Herbivores and plant defences affect selection on plant reproductive traits more strongly than pollinators.

Pollinators and herbivores can both affect the evolutionary diversification of plant reproductive traits. However, plant defences frequently alter antagonistic and mutualistic interactions, and therefore, variation in plant defences may alter patterns of herbivore- and pollinator-mediated selection on plant traits. We tested this hypothesis by conducting a common garden field experiment using 50 clonal genotypes of white clover (Trifolium repens) that varied in a Mendelian-inherited chemical antiherbivore defence-the production of hydrogen cyanide (HCN). To evaluate whether plant defences alter herbivore- and/or pollinator-mediated selection, we factorially crossed chemical defence (25 cyanogenic and 25 acyanogenic genotypes), herbivore damage (herbivore suppression) and pollination (hand pollination). We found that herbivores weakened selection for increased inflorescence production, suggesting that large displays are costly in the presence of herbivores. In addition, herbivores weakened selection on flower size but only among acyanogenic plants, suggesting that plant defences reduce the strength of herbivore-mediated selection. Pollinators did not independently affect selection on any trait, although pollinators weakened selection for later flowering among cyanogenic plants. Overall, cyanogenic plant defences consistently increased the strength of positive directional selection on reproductive traits. Herbivores and pollinators both strengthened and weakened the strength of selection on reproductive traits, although herbivores imposed ~2.7× stronger selection than pollinators across all traits. Contrary to the view that pollinators are the most important agents of selection on reproductive traits, our data show that selection on reproductive traits is driven primarily by variation in herbivory and plant defences in this system.

HERITABILITY OF CLOVER ROT RESISTANCE (SCLEROTINIA SPP.) IN RED CLOVER (TRIFOLIUM PRATENSE) POPULATIONS.

European red clover (Trifolium pratense) crops are susceptible to clover rot, a destructive disease caused by Sclerotinia trifoliorum or S. sclerotiorum. The lack of knowledge on the heritability of clover rot resistance is, among other reasons, responsible for the slow progress of resistance breeding. In this paper, we acquired insight in the heritability of clover rot resistance through divergent selection by our high-throughput bio-test on an experimental diploid population. The disease susceptibility indices of the first generation after selection for susceptibility and the first and the second generation after selection for resistance were compared with the susceptibility of the original population. The susceptible population (79.2%), the original population (70.5%) and the first generation resistant population (62.3%) differed significantly in susceptibility (p < 0.001). The first (62.3%) and second generation resistant population (60.0%) did not differ significantly in susceptibility. The heritability (h2) of clover rot resistance was low: 0.34 and 0.07 in the first and second cycle of selection respectively. This indicates that mass selection is not suitable to improve clover rot resistance. Family selection may allow a sustained increase in resistance for multiple generations.

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene.

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Infection processes and involvement of defense-related genes in the expression of resistance in cultivars of subterranean clover (Trifolium subterraneum) to Phytophthora clandestina.

Studies on infection processes and gene expression were done to determine differential responses of cultivars of Trifolium subterraneum resistant and susceptible to infection by races of Phytophthora clandestina. In the infection process study, one race was inoculated onto the roots of T. subterraneum cvs. Woogenellup and Junee (compatible or incompatible interactions, respectively). There were no differences in relation to the processes of cyst attachment, germination, and hyphal penetration. There were, however, major differences in infection progression observed post-penetration between compatible and incompatible interactions. In susceptible cv. Woogenellup, hyphae grew into the vascular bundles and produced intercellular antheridia and oogonia in the cortex and stele by 4 days postinoculation (dpi), oospores in the cortex and stele by 8 dpi, when sporangia were evident on the surface of the root. Infected taproots were discolored. Early destruction of taproots prevented emergence of lateral roots. Roots of resistant cv. Junee showed no oospores or sporangia and no disease at 8 dpi. In the gene expression studies, two races of P. clandestina were inoculated onto three cultivars of T. subterraneum. Results showed that three genes known to be associated with plant defense against plant pathogens were differentially expressed in the roots during compatible and incompatible interactions. Phenylalanine ammonia lyase and chalcone synthase genes were activated 4 h postinoculation (hpi) and cytochrome P450 trans-cinnamic acid 4-monooxygenase gene was activated 8 hpi in the incompatible interactions in cvs. Denmark and Junee following inoculation with Race 177. In contrast, in compatible interactions in cv. Woogenellup, there were no significant changes in the activation of these three genes following inoculation, indicating that these three genes were associated with the expression of resistance to Race 177 of the pathogen by the host. To confirm this result, in the second test, cv. Woogenellup was challenged by Race 000 of P. clandestina. In this incompatible interaction, cv. Woogenellup was resistant and expressed highly all three genes in the manner similar to the incompatible interactions observed in the first test.

Mapping candidate QTLs related to plant persistency in red clover.

Red clover (Trifolium pratense L.) is a diploid (2n = 14), self-incompatible legume that is widely cultivated as a forage legume in cold geographical regions. Because it is a short-lived perennial species, improvement of plant persistency is the most important objective for red clover breeding. To develop a marker-assisted selection (MAS) approach for red clover, we identified candidate QTLs related to plant persistency. Two full-sib mapping populations, 272 x WF1680 and HR x R130, were used for QTL identification. Resistance to Sclerotinia trifoliorum and Fusarium species, as well as to winter hardiness, was investigated in the laboratory and in field experiments in Moscow region (Russia), and Sapporo (Japan). With the genotype data derived from microsatellite and other DNA markers, candidate QTLs were identified by simple interval mapping (SIM), Kruskal-Wallis analysis (KW analysis) and genotype matrix mapping (GMM). A total of 10 and 23 candidate QTL regions for plant persistency were identified in the 272 x WF1680 and the HR x R130 mapping populations, respectively. The QTLs identified by multiple mapping approaches were mapped on linkage group (LG) 3 and LG6. The significant QTL interactions identified by GMM explained the higher phenotypic variation than single effect QTLs. Identification of haplotypes having positive effect QTLs in each parent were first demonstrated in this study for pseudo-testcross mapping populations in plant species using experimental data.