| High yield,good fibre quality and strong adversity resistance are important production goals in cotton(Gossypium hirsutum L.)which is a leading natural fibre crop worldwide.However,these traits are complex quantitative traits that are controlled by multiple genes and are affected by environmental conditions.Cotton breeding programs have entered the Breeding 3.0 stage,namely the molecular marker assisted breeding stage,especially in the developed countries.In the past decades,a series of cotton cultivar with high yield,goog quality and strong adversity resistance have been developed through the traditional breeding strategy,which played an important role in the cotton breeding in China,However,the cotton breeding programs are still at Breeding 2.0 stage compared with the developed countries.In addition,cotton is used as a pioneer crop of saline-alkali land for soil reclamation,which provides improved farmland for grain crops production.And with progress in spinning technology and increasing human demand,enhancing the lint yield and fibre quality has become an important breeding goal for cotton improvement.Therefore,a greater understanding of the genetic variants underlying agronomic traits are still required.In this study,a recombinant inbred line(RIL)population derived from a cross between cv.Nongdamian 13(NDM13)and the cv.Nongda 601(ND601)and a core collection were used as research materials and genotyped by resequencing strategy.We identified elite loci related to yield,fibre quality and adversity resistance traits(salt tolerance,phosphorus(P)deficiency tolerance).The aims of this study were to mine the excellent loci,to analyze the distribution of excellent loci on chromosome and to validate the function of candidate genes.The loci,genes and germplasms related to important agronomic traits in upland cotton could lay a foundation for marker-assisted selection breeding in this study.The main results are as follows:1.The highest density intraspecific genetic linkage map of upland cotton was constructed and new stable QTLs for yield and quality traits were discovered.In this study,a large scale population derived from an intraspecific cross between the upland cotton cv.NDM13 with good fiber quality,and ND601 with high yield,was genotyped by using 232,946 polymorphic single-nucleotide polymorphisms(SNPs)obtained via a wholegenome resequencing strategy,and 6737 bin markers was developed by a sliding window approach.We constructed a high-density bin linkage map containing 6,187 bin markers spanning 4,478.98 cM with an average distance of 0.72 cM.On the basis of the phenotypic data from five yield or eight fibre quality related traits across eight environments,we identified 152 QTLs,of which,119 belonged to novel QTLs.Importantly,the QTL number for fibre quality in the Dt subgenome was more than that in the At subgenome,and chromosome D02 harboured the greatest number of QTLs.Furthermore,we discovered 24 stable QTLs for fibre quality,and 12 stable QTLs for yield traits.Four novel stable QTLs(qFL-D02-1,qFL-D02-4,qFS-D11-1,qLP-A03-1)explaining more than 10%of the observed phenotypic(except qLP-A03-1)related to fibre length,fibre strength and lint percent,and seven previously unreported candidate genes with significantly differential expression between the two parents were identified and validated by RNA-seq.One elite recombinant line were selected harboring the above-mentioned four stable QTLs with good fibre quality and a high yield.2.The elite alleles and germplasms related to adversity resistance traits were identified.In this study,we conducted QTL analysis for salt tolerance trait under phytotron and filed conditions,using the seeds of RIL population.We identified 11 QTLs controlling relative germination rate(RGR)trait with approximately normal distribution on chromosome A01,A03,A04,A05,A07,A08,A13,D01 and D09 harboring one QTL except A03 and A13,of which four previously undescribed QTLs on chromosome A03,A04,A05 and A07 were observed.Among these,a novel stable QTL qRGR-A04-1 explaining 6.11%～12.63%of the observed phenotypic was selected to further analysis.Five elite germplasms with high salt tolerance were selected and can be utilized for cotton breeding.In addition,we evaluated the variation of ten P-deficiency related traits at the seedling stage and found that the shoot dry weight(SDW)with the highest coefficient of variation were significantly positively correlated with most of the other traits.So,the SDW could be considered as an indicator for screening P-tolerant germplasm resources,and 10 accessions were identified as P-tolerant accessions for future genetic research and breeding in cotton.A genome-wide association study was performed for SDW-related traits using 3.66 million highquality single-nucleotide polymorphisms.A total of 102 significantly associated SNPs was identified,of which,27 of the SNPs on chromosomes A01,A08,A10,A12,D07 and D11 were consistently detected for multiple traits or multiple environments.There were 64 and 37 SNPs in the At sub-genome and Dt sub-genome,respectively.Furthermore,we observed that the modern varieties had a lower frequency of elite SNPs than the early varieties,which indicated that low phosphorus tolerance traits were ignored in the breeding process,resulting in the loss of low phosphorus tolerance loci.3.Two salt tolerance and one P-starved tolerance genes were identified,and their functions were investigated.Two candidate genes with significantly differential expression between two parents were identified based on the RNA-seq and RT-PCR analysis.Further functional analysis showed that GhGASA1-and GhADC2-overexpression lines were more sensitive to salt stress than wild-type Arabidopsis with a lower RGR,survival rate and fresh weight based on the regulation of the transcript levels of GA-and PA-related genes in gibberellic acid(GA)and polyamine(PA)biosynthesis and the reduction in the accumulation of GA and PA,respectively,under salt stress.Virus-induced gene silencing(VIGS)analysis showed that TRV:GASA1 and TRV.ADC2 displayed more tolerance to salt stress by increasing the expression of GA-synthesis genes and decreasing the H2O2 content,respectively.Taken together,our results suggested that GhGASAl and GhADC2 were promising candidate genes responsible for the cotton salt tolerance breeding.A novel candidate gene,Gh_D11G2219,on the chromosome D11 was identified and validated by qRT-PCR analysis and the results suggested that the expression of Gh_D11G2219 is significantly higher in the P deficiency-tolerant varieties than in the P deficiency-sensitive varieties under P-starved stress.We further performed virus-induced gene silencing to test whether Gh_D11G2219 was required for cotton tolerance to P-deficient stress.The plant height,shoot dry weight and root dry weight of the VIGS plants were significantly lower than those of the controls.All of these results indicate that Gh_D11G2219 may play a role in the positive regulation of tolerance to P-deficiency stress in cotton.In conclusion,in this study,the highest genetic map was constructed based on a large scale RIL population with a clear genetic background and wide variations.Sixteen germplasms with high yield,good fibre quality and strong adversity tolerance were selected.A total of 28 novel loci for yield and fibre quality,one novel locus for salt tolerance,and 27 novel loci for P-starved tolerance were identified,from which four stable QTLs explaining more than 10%of the observed phenotypic was found.Chromosomes A03,A04,A05 and A07 harbored QTLs related to yield,fibre quality,salt tolerance and P-starved tolerance traits.A total of seven genes for yield and quality,two for salt tolerance,one for P-starved tolerance was identified and three genes related to adversity tolerance were analyse the function.These findings provided new resources for cotton breeding of high yield,good fibre quality and strong adversity tolerance through molecular marker assisted selection in the future. |
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