Genome assembly and analysis of Lactuca virosa: implications for lettuce breeding.

Lettuce (Lactuca sativa L.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. To breed resilient and resistant lettuce in the future, valuable genetic variation found in close relatives could be further exploited. Lactuca virosa (2x = 2n = 18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) than Lactuca sativa (2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present a de novo reference assembly of L. virosa with high continuity and complete gene space. This assembly facilitated comparisons to the genome of L. sativa and to that of the wild species L. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the 3 Lactuca species as core, accessory, and unique. In addition, we identified 3 interspecific chromosomal inversions compared to L. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using 3-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion of L. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases among Lactuca spp. and indicated the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation in L. virosa, which is beneficial for the breeding of improved lettuce varieties.

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae.

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibility (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad-spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen recognition receptors, including nucleotide-binding leucine-rich repeat (NLR) proteins and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia-infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidates for determinants of the NHR phenotype 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.

Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew.

KEY MESSAGE: The nonhost resistance of wild lettuce to lettuce downy mildew seems explained by four components of a putative set of epistatic genes. The commonplace observation that plants are immune to most potential pathogens is known as nonhost resistance (NHR). The genetic basis of NHR is poorly understood. Inheritance studies of NHR require crosses of nonhost species with a host, but these crosses are usually unsuccessful. The plant-pathosystem of lettuce and downy mildew, Bremia lactucae, provides a rare opportunity to study the inheritance of NHR, because the nonhost wild lettuce species Lactuca saligna is sufficiently cross-compatible with the cultivated host Lactuca sativa. Our previous studies on NHR in one L. saligna accession led to the hypothesis that multi-locus epistatic interactions might explain NHR. Here, we studied NHR at the species level in nine accessions. Besides the commonly used approach of studying a target trait from a wild donor species in a cultivar genetic background, we also explored the opposite, complementary approach of cultivar introgression in a wild species background. This bidirectional approach encompassed (1) nonhost into host introgression: identification of L. saligna derived chromosome regions that were overrepresented in highly resistant BC1 plants (F1 × L. sativa), (2) host into nonhost introgression: identification of L. sativa derived chromosome regions that were overrepresented in BC1 inbred lines (F1 × L. saligna) with relatively high infection levels. We demonstrated that NHR is based on resistance factors from L. saligna and the genetic dose for NHR differs between accessions. NHR seemed explained by combinations of epistatic genes on three or four chromosome segments, of which one chromosome segment was validated by the host into nonhost approach.

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.

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. 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 'more-than-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.

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

KEY MESSAGE: Three regions with quantitative resistance to downy mildew of non-host and wild lettuce species, Lactuca saligna , disintegrate into seventeen 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. sativa F 2 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.

Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.

Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid.

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis.

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 interfered.