Identification Of Candidate Genes And QTL Mapping Of Traits Related Fiber Length,Oil Content And Plant Height In Tetraploid Cotton

Cotton supplies the most important natural fiber for the textile industry and produces edible cottonseed oil for human consumption.Currently,developing new cotton cultivars with long fiber length,high oil content and optimal plant architecture is one of the main goals in cotton breeding.Marker-assisted selection is more efficient and accurate to improve the target traits than traditional phenotypic selection.In the past two decades,molecular markers such as RFLP,AFLP and SSR have contributed to cotton breeding to some extent.However,the low polymorphic levels of these markers cannot meet the need of genome wide association studies?GWAS?.With the rapid development of DNA sequencing technology,single nucleotide polymorphism?SNP?markers,with a high genetic diversity on the genome-wide scale,have been widely used to study the cotton species evolution,germplasm resources classification,GWAS,genetic linkage map construction,QTL mapping and gene identification.In the present study,83 upland cotton and 10 sea-island cotton germplasm lines were selected and genotyped with the Cotton 63K Illumina SNP array;and the 83 upland cotton germplasm lines for GWAS of oil content and dynamic fiber length.An interspecific backcross inbred line?BIL?population containing 250 lines which was derived from a cross and backcross between upland cotton?CRI36?as the recurrent parent and sea-island cotton?Hai7124?,was genotyped by SLAF-seq and QTL mapping for plant height?PH?.This study allowed to identify candidate genes for the traits,and detailed results are described as follows:1.The mean coefficients of variation?CVs?for fiber length?FL?at 10,15,20,and25 days post-anthesis?DPA?and maturity were 11.29,10.09,7.56,7.69 and 6.45%,respectively,indicating that the FL in the natural population showed higher variation at early developmental time points.A further partition of variances showed that the broad-sense heritability?H²?values of FL at 10,15,20,and 25 DPA and maturity were49.14,47.56,60.63,71.06,and 92.45%,respectively,suggesting that the H² increased as the fibers elongated and grew after 10-15 DPA and that the FL at maturity was highly inherited.However,only approximately?of the phenotypic variation in FL was accounted for by the genetic variation at 10 and 15 DPA,indicating that the FL is more influenced by environmental factors during early developmental time-points than at later time-points.2.By the GWAS analysis,a total of 25,38,57,89 and 88 SNPs showed significant correlations with FL at 10,15,20 and 25 DPA and maturity,respectively.In addition,60more promising SNPs were detected in at least two tests and two FL developmental time points.Among the 60 SNPs,47 SNPs were located within the confidence intervals of QTLs identified in previous studies,and the other 13 SNPs were first identified in this study.The above results suggested that examining the continual development of FL contributed to the identification of more QTLs for FL,and the results of our GWAS for developing fibers are reliable.A combined transcriptome and qRT-PCR analysis revealed that an alkane hydroxylase gene?GhMAH1?was predominantly expressed during the early-middle stage?10-15DPA?of fiber development.The virus-induced gene-silenced cotton suppressed90.68%of GhMAH1 gene expression and reduced the FL by 3.27 mm.This result indicated that GhMAH1 is likely involved in fiber elongation.3.The CVs of cottonseed OC ranged from 13.60 to 22.03%in the association mapping panel of 83 upland cotton accessions measured in four environments,and the H²was 90.74%.These results suggested that there existed a wide range phenotypic variation of OC in the 83 upland cotton accessions,and the OC is highly inherited.By the GWAS analysis,53 promising SNPs were detected in at least three environments.Among the 53SNPs,25 SNPs anchoring 7 OC QTLs were identified in previous studies.For a further analysis,QTL qOC-Dt5-1 contained 9 most significant SNPs,which overlapped the confidence interval for OC in another reported association studies.A combined transcriptome and qRT-PCR analysis revealed that a peroxidase gene?GhPRXR1?was predominantly expressed during the middle-late stage?20-35 DPA?of ovule development.The overexpression of GhPRXR1 in yeast significantly increased the OC by 20.01-37.25%.Suppression of GhPRXR1 gene expression in the virus-induced gene-silenced cotton reduced the OC by 18.11%.Our results contribute to identification of more OC QTLs and verifying a candidate gene GhPRXR1 that influences cottonseed oil biosynthesis.4.The BILs were produced by a cross between G.barbadense Hai7124 and G.hirsutum CRI36,using CRI36 as the recurrent parent for backcrossing with F₁ to produce BC₁F₁,followed by seven generations of selfing.An interspecific population of 250 BILs was utilized in this study.The SLAF-seq technology was utilized to sequencing the BILs and the two parents.The SLAF numbers of CRI36 and Hai7124 were 468,850 and 500,626,and their average sequencing depths were 25.03×and 21.20×with 5.46%and 5.81%of genome coverage,respectively.In the BIL population,the number of SLAFs ranged from275,045 to 527,319,with an average of 352,920.The average sequencing depth was 11.12×,with 4.14%of the average genome coverage.Through a further filtering,a total of 7,709high-quality SNP markers were mapped to 26 chromosomes and spanned a total genetic distance of 3,433.24-cM with a mean marker interval of 0.67 cM.5.The CVs of PH ranged from 13.03 to 21.42%in 250 BILs measured in seven environments,and the H² was 88.01%.These results suggested that there is a wide range of phenotypic variation of PH in the BIL population,and the PH is highly inherited.Furthermore,there was a transgressive segregation of PH in the BIL population when compared with the parents Hai7124 and CRI36.In total,10 PH QTLs were identified and each QTL explained 4.91-14.92%of the phenotypic variation,four of which were stable as they were mapped in at least two tests.A combined transcriptome and qRT-PCR analysis identified an IAA efflux transporter?GhPIN3?in a stable QTL region with four QTLs for PH.Suppression of the GhPIN3 gene expression in the virus-induced gene-silenced upland cotton CRI36 and Sea-island cotton Hai7124 increased the PH by 10.33 cm and 16.17 cm,respectively.These results indicate that GhPIN3 is likely involved in PH development in cotton.