To avoid contamination when handling anthers and pollen, I cleansed our fingers and tweezers by splashing fresh alcohol before and after every use. When possible, each of the mix pollen crosses was replicated at least twice on each plant. Seed paternity – We collected at most five young leaves when available or any leaves from all father, mother, and offspring plants. The tissue collected in the field or at the greenhouse was immediately packed inside labeled clear plastic envelopes, placed inside a cooler with dry ice and promptly transferred to be stored in -80° C until initiating the DNA extractions. We determined paternity by genotyping microsatellite loci or short tandem repeats previously used for Brassicaceae species . Total genomic DNA was extracted from 300 mg of leaf tissue collected using the DNAeasy Plant Mini kit . We followed the kit’s instructions only modifying the elution step by reducing the amount of buffer to 50 µl to yield 100 µl of final product. Ten pairs of previously developed primers for Brassicaceae were initially tested and screened for amplification and detection of polymorphism among the five fathers. DNA concentration was quantified using a micro-volume UV-vis spectophotometer Nanodrop 2000 . Among those ten primers I chose the four most informative and polymorphic comparatively among the five fathers .Polymerase chain reactions amplifications of the four loci were performed in a 20 µL total volume with X 0.3 U of Taq polymerase , 2 µL of 10X buffer , 10 mM dNTP, 10 µM/L primers, 10 µM M13 dye and 1.2 µL of ~5-40 ng total DNA. For each locus, procona London container the forward primer had a M13 tail labeled with a fluorescent dye .
A pigtail sequence was attached to each reverse primer to avoid scoring problems due to genotyping errors as a result of adenosine addition artifacts . Amplification was performed as follows: 94o C for 5 min, 30 cycles of 94o C for 30s, 56o C for 45s, and 72o C for 45s followed by 8 additional M13 tail cycles of 94o C for 30s, 53o C for 45s and 72o C for 45s and a final extension of 72o C for 10 min. Analysis of microsatellite fragment size for all four loci were done in a Big Dye Terminator v3.1 sequencing chemistry .Within-fruit seed characteristics – Data were normalized when needed and feasible with log-normal or Box Cox transformations . Significant probability values were adjusted a posteriori with sequential Bonferroni tests to adjust for type I error . One-way analyses of variance were used to compare seed weight at the lineage and population level. Significant results were followed by TukeyHSD post hoc tests for multiple paired comparisons of means at the lineage and populations levels. To compare within-fruit seed characteristics among different seed positions I used a Kruskal-Wallis tests to compare: seed weight, within-fruit seed weight percentage, and the relative within-fruit seed fecundity. For the purpose of these tests, I discerned among the three types of crosses: either mix or single hand pollination or the control open pollinated plants. Multiple regressions followed by the associated ANOVA were performed to assess the effect of lineage, population, type of cross, maternity, paternity and seed weight.
Comparisons among cross types and paternal siring frequencies at different fruit positions were assessed with goodness of fitness chisquare tests, which were followed by Pearson’s chi-square with 10000 permutations the differences among row were lower than 5. All statistical tests were implemented using the R statistical program and extra statistical R packages were downloaded from the Comprehensive R Archive Network . Paternity – We scored genotypes of father, mother and offspring individuals by visualization of the results in GeneMapper Software 3.7 . A genotype with a single PCR fragment was considered a homozygote having two identical alleles. Visual inspection of allele assignments and manual corrections were systematically done. We employed the exclusion parentage analysis to determine from the pool of fathers used, which one sired a particular seed by comparing the genotype of the three or four father candidates and the known mother to the focal progeny. We determined multiple paternity by comparing the siring fathers at different seed positions within the same fruits. We also assessed whether the fathering occurred in a non-random manner by measuring the frequency at which the siring occurred. Finally, I compared the performance of the fathers by calculating at the offspring siring times, seed weight, within-fruit seed weight percentage and within-fruit relative fecundity and per fathers.Knowing that lineages and populations have a significant influence on weight, I moved on to compare, within lineages, if the type of crosses and within-fruit seed position influence seed weight, within-fruit seed weight percentage and within-fruit seed fecundity . We tested this by using Kruskall-Wallis tests independently for each seed position and seed position bin. The tests were done for each type of cross individually within each lineage. Figures 3.2, 3.3, and 3.4 graphically show the average values from our data set. Within-fruit seed positioning has no statistical significant influence on weight. A trend for heavier seeds at peduncular positions for the hybrid derived lineage CAwr in control fruits can be visualized in figure 3.2. The opposite trend seems to be true for the wild radish Rr. The cultivar Rs has a sinusoidal trend. In the case of the percentage of within-fruit seed weight, seed position per se : influences significantly control, single and mixed crosses fruits of CAwr, and influences significantly mixed Rr and slightly single Rr fruits. Seed position bins only moderately influence within-fruit seed weight percentage in control CAwr fruits. No effects of seed position were found on within-fruit seed fecundity. One-way analysis of variance followed by Tukey post-hoc tests were performed to test if seed position bins for each lineage and type of cross had an effect on seed weight .
