The genetics of non-host resistance to the lettuce pathogen Bremia lactucae in Lactuca saligna

Plants are continuously exposed to a wide variety of pathogens. However, all plant species are non-hosts for the majority of the potential plant pathogens. The genetic dissection of non-host resistance is hampered by the lack of segregating population from crosses between host and non-host species, since hardly any non-host is crossable with a host. We have studied the non-host resistance in Lactuca saligna (wild lettuce) to lettuce downy mildew ( Bremia lactucae ). L. saligna is one of the few examples of a non-host species that is crossable with a related host species, L. sativa (lettuce). Based on this interspecific cross, segregating populations have been developed for genetical analysis of the non-host resistance. To map the resistance, we have used two strategies in which we make use of DNA markers to genotype plants. As no accurate linkage map was available for lettuce, we started with the construction of a linkage map of L. saligna´L. sativa . In Chapter 2, the development of an integrated linkage map, based on two populations, is described. To acquire DNA markers, AFLP analyses have been performed on the F 2 populations of the crosses L. saligna CGN 5271 ´L. sativa Olof and L. saligna CGN 11341´L. sativa Norden. Based on these AFLP analyses the polymorphism rate between L. saligna and L. sativa is estimated to be 81%. A linkage map was constructed that comprises 12 SSRs and 476 AFLP markers over 854 cM in nine linkage groups (n=9). Since the markers are randomly spread over all chromosomes, we assume this map is an accurate representative of both parental genomes and very useful for Marker Assisted Selection.The first mapping strategy for downy mildew resistance is described in Chapter 3. In that study, we have performed a QTL analysis on 126 F 2 plants of a cross between the resistant L. saligna CGN 5271 and the susceptible L. sativa Olof. For this QTL analysis all 126 F 2 plants have been tested for resistance in four disease tests with two complex Bremia races (NL14 and NL16). The F 2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. Evidence is presented for a quantitative resistance against both Bremia races as well as for a race-specific resistance against Bremia race NL16 and not against NL14. These disease test data sets have been combined with DNA marker data of all 126 F 2 plants that had already been obtained for the construction of the linkage map. QTL mapping revealed a qualitative gene ( R39 ) explaining the race-specific resistance and three QTLs ( RBQ1 , RBQ2 and RBQ3 ) explaining the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it shows features similar to Dm genes (dominant race specific genes that give a complete resistance to d owny m ildew). The three QTLs explain 51% of the quantitative resistance against NL14, which indicates that probably not all QTLs have been detected in this F 2 population.In addition to this rather classical F 2 mapping strategy, we have performed an alternative mapping strategy based on the development and characterization of a set of Backcross Inbred Lines (BILs). These BILs are genetically nearly completely like L. sativa but contain a single chromosome substitution segment of L. saligna CGN 5271 (Chapter 4). Starting from an F 1 plant, BILs have been developed by four to five generations of backcrosses and one generation of selfing. All backcrosses from F 1 to BC 4 were made randomly without intentional selection. Marker Assisted Selection was started in the BC 4 generation. Finally, a set of 29 lines was obtained that covers 95% of the L. saligna genome, comprising 16 lines with a single homozygous introgression (BILs), one line with two homozygous introgressions, five lines with heterozygous single introgressions and seven lines with two or more heterozygous introgressions. Several chromosome regions showed severe distorted segregation in the F 2 population. Based on segregation ratios in backcross lines, we were able to explain distorted segregations of three chromosome regions observed in the F 2 population by genetic loci that are involved in pollen- or egg cell fitness.When seed of the first developed BILs was available, a disease test had been set up to test if the BILs, which carried QTLs as identified in the F 2 population, showed enhanced levels of quantitative resistance indeed. Nine BILs (or nearly-BILs) have been tested for resistance to Bremia race NL16. They covered together 31% of the L. saligna parental genome. Two resistance loci detected in the F 2 population ( R39 and RBQ3 ) have been confirmed in the disease test on the BILs. R39 is a dominant gene, which gives a complete resistance against Bremia race NL16. RBQ3 reduces the infection severity of the susceptible L. sativa by 49% ten days post inoculation. The quantitative effects from the resistance genes in these BILs were higher than expected from the F 2 mapping results. No conclusive comparisons of RBQ2 could have been made, as the introgression in the backcross line was not homozygous. RBQ1 has not been tested. Most exciting, the BIL method revealed a new resistance locus on Chromosome 8 with a 77% reduction on the infection severity compared to the susceptible control ten days post inoculation. We conclude that the BIL mapping method can reveal new QTLs unnoticed in the F 2 mapping method and it enables a quantification of the resistance gene effect in a L. sativa background.To extend our knowledge about the non-host resistance of L. saligna to Bremia , we have compared the genetics of non-host resistance to Bremia in L. saligna CGN 5271 with another accession L. saligna CGN 11341. The two accessions show a 39% AFLP polymorphism rate. We have analyzed the non-host resistance of L. saligna CGN 11341 by disease tests and DNA marker analyses on an F 2 and BC 1 population. Disease tests with Bremia races NL14 and NL16 showed a wide range of infection severity scores from resistant to susceptible to both races. The majority of plants had a similar resistance level to both Bremia races. These findings imply that the resistance of L. saligna is quantitatively expressed and is probably race non-specific. A few F 2 and BC 1 plants were completely resistant against Bremia race NL16 and rather susceptible to race NL14. QTL mapping revealed that a major resistance gene that was located on Chromosome 9 explains this race-specific resistance. This gene is designated R39b , as it may be different from R39 .No additional QTLs have been detected in this small F 2 population (n= 54). However, F 2 plants with L. saligna CGN 11341 alleles at loci of RBQ1 , RBQ2 , RBQ3 and RBQ4 mapped in L. saligna CGN 5271, were more resistant than F 2 plants with L. sativa alleles at these loci. In conclusion, we state that it is very likely that the same genes explain the resistances to Bremia in both L. saligna accessions. A backcross program for a set of Backcross Inbred Lines (BIL) that cover R39b and loci for putative QTLs, is in progress.In the last chapter of this thesis the basic results of the study have been discussed. We adduce that non-host resistance in L. saligna is not explained by accumulation of race-specific major resistance genes ( Dm genes) but by a resistance mechanism based on QTLs. Further, we have made a comparison for efficiency of four breeding methods to introgress the resistance genes from L. saligna . Based on this study, we conclude that twice as many resistance genes are introgressed when Marker Assisted Selection is used. Finally, several recommendations concerning research on non-host resistance and the applications of Backcross Inbred Lines have been suggested

