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Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

ABSTRACT: Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield-limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome-wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific-locus amplified fragment sequencing (SLAF-seq) approach. Trait-SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^-5 (P = 1/85 630, -log₁₀ P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P-value (-log₁₀ P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372-kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR-NBS-LRR domains that may be involved in disease resistance response. Real-time quantitative PCR and virus-induced gene silencing (VIGS) analysis showed that CG02 was specific to up-regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.

SUBMITTER: Li T

PROVIDER: S-EPMC5698051 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

Li Tinggang T Ma Xuefeng X Li Nanyang N Zhou Lei L Liu Zheng Z Han Huanyong H Gui Yuejing Y Bao Yuming Y Chen Jieyin J Dai Xiaofeng X

Plant biotechnology journal 20170708 12

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield-limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome-wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific-locus amplified fragment sequencing (SLAF-seq) approach. Trait-SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 1 ...[more]

PMID: 28371164

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Project description:Cotton (Gossypium spp.) is the best plant fiber source in the world and provides the raw material for industry. Verticillium wilt caused by Verticillium dahliae Kleb. is accepted as a major disease of cotton production. The most practical way to deal with verticillium wilt is to develop resistant/tolerant varieties after cultural practices. One of the effective selections in plant breeding is the use of marker-assisted selection (MAS) via quantitative trait loci (QTL). Therefore, in this study, we aimed to discover the genetic markers associated with the disease. Through the association mapping analysis, common single nucleotide polymorphism (SNP) markers were obtained using 4730 SNP alleles. As a result, twenty-three markers were associated with defoliating (PYDV6 isolate) pathotype, twenty-one markers with non-defoliating (Vd11 isolate) pathotype, ten QTL with Disease Severity Index (DSI) of the leaves at the 50-60% boll opening period and eight markers were associated with DSI in the stem section. Some of the markers that show significant associations are located on protein coding genes such as protein Mpv17-like, 21 kDa protein-like, transcription factor MYB113-like, protein dehydration-induced 19 homolog 3-like, F-box protein CPR30-like, extracellular ribonuclease LE-like, putative E3 ubiquitin-protein ligase LIN, pentatricopeptide repeat-containing protein At3g62890-like, fructose-1,6-bisphosphatase, tubby-like F-box protein 8, endoglucanase 16-like, glucose-6-phosphate/phosphate translocator 2, metal tolerance protein 11-like, VAN3-binding protein-like, transformation/transcription domain-associated protein-like, pyruvate kinase isozyme A, ethylene-responsive transcription factor CRF2-like, molybdate transporter 2-like, IRK-interacting protein-like, glycosylphosphatidylinositol anchor attachment 1 protein, U3 small nucleolar RNA-associated protein 4-like, microtubule-associated protein futsch-like, transport and Golgi organization 2 homolog, splicing factor 3B subunit 3-like, mediator of RNA polymerase II transcription subunit 15a-like, putative ankyrin repeat protein, and protein networked 1D-like. It has been reported in previous studies that most of these genes are associated with biotic and abiotic stress factors. As a result, once validated, it would be possible to use the markers obtained in the study in Marker Assisted Selection (MAS) breeding.

Project description:Understanding the population structure and linkage disequilibrium in an association panel can effectively avoid spurious associations and improve the accuracy in association mapping. In this study, one hundred and fifty eight elite cotton (Gossypium hirsutum L.) germplasm from all over the world, which were genotyped with 212 whole genome-wide marker loci and phenotyped with an disease nursery and greenhouse screening method, were assayed for population structure, linkage disequilibrium, and association mapping of Verticillium wilt resistance. A total of 480 alleles ranging from 2 to 4 per locus were identified from all collections. Model-based analysis identified two groups (G1 and G2) and seven subgroups (G1a-c, G2a-d), and differentiation analysis showed that subgroup having a single origin or pedigree was apt to differentiate with those having a mixed origin. Only 8.12% linked marker pairs showed significant LD (P<0.001) in this association panel. The LD level for linked markers is significantly higher than that for unlinked markers, suggesting that physical linkage strongly influences LD in this panel, and LD level was elevated when the panel was classified into groups and subgroups. The LD decay analysis for several chromosomes showed that different chromosomes showed a notable change in LD decay distances for the same gene pool. Based on the disease nursery and greenhouse environment, 42 marker loci associated with Verticillium wilt resistance were identified through association mapping, which widely were distributed among 15 chromosomes. Among which 10 marker loci were found to be consistent with previously identified QTLs and 32 were new unreported marker loci, and QTL clusters for Verticillium wilt resistanc on Chr.16 were also proved in our study, which was consistent with the strong linkage in this chromosome. Our results would contribute to association mapping and supply the marker candidates for marker-assisted selection of Verticillium wilt resistance in cotton.

Project description:BackgroundThe receptor-like cytoplasmic kinases subfamily VII (RLCK-VII) is critical in regulating plant growth, development, and pattern-triggered immunity. However, a comprehensive exploration of these genes in the allotetraploid Gossypium hirsutum is still lacking. This study aimed to identify RLCK-VII genes in G. hirsutum and investigate their evolutionary history, structural features, expression patterns, and role in plant defense.ResultsSeventy-two RLCK-VII genes in the G. hirsutum genome were unveiled and classified into nine groups following their phylogenetic analysis with Arabidopsis thaliana. Group VII-1 was the largest, accounting for 28%, while Groups VII-2 and VII-3 had only one member each. The analysis using MCScanX revealed that these 72 genes formed 166 collinear gene pairs and were resided on 26 chromosomes of G. hirsutum, suggesting that they were derived from whole genome segmental duplication events. Their calculated Ka/Ks values were below one, implying the occurrence of purification selection during the evolution and inhibition of gene function differentiation/loss. All members of the RLCK-VII subfamily possessed two conserved domains, PKinase-Tyr and PKinase, and several conserved PBS1 kinase subdomains, individually included in one of the ten motifs identified using MEME. The RNA-Seq results showed that RLCK-VII genes exhibited different spatiotemporal expression, indicating their involvement in cotton growth, development, and defense responses to Verticillium dahliae. The transcription patterns of RLCK-VII genes found by RNA-Seq were further validated using qRT-PCR assays after inoculating "20B12" (cotton cultivar) with "V991" (V. dahliae). The virus-induced gene silencing (VIGS) assays uncovered that two RLCK-VII genes (Gohir.A13G227248 and Gohir.A10G219900) were essential to G. hirsutum resistance to Verticillium wilt.ConclusionsThese observations offer valuable insight into the attributes and roles of RLCK-VII genes in G. hirsutum, potentially enable the breeding of new cotton cultivars with enhanced resistance to Verticillium wilt.

Project description:Background:Genetically modified cotton accounts for 64% of the world's cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. Results:We developed a transient transformation system for gene characterization in upland cotton. Using ?-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3-10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. Conclusions:The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

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Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

Publications

Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

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