The results suggest that only in the case of CAwr control plants is there a significant effect of the bins on seed weight. In this particular case, the Tukey post-hoc test reveals that it is the stylar end bin compared to the peduncular end were the difference lies with a significant negative effect on seed weight. Fecundity and relative fitness – A total of 540 crosses formed viable fruits out of the 595 crosses that I performed. Among the 949 seeds found viable after extraction and first visual inspection, 312 seeds were transplanted to the common gardens at AgOps and among those 247 survived to the end of the experiment. Within lineages, cut flower transport bucket multiplicative fitness functions for mixed and single crosses reveal that total relative fitness was not significantly different among seeds from either cross for both progenitors and marginally different for the hybrid-derived lineage . For Rs the difference was significant as a result of lower viable seed/pod. When the fitness functions for mix and single crosses were compared within each lineage differences were found in all three cases. Surprisingly, mix crosses had lower fitness than single crosses for Rr and CAwr, and the opposite is true for Rs. Differences in fecundity, number of viable pods per pollination and seed viability, while not always significant, affect the overall fitness. Paternity – We found evidence of within-fruit multiple paternity, i.e. seed sired by different fathers within the same fruit, for all three lineages: 11 out of 11 fruits for CAwr, 9 out of 11 for Rr and 1 out of 2 for Rs. Fruits from single crosses had all seed sired by the chosen father. As mentioned before, seed viability was an issue for our experiment. Very few fruits with all or most seeds survived to the end of the experiment, in particular for Rs . For this reason I cannot accurately assess if the multiple paternity is or is not random. Also, because I had so few plants from Rs that survived until the end of the experiment, I eliminated them from the rest of the analysis . The percentage of siring, seed weight average, within-fruit seed percentage average, and within-fruit seed fecundity average for CAwr and Rr for mixed and single crosses are represented in appendices I.1, I.2, J.1, and J2. The siring success of the five CAwr fathers is provided in table 3.7. Each column contains the results by individual father, and each row within a division is the value per seed position bin. All of the values in table 3.7 pertain only to mixed crosses with CAwr and Rr mothers because there was insufficient sample size to analyze the crosses with Rs mothers.
The number of seeds sired in each of the three seed position bins did not differ significantly by father. However, when the number of seeds sired by each father was expressed relative to the number of times a particular father was included in a mixed pollination, the percentages of siring were significantly different. Father 1 and father 2 had higher success siring than the other 3 fathers . Their total percentage of siring exceeds 100% because they sired seeds at least twice within same fruits. Father 2 was the most successful siring at both stylar and peduncular portions of the fruits. Seed weight average and within-fruit seed weight did not differ significantly among fathers. In contrast, the highest average within-fruit seed weight was found on seeds sired by father 2 at peduncular position. The within-fruit fecundity did differ significantly among fathers. Father 2 had higher fecundity relative to the other fathers, at all seed position bins . In mixed crosses, when I assessed each father at different seed position bins, I found that father 2 has higher and moderately to significantly different: siring percentage, average seed weight and percentage of seed weight at peduncular portions of the fruits However its within-fruit relative fecundity was the same in all seed position bins. Father 3 has a moderately higher average seed weight at stylar positions and significantly higher within-fruit relative fruit of seed at peduncular positions. In single crosses, the only significantly different performances across seed position bins happened for average seed weight of seeds sired by father 4 at middle positions and within-fruit relative fecundity of seeds sired by father 4 and father B at peduncular positions. Father performances vary when pollinating in single and mixed pollen crosses. When I assessed lineage-by-lineage, fathers and seed position bins, I found that CAwr offspring resulting from mixed crosses fruits, father 2 appears to have the highest siring percentage at seeds in peduncular ends, with highest average within-fruit seed weight and fecundity at all sections of the fruit. Father 3 has the highest seed weight at stylar and peduncular ends but high average fecundity only at stylar end seeds . These results are not replicated in the case of single pollen crosses. In the case of Rr offspring resulting from mixed crosses, father 2 also sired the most seeds but this time at the stylar end with highest average within-fruit seed fecundity. Here also the results were not replicated at the single pollen crosses. Allele frequencies in father, mother and offspring are compiled in appendix K. Maternal and paternal effects – Maternal effects are significant at phenological life cycle level with significant effect on days to germination, days to first true leaf emergence, and final plant weight. Paternal effects significantly influence reproductive output such as total fruit production and potential reproduction as well as offspring seed weight . Fathers also marginally influence cotyledon width and days to flower buds emergence. Lineage and population influence both morphological as well as fitness related characters including seed weight, which is consistent with our previous results. Seed weight is also influenced by the type of cross but not by seed position bins. Seed weight influences cotyledon width and days to first true leaf emergence.Previous studies have demonstrated non-radom multiple paternity for the hybrid derived CAwr fruits . Our results show that multiple paternity also occurs in both progenitor lineages. Because very few whole fruits were represented in the offspring that survived until the end of the experiment, I were unable to determine if the distribution of paternal DNA is non-random with respect to seed position within the pod. Across lineages, for mixed crosses only, the father that sired most seeds was the one from which offspring were the most fecund. Mixed and single hand pollinations gave different results for individual fathers at different sections of the fruits.