Factores ambientales

Harvested seed yields from the designated maternal genotypes of the bi-parental crosses were in the expected range of 0.5-2 g plant-1 _{(Ford, 2013). Polycross seed yields on the other hand}

were lower than expected. The lower than expected seed yields may have been due to a number of contributing factors including but not limited to: higher than average rainfall and lower than average temperatures for the month of December 2011, insufficient number of pollinators, crossing duration and residue insecticides. Mortality rates for bumble bees inside the isolation cages were not recorded, although multiple bees had to be replaced over the crossing period; indicating environmental conditions were not favourable. All parental clones had ample numbers of inflorescences to reach expected seed yields (Appendix A.2), and thus inflorescence density was unlikely to be a factor.

Despite the lower than expected polycross seed yields, the technique of using wild bumble bees for pollination was successful. The bumble bees generated half and full sibling families with low and no detectable levels of self-fertilized progeny (Tables 3.1 and 3.3). The low levels of self-fertilization are consistent with the reported strong self-incompatibility of white clover (Atwood, 1940; Wright, 1939). It is important to note that no detectable levels of self-

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fertilized progeny, based on SSR marker analyses, were present in any of the sampled bi- parental progenies. Selfed progeny can alter the co-variances among families and

consequently bias estimated genetic variance components. Manual emasculation and hand crossing can be used instead of bees to ensure purity of full-sib families as reported by Jahufer (1999), however a drawback associated with this technique is the restriction on seed quantity and number of families generated due to its labour intensive requirements.

Due to seed availability for each clone and unsuccessful paternal assignments, there was an uneven number of progeny per maternal clone analysed. An unbalanced number of progeny per maternal clone may lead to biased results (Riday et al., 2013), and therefore progeny counts for each paternal genotype were adjusted accordingly to remove the effect of unequal maternal sampling. In the presence of random mating, progeny counts should be equal among paternal parents; however paternal counts deviated from random mating ratios in both

isolation cages (Table 3.2). The fluctuating range of paternal progeny counts highlights the extent of non-random mating within the isolation cages. It is interesting to note that genotype 18 had only two paternal progeny among both isolation cages, indicating a degree of male infertility. The cause of the partial male infertility is yet to be diagnosed, but it may provide a valuable germplasm source for future white clover breeding programmes.

The linear relationship between polycross maternal seed yield and paternal progeny counts suggests high seed yielding maternal parents also tended to have higher paternal progeny counts (Figure 3.4). A similar pattern was observed by Riday (2013) in a 15 parent lucerne

(Medicago sativa L.) polycross, although the function of that relationship was primarily due

to self-fertilization. Due to the minimal self-fertilization among parents (<1%) within this experiment, a likely explanation for the positive correlation in Figure 3.4 is the effect of higher seed yielding maternal parents having more harvested inflorescences (Appendix A.2). Assuming the number of inflorescences harvested at seed maturation is representative of the actual number of inflorescences during pollination (no counts during pollination were carried out) there was a positive correlation between number of inflorescences at pollination and success of siring offspring. In other plant species, more flowers per plant have been shown to increase pollinator visits (Galloway et al., 2002; Mitchell, 1994) and in some instances, increasing their siring success (Broyles and Wyatt, 1990). It is likely that in this experiment, genotypes with more inflorescences attracted more pollinators which in turn increased the frequency of pollen transfers between donor and recipient plants, thereby increasing their paternal outcross count.

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Genotypes with more harvested inflorescences did not have a higher frequency of selfed progeny. In lucerne (Medicago sativa L.), which is similar to white clover as an outcrossing insect-pollinated forage species (De Lucas et al., 2012), Strickler and Vinson (2000) reported that genotypes with a greater number of flowers were also inclined to have an increased level of self-fertilized progeny, as pollinators are more likely to move between flowers on the same plant. The lack of observed self-fertilized progeny in this population again highlights the strength of the self-incompatibility system in white clover. Paternity testing also confirmed the absence of any self-fertile genotypes, which have been shown to occur at low frequencies within white clover populations (Atwood, 1941).

The nil detection of foreign alleles in both the bi-parental and polycross progeny suggests the introduced bumble bees were clean of residual pollen, which is a reassuring result for field breeders using the technique described by Williams (1987). The result also confirms no handling contaminations or mistakes were made during crossing, harvest, seed cleaning and sowing. The above demonstrates the strength of paternity testing for not only increasing the efficiency of breeding methods (Chapter 5) but for also ensuring purity and diversity of cross- pollinated breeding pools.

3.4.2 Pollen distribution

The relationship between full-sib family progeny counts and distance of pollen donors from recipient maternal parents (inter-full-sib parent pollination distance) clearly indicates that positioning of pollen donor parents is imperative for successful outcrossing with recipient maternal parents (Appendix A.1, Figures 3.5 and 3.6). Similar leptokurtic patterns of pollen distribution have been described in other studies of white clover under field conditions (Clifford et al., 1996; De Lucas et al., 2012; MichaelsonYeates et al., 1997; Weaver, 1965). The exponential decay in pollinator success at increasing recipient parent distances appears to be a function of the small movements made by pollinators between succeeding inflorescences (Weaver, 1965) and a pollen dilution effect, whereby effective pollen came mostly from the last compatible inflorescence visited (MichaelsonYeates et al., 1997). Although all of these studies have investigated pollen transfer, the majority have been investigated under field conditions. Due to the requirement of isolated cages for rapid and consistent development of breeding populations, it is also imperative that pollen distribution is understood within isolation cages for white clover.

In lucerne, Riday (2013) reported a negative power function relationship between full-sib family progeny counts and inter-fullsib parent pollination distance in a 15 parent isolated cage

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polycross. Michaelson-Yates et al. (1997) appears to be the sole study of pollination

distribution within isolated polycross cages for white clover. Their results again confirm the significant relationship between parent distance and siring success. The results of this study, while similar to Michaelson-Yates et al. (1997), show the foraging behaviour of bumble bees in an actual white clover breeding pool with a wider range of phenotypically diverse parents as encountered in breeding programmes.

Despite previous reports in the literature, it was alarming to see such a large neighbour effect in a small 20 parent polycross, where even the furthest pollen donor plant was within a close proximity of the recipient maternal parent (179 cm). As illustrated in Figures 3.5 and 3.6, a large proportion of this nearest neighbour pollination affect was due to the unequal

frequencies of potential full-sibs at closer rather than further distances. Figure 3.5 shows the likely pollination pattern in an unrestricted sized cage where full-sib frequencies are similar at various inter-full-sib parent pollination distances, whereas Figure 3.6 shows the pollination pattern in smaller cages like that used in this experiment. An unbiased relationship between full-sib progeny counts and inter-full-sib parent pollination distance could only be achieved in a wagon wheel like design, where each distance has the same number of potential outcrossing parents as presented in a field study by De Lucas et al. (2012).

It is interesting to note that despite the strong influence of inter-full-sib parent pollination distance on full-sib family progeny counts, there still seems to be variation in full-sib progeny counts between paternal parents within the same identical inter-full-sib parent pollination distance for any given maternal parent (Appendix A.1). For example, the progeny counts within a maternal half-sib family were not distributed evenly amongst all potential paternal parents at any given pollination distance. This non-uniformity warrants investigation as to whether there is a pollination pattern among paternal parents at the same distance. If a clear pattern emerges, candidate traits such as flower number, nectar flow and flower colour could be monitored, which have been shown to influence pollinators in legume species (Bosch et al., 1996; Clement, 1965).