Whole‐genome sequencing bulked segregant analysis uncovered FW7, a Fusarium wilt resistance gene masked by epistasis in octoploid strawberry

Genetic analysis and molecular resistance to race 2 of Fusarium wilt in pigeonpea [Cajanus cajan (L.) Millsp.

Fusarium wilt (FW) is widespread in global cotton production, but the mechanism underlying FW resistance in superior-fiber-quality Sea Island cotton is unclear. This study reveals that FW resistance has been the target of genetic improvement of Sea Island cotton in China since the 2010s. The key nonsynonymous single nucleotide polymorphism (SNP, T/C) of gene Gbar_D03G001670 encoding protein phosphatase 2C 80 (PP2C80) results in an amino acid shift (L/S), which is significantly associated with FW resistance of Sea Island cotton. Silencing GbPP2C80 increases FW resistance in Sea Island cotton, whereas overexpressing GbPP2C80 reduces FW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 exist synergistically in Sea Island cotton accessions with haplotype forms "susceptible-susceptible" (TA) and "resistant-resistant" (CC), and interact with each other. CRISPR/Cas9-mediated knockout of GbWAKL14 enhances FW and Verticillium wilt (VW) resistance in upland cotton and overexpression of GbWAKL14 and GbPP2C80 weakens FW and VW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 respond to FW and VW by modulating reactive oxygen species (ROS) content via affecting MPK3 expression. In summary, two tandem genes on chromosome D03, GbPP2C80, and GbWAKL14, functions as cooperative negative regulators in cotton wilt disease defense, providing novel genetic resources and molecular markers for the development of resistant cotton cultivars.

Key MessageSeveral Fusarium wilt resistance genes were discovered, genetically and physically mapped, and rapidly deployed via marker-assisted selection to develop cultivars resistant toFusarium oxysporumf. sp.fragariae, a devastating soil-borne pathogen of strawberry.Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae, poses a significant threat to strawberry (Fragariatimesananassa) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene (FW1) on chromosome 2B that confers resistance to race 1 of the pathogen, and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here, we show that resistance to both races is widespread in natural and domesticated populations and that resistance to race 1 is conferred by partially to completely dominant alleles among loci (FW1, FW2, FW3, FW4, and FW5) found on three non-homoeologous chromosomes (1A, 2B, and 6B). The underlying genes have not yet been cloned and functionally characterized; however, plausible candidates were identified that encode pattern recognition receptors or other proteins known to confer gene-for-gene resistance in plants. High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1-FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5, in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.

Fusarium wilt (FW) is one of the most economically damaging cotton diseases worldwide, causing yellowing, wilting, defoliation, vascular tissue damage and ultimately death. Identification of molecular markers linked to FW genes is vital to incorporate resistance into elite cotton cultivars. An intraspecific F(2) in Gossypium hirsutum L. was developed by crossing with a highly resistant cultivar Zhongmiansuo 35 (ZMS35) and a susceptible cultivar Junmian 1 to screen simple sequence repeats (SSRs) closely linked to the FW resistance gene. FW was identified in F(2:3) families by evaluating seedling leaf symptoms and vascular tissue damage at plant maturity under natural field infection conditions over 2 years. The results showed that FW resistance segregated in a 3:1 ratio as a simple monogenic trait in F(2:3) families. Molecular mapping identified a FW resistance gene closely linked with the SSR marker JESPR304(-280) in chromosome D3(c17). We proposed to name this gene FW ( R ). A composite interval mapping method detected four QTLs for FW resistance in Chr.A7(c7), D1(c15), D9(c23) and D3, respectively. Among them, one major QTL (LOD > 20) was tagged near marker JESPR304 within an interval of 0.06-0.2 cM, and explained over 52.5-60.9% of the total phenotypic variance. The data confirmed the existence of a major gene in Chr.D3. This is the first report of molecular mapping of a major gene contributing FW resistance in cotton. The present research therefore provides an opportunity to understand the genetic control of resistance to FW and conduct molecular marker-assisted selection breeding to develop FW resistant cultivars.

