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Downy mildew, caused by the oomycete Bremia lactucae, is a devastating disease in lettuce (Lactuca sativa) cultivation, leading to high yield losses. An effective method of downy mildew control is the deployment of resistant lettuce cultivars. Over 50 monogenic, dominant resistance (R) genes have been deployed in lettuce breeding. R genes provide high levels of resistance, but are continually rendered ineffective by the occurrence of pathogenic strains with new virulence characteristics. This demonstrates the presence of an arms race between plant and pathogen. R genes may be an effective but short-term solution to control downy mildew in lettuce, as long as sufficient resistance sources can continuously provide novel R genes. However, the continuous introgression of new R genes in elite breeding material demands much time of breeders and is not a durable solution. Until now, most resistance genes have been derived from the primary gene pool of cultivated lettuce. The wild lettuce species L. saligna is a member of the secondary gene pool, completely resistant to all B. lactucae isolates and considered as a nonhost for downy mildew. Despite its potential attractiveness as a resistance source, L. saligna has hardly been exploited for lettuce breeding.
In chapter 2, we explored the potential of screening for R genes in L. saligna by use of pathogen candidate effectors. Effectors are pathogenic proteins to aid infection of specific plant species. We transiently expressed 16 candidate effectors from B. lactucae in a diverse Lactuca germplasm set (n=150). Accessions that react with a hypersensitive response (HR) are supposed to carry an R gene that recognizes the effector as an avirulence factor. Two candidate effectors (BLN08 and BLR31) induced an HR in L. saligna accessions. BLN08 triggered an HR in 55% of the accessions, but in segregating populations responsiveness did not co-segregate with resistance to Bl:24, the B. lactucae race from which the effectors were cloned. BLR31 induced an HR in 5% of the accessions, and revealed a novel R gene providing resistance to isolate Bl:24. Consequently, we identified a candidate avirulence effector of B. lactucae and its cognate R gene in L. saligna. Additionally, resistant backcross plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. Our results suggested that R genes against B. lactucae seem common in L. saligna, but they are not essential for nonhost resistance (NHR).
As a nonhost for downy mildew, L. saligna harbours potentially durable resistance genes. The genetics of NHR are poorly understood. Inheritance studies of NHR are uncommon, because in general host and nonhost species are so much diverged that they are not cross-fertile anymore. The plant-pathosystem of lettuce and downy mildew provides a rare opportunity to study the inheritance of NHR, as the nonhost species L. saligna is cross-compatible with the host species L. sativa. In previous genetic studies on backcross inbred lines (BILs) and F2 populations, multiple individual, often plant stage dependent QTLs for resistance were identified in L. saligna CGN05271. However, stacking eight combinations of two race-nonspecific QTLs by intercrossing of BILs did not result in greatly elevated levels of resistance. These findings indicated that NHR in L. saligna may be due to a combination of epistatic genes, which individually show no or only small effects.
To identify such a combination of epistatic genes that together confer NHR, we used a bi-directional backcross approach in chapter 3. All previous NHR studies in L. saligna were based on one accession, CGN05271. Here, we selected nine L. saligna accessions from a wide geographic origin, to study NHR in L. saligna as a species. Variation in infection severity levels among F1 and backcross (F1 x L. sativa) populations of these nine accessions, suggested genetic variation for gene dose of NHR. Two accessions probably harbour a lower genetic dose for NHR than the other accessions. Selective genotyping of highly resistant backcross (F1 x L. sativa) plants of the multiple accessions, showed four loci with an overrepresentation of L. saligna alleles. Confirmation of one of these four loci (LG8) was found in inbred offspring of F1 plants backcrossed to the L. saligna parent with a homozygous L. sativa introgression on LG8 and relatively high infection levels. Combinations of three out of these four L. saligna loci seem to lead to complete resistance and could represent a core set of essential NHR genes.
Interspecific crosses are often hampered by reproductive barriers, which can lead to inviability, infertility and non-Mendelian skewed segregation of alleles (transmission ratio distortion, TRD) in hybrids and their derived progeny. This is due to genetic incompatibilities between species, a phenomenon known as hybrid incompatibility (HI). In chapter 4, we studied postzygotic reproductive barriers between L. saligna and L. sativa. Genome-wide analysis of TRD in F2 and two BC1 populations gave an indication of HI genes between wild and cultivated lettuce. Ten TRD loci (TRDL) and subsets of these were detected in the F2, and in the BC1 respectively. Four out of eight TRDL with a L. sativa allele preference in the F2 were associated with an absolute HI in the BILs, meaning that no single homozygous L. saligna introgression could be retrieved in a L. sativa background. Three HI loci were due to a heterospecific two-locus interaction. One of the deleterious digenic interactions leading to HI was characterized in detail and was caused by non-transmission of one heterospecific allele combination, in both the male and female gametophyte.
In chapter 5 the main results of this thesis are evaluated in a broader perspective. The contribution of these results to our understanding of NHR in L. saligna are addressed. A genetic model for NHR is proposed in which a core set of epistatic loci is possibly supplemented with R genes or individually effective QTLs. Three scenarios for the evolution of NHR in L. saligna are suggested and some future perspectives for lettuce and downy mildew research are discussed.
|Qualification||Doctor of Philosophy|
|Award date||18 Apr 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|