Downy mildew resistance
Downy mildew infestation reduces the yield and quality of fresh-market spinach as a low threshold (< 5%) of infected leaves makes the crop unmarketable and involves an additional labor cost to manually remove infested plants in the commercial field [19]. Utilization of genetic resistance offers an efficient disease control method and has been adopted to control downy mildew in spinach [15]. However, the rapid emergence of the new P. effusa races is continually overcoming the genetic resistance deployed in the newly released cultivars [15, 19]. Spinach breeding relies on the planned deployment of resistance genes from two parents in a hybrid cultivar. For any new P. effusa races, breeding involves screening the resistance sources to identify race-specific resistance loci to incorporate in the new cultivars [15]. The need for stable resistant cultivars against all known DM races is vital, and hence identification and mapping of downy mildew resistance loci from the cultivars, germplasm collections, and wild species are prioritized to combat rapidly evolving virulent races. Genetic characterization of the available resistance sources and the identification of tightly linked markers allow adopting a marker-based selection system to develop new cultivars. Traditional screening and selection methods based on evaluating the whole plants are labor-intensive, P. effusa being an obligate biotroph requires living tissue for sporangia production, and the downy mildew phenotyping requires environment-controlled facilities [12]. Identifying markers and adopting marker assisted selection (MAS) would expedite and ease the selection of downy mildew resistant spinach lines, and the marker based selection would be more efficient in terms of cost and resource needed. Thus, molecular markers are being developed in spinach [19,20,21, 24]. Characterizing each RPF locus and identifying gene-based markers will enhance the efficiency and precision of selection in developing new downy mildew resistance cultivars. Indeed identification of new resistance loci with a different mechanism and linked DNA markers will make the pyramiding or stacking of multiple resistance loci (RPFs) into a single cultivar feasible. Cultivars stacked with multiple resistant genes are attractive options for the spinach industry as they are considered to be durably resistant because the simultaneous evolution of new virulent races against multiple resistant genes is less likely to occur [39]. Current commercial spinach cultivars are hybrids combining multiple resistant loci effective against different sets of P. effusa races.
Following the downy mildew evaluation on a panel of seedling population segregating from cultivar Whale, around 71% of genotypes were identified as resistant to race 16 of P. effusa. Whale and Lazio are commercially available hybrid spinach cultivars containing different RPF genes, and around 60 plants of each cultivar were kept together in an isolation chamber and allowed to cross. As a result, phenotype and marker data did not fit a 1:1 ratio in this panel as expected for the regular pseudo-F2 population generated from a cross between expected heterozygous resistant (Rr) and non-resistant (rr) parents. In addition, the resistant F1 lines that were used for the cross might contain a mixture of Rr and rr plants as the plants were not tested with P. effusa or RPF markers. Spinach is commonly a dioecious crop having separate male and female plants, although some monoecious plants are found [18]. Both males and females were present in the crossing chamber, and thus selfing between male and female Lazio plants (Rr x Rr) might have occurred, resulting in inbreeds in the seeds lots, and some crosses may have been more successful than others leading to such segregation ratio. In this regard, association studies offer a good alternative as no biparental populations were required to map the locus; and thus, association analysis was pursued to map the resistance loci in this study.
GWAS analysis for downy mildew resistance
Downy mildew disease response showed a bimodal distribution with 123 resistant and 49 susceptible genotypes. A major gene governs resistance to downy mildew in spinach. The resistant parent Whale carries the RPF3 locus and their response to available races of P. effusa have are known [5, 6]. Hence, association analysis was conducted using a binary or qualitative disease score to extend our understanding of the genes providing resistance at a higher resolution in spinach cultivar Whale. Principal components analysis was conducted to identify population structure and use the PCA covariates to correct the sub-population structure. The spinach association panel was subdivided into two subpopulations in both principal component analysis and the ADMIXTURE analysis. However, the resistance and susceptible genotypes were present in both groups, and no strong relationship between genetic structure and the resistance phenotype was observed (Fig. 3). PCA and relationship matrices were used as covariates in the mixed model analysis in TASSEL, PLINK, and GENESIS programs to account for the population structure and relatedness effect. The effect of population stratification was corrected using PCA covariates in all tested association models, as evidenced by QQ plots (Figs. 4, 5, 6). Furthermore, Bonferroni correction (LOD value > 5.34) was used to control the spurious association.