Lactuca saligna, a non-host for lettuce downy mildew (Bremia lactucae), harbors a new race-specific Dm gene and three QTL's for resistance

Lactuca sativa (lettuce) is susceptible to Bremia lactucae (downy mildew). In cultivated and wild Lactuca species, Dm genes have been identified that confer race-specific resistance. However, these genes were soon rendered ineffective by adaptation of the pathogen. Lactuca saligna (wild lettuce) is resistant to all downy mildew races and can be considered as a non-host. Therefore, L. saligna might be an alternative source for a more-durable resistance to downy mildew in lettuce. In order to analyze this resistance, we have developed an F2 population based on a resistant L. saligna 2 susceptible L. sativa cross. This F2 population was fingerprinted with AFLP markers and tested for resistance to two Bremia races NL14 and NL16. The F2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. By comparison of disease tests, we observed a quantitative resistance against both Bremia races as well as a race-specific resistance to Bremia race NL16 and not to NL14. QTL mapping revealed a qualitative gene (R39) involved in the race-specific resistance and three QTLs (RBQ1, RBQ2 and RBQ3) involved in the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it showed features similar to Dm genes. The three QTLs explained 51% of the quantitative resistance against NL14, which indicated that probably only the major QTLs have been detected in this F2 population. The perspectives for breeding for durable resistance are discusse

The development of lettuce backcross inbred lines (BILs) for exploitation of the Lactuca saligna (wild lettuce) germplasm