Starch content and its components are important for determining wheat end-use quality and yield. However, little information is available about their interactions at the QTL/gene level in more than one population using different QTL mapping methods. Therefore, to dissect these interactions, two mapping populations from two locations over 2 years were used. The QTLs for the populations were analyzed by unconditional and conditional QTL mapping by two different analysis methods. In the two populations, there were a total of 24 unconditional additive QTLs detected for flour amylose (FAMS), flour amylopectin (FAMP), flour total starch (FTSC), and the ratio of FAMS to FAMP using ICIMapping4.1 methods, but 26 unconditional QTLs were found using QTLNetwork2.0 methods. Of these QTLs, 10 stable major additive QTLs were identified in more than one environment, mainly distributed on chromosomes 3B, 4A, 5A, and 7D. The maximum percentage of phenotypic variance explained (PVE) reached 54.31%. Two new unconditional major additive QTLs on chromosome 3B (Qftsc3B and Qfamp3B) were found. A total of 23 and 19 conditional additive QTLs were identified in the two populations using two different methods, respectively. Of which, eight and six stable major conditional QTLs were detected on chromosomes 3B, 4A, and 7D, respectively. New repressed QTLs were identified, such as Qftsc/fams5B-1 and Qftsc/fams5B-2. There were 20 epistatic unconditional and 15 conditional QTLs detected. In all, important QTLs on chromosomes 3B, 4A, and 7A were found in both populations. However, the number of important QTLs in the special recombinant inbred line (RIL) population was higher than that in the double haploid (DH) population, especially on chromosomes 7D and 5B. Moreover, the QTLs on chromosomes 4A, 7A, and 7D were close to the Wx-1 loci in the RIL population. These indicated better results can be obtained by a special population to target traits than by a common population. The important QTLs on key chromosomes can always be detected no matter what kinds of populations are used, such as the QTLs on chromosome 4A. In addition, QTL clusters were found on chromosomes 4A, 3B, 7A, 7D, and 5A in the two populations, indicating these chromosome regions were very important for starch biosynthesis.

Fusarium wilt (FW), caused by Fusarium oxysporum Schlechtend f. sp. melonis (Leach & Currence) Snyd. & Hans (Fom) is a substantial threat to muskmelon (Cucumis meloL.) cultivation around the world. Identification of new sources of resistance and transfer of the resistance genes to elite cultivars is the effective breeding strategy to reduce the FW-associated crop losses in muskmelon. In the present study, inheritance studies and molecular mapping for FW resistance were conducted in a muskmelon inbred line KP4HM-15 with FW resistance introgressed from snapmelon. Inheritance studies in the F2 population derived from the cross Punjab Sunehri//KP4HM-15 indicated that FW resistance in KP4HM-15 is governed by a single dominant gene. Bulked segregant analysis was conducted to map the Fom-5(t) gene in KP4HM-15 using 527 SSR primers. Four primer pairs, CMCTN35, DM0096, CSWCTT02, and ECM181, were found to show differential polymorphism in the resistant and susceptible bulks and were analysed in the whole of the population. Two SSR markers, DM0096 and CSWCTT02, mapped close to the Fom-5(t) gene at a genetic distance of 1.4 and 2.5 cM, respectively. These markers can potentially be used to transfer Fom-5(t) in elite muskmelon genotypes through marker-assisted backcross breeding until more tightly linked markers are available.

Genetic diversity and identification of simple sequence repeat markers correlated with Fusarium wilt resistance was performed in a set of 36 elite cultivated pigeonpea genotypes differing in levels of resistance to Fusarium wilt. Twenty-four polymorphic sequence repeat markers were screened across these genotypes, and amplified a total of 59 alleles with an average high polymorphic information content value of 0.52. Cluster analysis, done by UPGMA and PCA, grouped the 36 pigeonpea genotypes into two main clusters according to their Fusarium wilt reaction. Based on the Kruskal-Wallis ANOVA and simple regression analysis, six simple sequence repeat markers were found to be significantly associated with Fusarium wilt resistance. The phenotypic variation explained by these markers ranged from 23.7 to 56.4%. The present study helps in finding out feasibility of prescreened SSR markers to be used in genetic diversity analysis and their potential association with disease resistance.

Fusarium wilt (FW) is a very serious soil-borne disease worldwide, which usually results in huge yield losses in cucumber production. However, the inheritance and molecular mechanism of the response to FW are still unknown in cucumber (Cucumis sativus L.). In this study, two inbred cucumber lines Superina (P1) and Rijiecheng (P2) were used as the sensitive and resistant lines, respectively. A mixed major gene plus polygene inheritance model was used to analyze the resistance to FW in different generations of cucumber, namely, P1, P2, F1 (P1×P2), B1, and B2, obtained by backcrossing F1 plants with Superina (B1) or Rijiecheng (B2), and F2, obtained by self-crossing the F1 plants. After screening 18 genetic models, we chose the E-1 model, which included two pairs of additive-dominance-epistatic major genes and additive-dominance polygenes, as the optimal model for resistance to FW on the basis of fitness tests. The major effect quantitative trait locus (QTL) fw2.1 was detected in a 1.91-Mb-long region of chromosome 2 by bulked-segregant analysis. We used five insertion/deletion markers to fine-map the fw2.1 to a 0.60 Mb interval from 1,248,093 to 1,817,308 bp on chromosome 2 that contained 80 candidate genes. We also used the transcriptome data of Rijiecheng inoculated with Fusarium oxysporum f. sp. cucumerinum (Foc) to screen the candidate genes. Twelve differentially expressed genes were detected in fw2.1, and five of them were significantly induced by FW. The expression levels of the five genes were higher in FW-resistant Rijiecheng inoculated with Foc than in the control inoculated with water. Our results will contribute to a better understanding of the genetic basis of FW resistance in cucumber, which may help in breeding FW-resistant cucumber lines in the future.

Quantitative Trait Loci for Resistance Against Fusarium Wilt Based on Three Cotton F 2 Populations

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melongena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F(2) and BC(1) populations of eggplant segregating for fusarium wilt resistance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F(2,) BC(1) and F(2) of BC(3) generations of the same cross. These markers provide a starting point for mapping the eggplant FOM resistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted selection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.

Inheritance of resistance to Fusarium wilt (FW) disease caused by Fusarium udum was investigated in pigeonpea using four different long duration FW resistant genotypes viz., BDN-2004-1, BDN-2001-9, BWR-133 and IPA-234. Based on the F2 segregation pattern, FW resistance has been reported to be governed by one dominant gene in BDN-2004-1 and BDN-2001-9, two duplicate dominant genes in BWR-133 and two dominant complimentary genes in resistance source IPA-234. Further, the efficacy of six simple sequence repeat (SSR) markers namely, ASSR-1, ASSR-23, ASSR-148, ASSR-229, ASSR-363 and ASSR-366 reported to be associated with FW resistance were also tested and concluded that markers ASSR-1, ASSR-23, ASSR-148 will be used for screening of parental genotypes in pigeonpea FW resistance breeding programs. The information on genetics of FW resistance generated from this study would be used, to introgress FW resistance into susceptible but highly adopted cultivars through marker-assisted backcross breeding and in conventional breeding programs.

SUMMARYThis study was undertaken in the variety Yaqui-53 to find the chromosomes carrying genes for resistance to three races of black rust. Critical monosomic analysis indicated (a) that resistance to race 15 is governed by a single factor located in chromosome 3B and a minor gene conferring additional resistance is present in chromosome 6D (b) Yaqui-53 carries two dominant genes for resistance to race 34 in chromosome 2A and 7B (c) Resistance to race 122 is conditioned by three genes located in chromosome 3A, 3B and 6D.Genes for resistance are mostly confined to A and B genomes of wheat. In the present studies five different chromosomes 2A, 3A, 3B, 7B and 6D are involved for resistance to only three races, which suggest that the number of genes conferring resistance to all the races of rusts may not be restricted to few.

Fusarium wilt is a destructive soil-borne disease that threatens the production of mung bean. Mung bean lines Zheng8-4 and Zheng8-20 show high resistance and high susceptibility to Fusarium wilt, respectively. Transcriptome analysis was carried out to identify candidate genes involved in Fusarium wilt resistance using Zheng8-4 and Zheng8-20 at 0, 0.5, 1, 2, and 4 days post inoculation (dpi). Differential expression analysis showed that 3,254 genes responded to pathogen infection and were differentially expressed in the resistant and susceptible lines. Weighted gene co-expression network analysis (WGCNA) was also performed to identify five modules highly correlated with Fusarium wilt resistance, in which 453 differentially expressed genes (DEGs) were considered likely to be involved in Fusarium wilt resistance. Among these DEGs, we found 24 genes encoding resistance (R) proteins, 22 encoding protein kinases, 20 belonging to transcription factor families, 34 encoding proteins with oxidoreductase activity, 17 involved in stimulation/stress responses, and 54 annotated to pathogen resistance-related pathways. Finally, 27 annotated genes were further selected as candidate genes of Fusarium wilt resistance in mung bean. This study identifies novel potential resistance-related genes against Fusarium wilt and provides a theoretical basis for further investigation of Fusarium wilt resistance in mung bean breeding.

Inheritance of resistance to downy mildew by F1 and F2 sunflower hybrids

Fusarium wilt (FW) and sterility mosaic diseases (SMD) are key biotic constraints to pigeonpea production. Occurrence of these two diseases in congenial conditions is reported to cause complete yield loss in susceptible pigeonpea cultivars. Various studies to elucidate genomic architecture of the two traits have revealed significant marker–trait associations for use in breeding programs. However, these DNA markers could not be used effectively in genomics-assisted breeding for developing FW and SMD resistant varieties primarily due to pathogen variability, location or background specificity, lesser phenotypic variance explained by the reported QTL and cost-inefficiency of the genotyping assays. Therefore, in the present study, a novel approach has been used to develop a diagnostic kit for identification of suitable FW and SMD resistant lines. This kit was developed with 10 markers each for FW and SMD resistance. Investigation of the diversity of these loci has shown the role of different alleles in different resistant genotypes. Two genes (C.cajan_03691 and C.cajan_18888) for FW resistance and four genes (C.cajan_07858, C.cajan_20995, C.cajan_21801 and C.cajan_17341) for SMD resistance have been identified. More importantly, we developed a customized and cost-effective Kompetitive allele-specific PCR genotyping assay for the identified genes in order to encourage their downstream applications in pigeonpea breeding programs. The diagnostic marker kit developed here will offer great strength to pigeonpea varietal development program, since the resistance against these two diseases is essentially required for nominating an improved line in varietal release pipeline.