Association analysis was performed using multiple models and programs to sort consensus sets of SNPs and increase the confidence of the detected SNPs. The single marker regression model without any covariates identified the same set of markers as the general linear model that includes PCA covariates in TASSEL, which was slightly more than the mixed linear model in TASSEL that accounts for population structure and kinship (Table 1). The principal component covariates-based logistic regression model in PLINK detected only one SNP that passed the Bonferroni threshold. Similarly, GENESIS’s mixed linear model detected an additional three SNPs not identified in the TASSEL mixed model. In general, association analysis from multiple programs detected three hotspot regions on chromosome 3 associated with P. effusa race 16 resistance. The genomic position of the significant SNPs detected in multiple models was examined to identify nearby disease-resistant candidate genes. Downy mildew resistance gene in Whale maps to a 0.57 Mb interval containing four plant defense regulating genes in three nearby zones. Association results from this study falls in the same region as the previously mapped RPF genes in spinach [19, 23, 24], illustrating that the use of a small panel of breeding population (bi-parental, multi-parent, or mixed progenies) were efficient to identify the association with the qualitative traits and map the locus in spinach.
Selection accuracy and efficiency were calculated for significantly associated SNPs from multiple association models (Table 3). The selection accuracy and efficiency were medium to high (> 50%), and these markers were suitable to search for candidate genes. A very high selection efficiency is expected for the lines generated from a cross of two-parent cultivars; however, phenotyping errors from disease escape and contamination with multiple races impose a lower marker-trait association and selection efficiency. The phenotype score of seedlings and their corresponding RPF3–5 predicted phenotype score shows an 80% match indicating some inconsistencies between marker and disease scores in this population. Although uncommon in our experiments, such deviating disease response may have arisen from the disease escape, a condition where the susceptible plants do not show disease signs and symptoms. The other scenario may be the mixed pathogen races and isolates. Visualization of the SNP genotypic data among the selected resistant and susceptible progeny populations showed the susceptible panels were fixed for alleles compared to resistant panels in this study (Fig. 8) that may facilitate selection using associated markers. However, some of the alleles associated with the resistance are present in reference cultivar Sp75 [22], which does not contain the RPF3 locus. Therefore, the RPF3 associated SNPs reported here should be tested in broad genetic backgrounds to confirm their potential to differentiate the RPF3 locus before implementing for breeding and selection purposes. We have performed resequencing for 480 worldwide spinach germplasm accessions, including the Whale, Lazio, and around 40 commercial cultivars. The GWAS analysis in the diverse panel will provide more insights on alleles associated with P. effusa, and the RPF3 associated SNP markers identified in this study will be used to search for their potential association in the resequenced panel.
Resistance to downy mildew disease in spinach is hypothesized to be governed by a major gene with a substantial effect on phenotype. Despite expected high LOD and R2 values for the resistant locus, medium LOD and R2 values, on average of 5.34–9.6 and 20%, were observed for the SNP markers associated with the Pfs 16 resistance. The low LOD might be because the associated SNPs are far from the candidate genes, the population investigated here was developed from a cross of multiple male parent lines providing susceptible inbreeds, and the disease escapes. Spinach is an open-pollinated and highly heterozygous species, and the linkage disequilibrium decay is faster and is estimated at around 4 Kb in spinach [22]. On the other hand, multiple minor effect genes or a gene with multiple alleles might control the resistance, so the associated SNP region has shown lower values of association (LOD values). Despite the moderate LOD and R2 values observed, the current result provided a high-resolution characterization of the RPF3 resistance locus. Additional information from this report and further understanding of the genetic mechanism underlying the resistance may help downy mildew resistance breeding and deploy the resistance alleles.
Candidate genes associated with P. effusa race 16
Polymorphisms in the causal genes regulating the phenotypic differences are of biological interest. Identification of candidate genes aids in functional characterization and identification of polymorphisms within the functional genes. Common SNPs identified from multiple association models were pursued to search for disease resistance candidate genes. None of the associated SNPs fell on the gene region responsible for disease resistance; however, most of the associated SNPs were within 10 Kb of the gene with functions pertinent to disease resistance. SNPs S3_658,306 and S3_692697 associated with Pfs 16 resistance in this study were in an LD block and harbored the two disease resistance genes Spo12736 and Spo12784. Another SNP S3_1050601 associated with P. effusa race 16 resistance was close to disease resistance gene Spo12908, but this SNP was not in LD with other nearby SNPs. Similarly, three SNPs (S3_1227787, S3_1227802, S3_1231197) in an LD block were less than 8 Kb from the disease resistance gene Spo12821.
The proximal end of chromosome 3 contains 14 annotated disease resistance genes [22,23,24], and the markers for RPF1, RPF2, and RPF3 were mapped in the same region [19,20,21,22,23,24]. The RPF1 locus was narrowed to a 1.5 Mb [19], 0.89 Mb [23], and 0.84 Mb region [24]. Based on the NBS-LRR domain in the spinach genome, five genes (Spo12736, Spo12784, Spo12903, Spo12905, and Spo12821) were predicted as potential downy mildew resistance candidate genes [22]. Recently, amino acid conserved domain analysis between the RPF1 resistant and susceptible lines identified Spo12729, Spo12784, and Spo12903 as the candidate genes [23]. And following the association analysis performed in the segregating population generated from a cross of multiple resistant parents reported Spo12784, Spo12903, Spo12905, and Spo12821 as the potential candidate genes involved in providing resistance against the downy mildew pathogen [24].