Backcross inbred lines (BILs) were developed in which chromosome segments of Lactuca saligna (wild lettuce) were introgressed into L. sativa (lettuce). These lines were developed by four to five backcrosses and one generation of selfing. The first three generations of backcrossing were random. Marker-assisted selection began in the BC4 generation and continued until the final set of BILs was reached. A set of 28 lines was selected that together contained 96% of the L. saligna genome. Of these lines, 20 had a single homozygous introgression (BILs), four had two homozygous introgressions (doubleBILs) and four lines had a heterozygous single introgression (preBILs). Segregation ratios in backcross generations were compared to distorted segregation ratios in an F-2 population, and the results indicated that most of the distorted segregations can be explained by genetic effects on pollen- or egg-cell fitness. By means of BIL association mapping we were able to map 12 morphological traits and hundreds of additional amplified fragment length polymorphic (AFLP) markers. The total AFLP map now comprises 757 markers. This set of BILs is very useful for future genetic studies

Genetic dissection of Lactuca saligna nonhost resistance to downy mildew at various lettuce developmental stages

This study used the pathosystem of lettuce (Lactuca spp.) and downy mildew (Bremia lactucae) as a model to investigate the inheritance of nonhost resistance, and focused on the contribution of quantitative trait loci (QTLs) to nonhost resistance at various developmental stages in the lettuce life cycle. A set of 28 backcross inbred lines (BILs) of L. saligna CGN05271 (nonhost) introgressions in a L. sativa cv. Olof (host) background identified 16 introgressions that contributed to resistance at various plant developmental stages: seedlings, young plants, adult plants in the greenhouse and adult plants in the field. This paper provisionally considered these introgressions to be 16 QTLs. Of these 16 QTLs, seven were identified previously and nine were new. For 15 QTLs (Rbq1, Rbq2, rbq3-7 and Rbq8-15), the resistance alleles were derived from the nonhost L. saligna; the resistance allele of the other QTL (Rbq16) was from the susceptible L. sativa cv. Olof. Of the 15 QTLs in L. saligna, only two, rbq5 and rbq7, were found to be effective at every plant developmental stage; the other 13 QTLs were only effective at certain developmental stages. Experiments with seven B. lactucae races did not provide evidence that any QTL was race-specific. The data suggest that nonhost resistance in L. saligna is the result of cumulative effects of many resistance QTLs operating at various developmental stage

Three Combined Quantitative Trait Loci from Nonhost Lactuca saligna Are Sufficient to Provide Complete Resistance of Lettuce Against Bremia lactucae

The nonhost resistance of wild lettuce (Lactuca saligna) to downy mildew (Bremia lactucae) is based on at least 15 quantitative trait loci (QTL), each effective at one or more plant developmental stages. We used QTL pyramiding (stacking) to determine how many of these QTL from L. saligna are sufficient to impart complete resistance towards B. lactucae to cultivated lettuce, L. sativa. The alleles of four of the most promising QTL, rbq4, rbq5, rbq6+11, and rbq7 are effective at both the young and adult plant stages. Lines with these four QTL in all possible combinations were generated by crossing the respective backcross inbred lines (BIL). Using the 11 resulting lines (combiBIL), we determined that combinations of three QTL, rbq4, rbq5, and rbq6+11, led to increased levels of resistance; however, one QTL, rbq7, did not add to the resistance level when combined with the other QTL. One line, tripleBIL268, which contains the three QTL rbq4, rbq5, and rbq6+11, was completely resistant to B. lactucae at the young plant stage. This suggests that these three QTL are sufficient to confer the complete resistance of the nonhost L. saligna and any additional QTL in L. saligna are redundant. Histological analysis of B. lactucae infection in L. saligna, the BIL, and the combiBIL 48 h after inoculation revealed different microscopical phenotypes of resistance. The QTL differed with respect to the stage of the infection process with which they interfere

Fine mapping quantitative resistances to downy mildew in lettuce revealed multiple sub-QTLs with plant stage dependent effects reducing or even promoting the infection

