Dead (should be 1/1)**** Offtypes 1/3 (D Hybrid 3, 4 and 8) D Hybrid 3 and 4 1/3 1/1 D Hybrid 8 1/3 101