The RPF3 locus was characterized in this study using association analysis in the breeding population derived from cultivar Whale. The resistance locus was mapped to the three genomic regions (0.66–0.69 Mb, 1.05 Mb, 1.23 Mb) of chromosome 3. Four genes (Spo12736, Spo12784, Spo12908, and Spo12821) in the vicinity of peak SNPs were identified as the most probable candidate genes. The candidate genes were annotated as NB-ARC leucine-rich-repeat (LRR) and CC-NBS-LRR disease resistance protein (Table 1). The NBS-LRR domains are the most common plant disease resistance genes acting as a receptor of pathogen effectors and activating the signaling cascades for defense [40]. RPF3 gene postulated from the current mapping effort falls in the same region as reported in earlier work, but the region (0.66–1.23 Mb) contains more than ten disease resistance genes [22, 23]. Three of the four candidate genes identified in this work except Spo12908 were reported as downy mildew resistance candidate genes in [22]. Candidate gene Spo12784 identified for the RPF3 locus in this study was also reported as a candidate gene for the RPF1 locus by She et al. [23]. Candidate genes Spo12784 and Spo12821 identified here were also reported in the study of Bhattarai et al. [24].
The RPF locus (RPF1 through RPF6) has been established in spinach, and these loci are being characterized at the genetic level [19, 24]. Effort and emphasis have been proposed to clone the RPF1 gene and validate the functions in disease resistance. Additional characterization and discovering major and minor downy mildew resistance genes are essential as the downy mildew pathogen with a high potential to evolve with new virulences may quickly overcome the known resistances deployed in the commercial cultivars. Detailed genetic characterization of the resistance genes and identification of breeder-friendly diagnostic markers will enable an increased selection efficiency to introgress resistant alleles in cultivar development. In addition, functional characterization of the R genes will explain the genetic and regulatory mechanism of host-pathogen interaction, disease development, mechanism of evolution of the new virulent races, and their strategy to break down the R genes. Such information on host-pathogen interactions may help formulate an improved strategic approach in spinach breeding and cultivar development.
Identification and development of functional markers residing on the gene are most desirable, but it warrants identification and cloning of genes with explained functions of the domains towards resistance-susceptibility. Alternatively, genetically linked and associated markers to the traits are commonly used in plant breeding programs to select plants with expected phenotypes based on the marker genotype data. And a large number of SNPs [41] and SSR markers [42] are available in spinach. With recent advancements in sequencing platforms and continuously reducing sequencing costs, the genome or transcriptome of large plant panels can be sequenced at a lower price. Whole-genome resequencing of spinach core collections has been recently completed, and the sequence-based genomic resources and millions of SNP of the core collections are available. The new sequence resources are expected to expand our current understanding of genetics, genomics, and biology of commercially important traits, including the resistance to downy mildew pathogen. In recent years, field trials were performed to evaluate USDA spinach core collections for tolerance to downy mildew in the commercial growing regions under natural inoculum pressure [27]. The GWAS analysis performed with the field tolerance data identified several associated SNP regions that could breed downy mildew tolerant lines [27, 43]. The qualitative and quantitative screening and mapping efforts are aimed to identify the diverse genetic mechanism and desirable alleles contributing to resistance and use them in pyramiding the race-specific major genes and minor genes in a single cultivar.
A GWAS analysis was performed in a set of 172 spinach genotypes and mapped a major locus resistant to race 16 of P. effusa to a 0.57 Mb interval of chromosome 3, and identified a set of SNP markers statistically associated with the resistance to P. effusa. The SNP loci are close to the candidate genes that govern disease resistance. The beneficial allele can be used in spinach breeding programs to select the resistance genotypes through MAS approaches. The set of SNP markers identified in this study and others identified from several ongoing studies will be re-tested and validated in multiple populations to extend their use as a KASP marker for their potential use in MAS and narrow down the downy mildew resistance RPF3 and other RPF locus.
Furthermore, validation of candidate genes Spo12736, Spo12784, Spo12908, and Spo12821 via gene-knockout and gene-expression experiments may confirm their involvement in providing resistance to downy mildew and explaining the molecular mechanism of resistance. Research and investigations are ongoing to expand the current understanding of host-pathogen interaction in spinach downy mildew, including identifying and mapping multiple resistance sources, a functional test of the RPF genes, and characterizing functions of the effector genes. From the perspective of rapidly emerging races that are breaking down the resistance deployed in commercial cultivars, the host-pathogen battle in spinach downy mildew system offers a model to understand and explore the continued host-pathogen win-lose interaction, and a newer understanding may help in formulating and adopting an improved downy mildew resistance breeding strategy. Future reports on an expanded knowledge of spinach-downy mildew host-pathogen interaction and functional characterization of genetic resistance will be of high value to the scientific community and implement genetic resistance against the downy mildew.