Previous studies on the genetic dissection of the complete resistance of wild lettuce, Lactuca saligna, to downy mildew revealed 15 introgression regions that conferred plant stage dependent quantitative resistances (QTLs). Three backcross inbred lines (BILs), carrying an individual 30–50 cM long introgression segment from L. saligna in a cultivated lettuce, L. sativa, background, reduced infection by 60–70 % at young plant stage and by 30–50 % at adult plant stage in field situations. We studied these three quantitative resistances in order to narrow down their mapping interval and determine their number of loci, either single or multiple. We performed recombinant screenings and developed near isogenic lines (NILs) with smaller overlapping L. saligna introgressions (substitution mapping). In segregating introgression line populations, recombination was suppressed up to 17-fold compared to the original L. saligna × L. sativaF2 population. Recombination suppression depended on the chromosome region and was stronger suppressed at the smallest introgression lengths. Disease evaluation of the NILs revealed that the resistance of all three BILs was not explained by a single locus but by multiple sub-QTLs. The 17 L. saligna-derived sub-QTLs had a smaller and plant stage dependent resistance effect, some segments reducing; others even promoting downy mildew infection. Implications for lettuce breeding are outlined

Effects of stacked quantitative resistances to downy mildew in lettuce do not simply add up

Key message In a stacking study of eight resistance QTLs in lettuce against downy mildew, only three out of ten double combinations showed an increased resistance effect under field conditions. Abstract Complete race nonspecific resistance to lettuce downy mildew, as observed for the nonhost wild lettuce species Lactuca saligna, is desired in lettuce cultivation. Genetic dissection of L. saligna’s complete resistance has revealed several quantitative loci (QTL) for resistance with field infection reductions of 30–50 %. To test the effect of stacking these QTL, we analyzed interactions between homozygous L. saligna CGN05271 chromosome segments introgressed into the genetic background of L. sativa cv. Olof. Eight different backcross inbred lines (BILs) with single introgressions of 30–70 cM and selected predominately for quantitative resistance in field situations were intercrossed. Ten developed homozygous lines with stacked introgression segments (double combinations) were evaluated for resistance in the field. Seven double combinations showed a similar infection as the individual most resistant parental BIL, revealing epistatic interactions with ‘less-than-additive’ effects. Three double combinations showed an increased resistance level compared to their parental BILs and their interactions were additive, ‘less-than-additive’ epistatic and ‘more-than-additive’ epistatic, respectively. The additive interaction reduced field infection by 73 %. The double combination with a ‘morethan-additive’ epistatic effect, derived from a combination between a susceptible and a resistant BIL with 0 and 30 % infection reduction, respectively, showed an average field infection reduction of 52 %. For the latter line, an attempt to genetically dissect its underlying epistatic loci by substitution mapping did not result in smaller mapping intervals as none of the 22 substitution lines reached a similar high resistance level. Implications for breeding and the inheritance of L. saligna’s complete resistance are discussed

Effector-mediated discovery of a novel resistance gene against Bremia Lactucae in a nonhost lettuce species

Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR). We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance. BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna

Patterns of Transmission Ratio Distortion in Interspecific Lettuce Hybrids Reveal a Sex-Independent Gametophytic Barrier

Interspecific crosses can result in progeny with reduced vitality or fertility due to genetic incompatibilities between species, a phenomenon known as hybrid incompatibility (HI). HI is often caused by a bias against deleterious allele combinations, which results in transmission ratio distortion (TRD). Here, we determined the genome-wide distribution of HI between wild lettuce, Lactuca saligna, and cultivated lettuce, L. sativa, in a set of backcross inbred lines (BILs) with single introgression segments from L. saligna introgressed into a L. sativa genetic background. Almost all BILs contained an introgression segment in a homozygous state except a few BILs, for which we were able to obtain only a single heterozygous introgression. Their inbred progenies displayed severe TRD with a bias toward the L. sativa allele and complete nontransmission of the homozygous L. saligna introgression, i.e., absolute HI. These HI might be caused by deleterious heterospecific allele combinations at two loci. We used an multilocus segregating interspecific F2 population to identify candidate conspecific loci that can nullify the HI in BILs. Segregation analysis of developed double-introgression progenies showed nullification of three HI and proved that these HI are explained by nuclear pairwise incompatibilities. One of these digenic HI showed 29% reduced seed set and its pattern of TRD pointed to a sex-independent gametophytic barrier. Namely, this HI was caused by complete nontransmission of one heterospecific allele combination at the haploid stage, surprisingly in both male and female gametophytes. Our study shows that two-locus incompatibility systems contribute to reproductive barriers among Lactuca species

Effector-mediated discovery of a novel resistance gene against Bremia Lactucae in a nonhost lettuce species

Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR). We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance. BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna