Key message

A population of lettuce that segregated for photoperiod sensitivity was planted under long-day and short-day conditions. Genetic mapping revealed two distinct sets of QTLs controlling daylength-independent and photoperiod-sensitive flowering time. The molecular mechanism of flowering time regulation in lettuce is of interest to both geneticists and breeders because of the extensive impact of this trait on agricultural production. Lettuce is a facultative long-day plant which changes in flowering time in response to photoperiod. Variations exist in both flowering time and the degree of photoperiod sensitivity among accessions of wild (Lactuca serriola) and cultivated (L. sativa) lettuce. An F₆ population of 236 recombinant inbred lines (RILs) was previously developed from a cross between a late-flowering, photoperiod-sensitive L. serriola accession and an early-flowering, photoperiod-insensitive L. sativa accession. This population was planted under long-day (LD) and short-day (SD) conditions in a total of four field and screenhouse trials; the developmental phenotype was scored weekly in each trial. Using genotyping-by-sequencing (GBS) data of the RILs, quantitative trait loci (QTL) mapping revealed five flowering time QTLs that together explained more than 20% of the variation in flowering time under LD conditions. Using two independent statistical models to extract the photoperiod sensitivity phenotype from the LD and SD flowering time data, we identified an additional five QTLs that together explained more than 30% of the variation in photoperiod sensitivity in the population. Orthology and sequence analysis of genes within the nine QTLs revealed potential functional equivalents in the lettuce genome to the key regulators of flowering time and photoperiodism, FD and CONSTANS, respectively, in Arabidopsis.

Fresh-cut lettuce is popular, but highly perishable product. Genetic studies of two bi-parental populations derived from crossing parents with rapid and slow rates of deterioration showed that the deterioration rate is a heritable trait (broad spectrum heritability, H2 of 0.56-0.87). The major genetic determinant of the deterioration rate in both populations was the quantitative trait locus (QTL), qSL4, located on linkage group 4. This QTL explained 40-74% of the total phenotypic variation of the trait in the two populations. Saturating the qSL4 region with single-nucleotide (SNP) markers allowed detection of six haplotypes in a set of 16 lettuce accessions with different rates of deterioration. Three of the haplotypes were always associated with very rapid rates of deterioration, while the other three haplotypes were associated with slow rates of deterioration. Two SNPs located 53 bp apart were sufficient to separate the 16 accessions into two groups with different rates of deterioration. The accuracy of markers-trait association was subsequently tested on 350 plants from seven F2 families that originated from crossing parents with different rates of deterioration. The H2 of deterioration rate in these seven families ranged from 0.64 to 0.90. The SNP-based analysis accurately identified individuals with rapid, intermediate, and slow rates of deterioration in each family. Intermediate rate of deterioration was found in individuals having heterozygous alleles at qSL4, indicating an additive effect of the alleles. The assay can be used for fast, accurate, and reliable identification of deterioration rate after processing for salad.

Key message

Eleven new major resistance genes for lettuce downy mildew were introgressed from wild Lactuca species and mapped to small regions in the lettuce genome. Downy mildew, caused by the oomycete pathogen Bremia lactucae Regel, is the most important disease of lettuce (Lactuca sativa L.). The most effective method to control this disease is by using resistant cultivars expressing dominant resistance genes (Dm genes). In order to counter changes in pathogen virulence, multiple resistance genes have been introgressed from wild species by repeated backcrosses to cultivated lettuce, resulting in numerous near-isogenic lines (NILs) only differing for small chromosome regions that are associated with resistance. Low-pass, whole genome sequencing of 11 NILs was used to identify the chromosome segments introgressed from the wild donor species. This located the candidate chromosomal positions for resistance genes as well as additional segments. F₂ segregating populations derived from these NILs were used to genetically map the resistance genes to one or two loci in the lettuce reference genome. Precise knowledge of the location of new Dm genes provides the foundation for marker-assisted selection to breed cultivars with multiple genes for resistance to downy mildew.

Plants undergo profound physiological changes when transitioning from vegetative to reproductive growth. These changes affect crop production, as in the case of leafy vegetables. Lettuce is one of the most valuable leafy vegetable crops in the world. Past genetic studies have identified multiple quantitative trait loci (QTLs) that affect the timing of the floral transition in lettuce. Extensive functional molecular studies in the model organism Arabidopsis provide the opportunity to transfer knowledge to lettuce to explore the mechanisms through which genetic variations translate into changes in flowering time. In this review, we integrated results from past genetic and molecular studies for flowering time in lettuce with orthology and functional inference from Arabidopsis. This summarizes the basis for all known genetic variation underlying the phenotypic diversity of flowering time in lettuce and how the genetics of flowering time in lettuce projects onto the established pathways controlling flowering time in plants. This comprehensive overview reveals patterns across experiments as well as areas in need of further study. Our review also represents a resource for developing cultivars with delayed flowering time.

Background

Water supply limits agricultural productivity of many crops including lettuce. Identifying cultivars within crop species that can maintain productivity with reduced water supply is a significant challenge, but central to developing resilient crops for future water-limited climates. We investigated traits known to be related to water-use efficiency (WUE) and yield in lettuce, a globally important leafy salad crop, in a recombinant inbred line (RIL) lettuce mapping population, produced from a cross between the cultivated Lactuca sativa L. cv. Salinas and its wild progenitor L. serriola L.

Results

Wild and cultivated lettuce differed in their WUE and we observed transgressive segregation in yield and water-use traits in the RILs. Quantitative trait loci (QTL) analysis identified genomic regions controlling these traits under well-watered and droughted conditions. QTL were detected for carbon isotope discrimination, transpiration, stomatal conductance, leaf temperature and yield, controlling 4-23 % of the phenotypic variation. A QTL hotspot was identified on chromosome 8 that controlled carbon isotope discrimination, stomatal conductance and yield under drought. Several promising candidate genes in this region were associated with WUE, including aquaporins, late embryogenesis abundant proteins, an abscisic acid-responsive element binding protein and glutathione S-transferases involved in redox homeostasis following drought stress were also identified.

Conclusions

For the first time, we have characterised the genetic basis of WUE of lettuce, a commercially important and water demanding crop. We have identified promising candidate genomic regions determining WUE and yield under well-watered and water-limiting conditions, providing important pre-breeding data for future lettuce selection and breeding where water productivity will be a key target.

Tipburn is an important physiological disorder of lettuce, Lactuca sativa L., related to calcium deficiency that can result in leaf necrosis and unmarketable crops. The major quantitative trait locus (QTL), qTPB5.2, can account for up to 70% of the phenotypic variance for tipburn incidence in the field. This QTL was genetically dissected to identify candidate genes for tipburn by creating lines with recombination events within the QTL and assessing their resistance to tipburn. By comparing lines with contrasting haplotypes, the genetic region was narrowed down to ∼877 Kb that was associated with a reduction of tipburn by ∼60%. Analysis of the lettuce reference genome sequence revealed 12 genes in this region, one of which is a calcium transporter with a single nucleotide polymorphism in an exon between haplotypes with contrasting phenotypes. RNA-seq analysis of recombinants revealed two genes that were differentially expressed between contrasting haplotypes consistent with the tipburn phenotype. One encodes a Teosinte branched1/Cycloidea/Proliferating Cell factor transcription factor; however, differential expression of the calcium transporter was detected. The phenotypic data indicated that there is a second region outside of the ∼877 Kb region but within the QTL, at which a haplotype from the susceptible parent decreased tipburn by 10-20%. A recombinant line was identified with beneficial haplotypes in each region from both parents that showed greater tipburn resistance than the resistant parent; this line could be used as the foundation for breeding cultivars with more resistance than is currently available.

Lettuce yields can be reduced by the disease bacterial leaf spot (BLS) caused by the pathogen Xanthomonas campestris pv. vitians (Xcv) and host resistance is the most feasible method to reduce disease losses. The cultivars La Brillante, Pavane and Little Gem express an incompatible host-pathogen interaction as a hypersensitive response (HR) to California strains of Xcv resulting in resistance. Little was known about the inheritance of resistance; however, resistance to other lettuce pathogens is often determined by resistance gene candidates (RGCs) encoding nucleotide-binding leucine-rich repeat (NB-LRR) proteins. Therefore, we determined the inheritance of BLS resistance in the cultivars La Brillante, Little Gem and Pavane and mapped it relative to RGCs. The reaction to Xcv was analyzed in nine F1, F2 and recombinant inbred line populations of lettuce from HR×compatible or HR×HR crosses. The HR in La Brillante, Pavane and Little Gem is conditioned by single dominant genes, which are either allelic or closely linked genes. The resistance gene in La Brillante was designated Xanthomonas resistance 1 (Xar1) and mapped to lettuce linkage group 2. Xar1 is present in a genomic region that contains numerous NB-LRR encoding RGCs and functional pathogen resistance loci in the RGC2 family. The Xar1 gene confers a high level of BLS resistance in the greenhouse and field that can be introgressed into commercial lettuce cultivars to reduce BLS losses using molecular markers.

Our ability to assemble complex genomes and construct ultradense genetic maps now allows the determination of recombination rates, translocations, and the extent of genomic collinearity between populations, species, and genera. We developed two ultradense genetic linkage maps for pepper from single-position polymorphisms (SPPs) identified de novo with a 30,173 unigene pepper genotyping array. The Capsicum frutescens × C. annuum interspecific and the C. annuum intraspecific genetic maps were constructed comprising 16,167 and 3,878 unigene markers in 2108 and 783 genetic bins, respectively. Accuracies of marker groupings and orders are validated by the high degree of collinearity between the two maps. Marker density was sufficient to locate the chromosomal breakpoint resulting in the P1/P8 translocation between C. frutescens and C. annuum to a single bin. The two maps aligned to the pepper genome showed varying marker density along the chromosomes. There were extensive chromosomal regions with suppressed recombination and reduced intraspecific marker density. These regions corresponded to the pronounced nonrecombining pericentromeric regions in tomato, a related Solanaceous species. Similar to tomato, the extent of reduced recombination appears to be more pronounced in pepper than in other plant species. Alignment of maps with the tomato and potato genomes shows the presence of previously known translocations and a translocation event that was not observed in previous genetic maps of pepper.

Thermoinhibition, or failure of seeds to germinate when imbibed at warm temperatures, can be a significant problem in lettuce (Lactuca sativa L.) production. The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures. Genes encoding germination- or dormancy-related proteins were mapped in a recombinant inbred line population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci (QTLs) associated with temperature and light requirements for germination. In particular, LsNCED4, a temperature-regulated gene in the biosynthetic pathway for abscisic acid (ABA), a germination inhibitor, mapped to the center of a previously detected QTL for high temperature germination (Htg6.1) from UC96US23. Three sets of sister BC(3)S(2) near-isogenic lines (NILs) that were homozygous for the UC96US23 allele of LsNCED4 at Htg6.1 were developed by backcrossing to cv. Salinas and marker-assisted selection followed by selfing. The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2-3°C when compared with sister NIL seed lots lacking the introgression. In addition, the expression of LsNCED4 was two- to threefold lower in the former NIL lines as compared to expression in the latter. Together, these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg6.1 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seeds.

Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes.