Inter-specific High-density Genetic Map Construction And Major Agronomic Traits QTL Mapping For Upland Cotton(Gossypium Hirsutum L.)

Gossypium hirsutum L.is one of the most important cash crops,offering people with natural fiber and edible oil,and has vital status in national economy and people's daily life.With the increasing of industrial economy and adjustment of country construction,arable land is decreasing,which stimulated the contradiction between food crop and cotton,resulting in cutting down of land for cotton cultivation.The current situation of cotton production is pessimistic.Short season cotton is one type of upland cotton,with the outstanding characteristic of shorter growth period,and has been regarded as one of the most practicable choices for remitting the difficulty of cotton production.And earliness has been one of the most important aims in cotton breeding.Focusing on earliness and fiber quality of early maturity cotton,we performed high-density genetic map construction and QTL mapping,and got the following main results:1.We developed an intra-specific permanent recombinant inbred line?RIL?population by crossing CCRI36 with G2005.CCRI36 is an early maturity cultivar,G2005 is an introgression inbred line with G.barbadense introgressed in G.hirsutum.By RAD-seq,we got 14,109,670 and 18,558,010 paired-end?PE?reads for CCRI36 and G2005,and the sequencing depth of CCRI36 and G2005 reached 6.43× and 10.57×,respectively.An average of 7,293,849 PE reads was obtained for per RIL line,with an average of 4.59×sequencing depth.The average Q30 scores of the sequencing data for CCRI36,G2005 and RIL population were 89.75%,90.95% and 86.38%,respectively,stating that the sequencing data was with enough quality for further analysis.By mapping back on the reference genome sequences of G.hirsutum L.,a total of 369,223 SNPs were identified,with 78,127 polymorphic between the parents,which accounted for 21.16% polymorphism.2.For the genetic map construction,we selected simple-SNPs between the parents.Combined with the results of polymorphic SSR markers,we constructed a genetic map containing 6,434 loci,including 6,295 SNP and 139 SSR loci.Total length of the genetic map was 4,071.98 cM,with an average of 0.63 cM between adjacent markers,making it one of the densest genetic maps in upland cotton.This also was the first SNP-based genetic map for short season cotton,with the densest markers up to now.The quality of the genetic map was evaluated by marker collinearity analysis between the genetic map and the reference genome sequences,heat map analysis of 26 linkage groups,as well as common SSR merkers with published authoritative genetic maps.3.We surveyed six earliness-related traits from 2010 to 2016,including flowering timing?FT?,whole growth period?WGP?,flowering to boll-opening period?FBP?,plant height?PH?,node of first fruiting branch?NFFB?and height of NFFB?HNFFB?.Relying on the constructed high-density genetic map,a total of 247 quantitative trait loci?QTL?were identified,including 39 for FT,47 for WGP,40 for FBP,33 for PH,43 for NFFB and 45 for HNFFB.Among the 247 QTLs,52 could be detected repeatedly in at least two environments,and 52 contributed more than 10% for the phenotypic variation?PV?.The most repeatable QTL was qFT-D3-3 that could be detected in six environments,and could explain as much as 29.37% of the observed PV.This study ranked the first for cotton earliness genetic excavation with the largest number of QTL,better accuracy of confidence interval,and could supply more references for further studies in cotton earliness genetics.Five fiber quality traits were observed in five years from 2011 to 2015.A total of 186 QTLs were identified,including 28 for fiber elongation?FE?,39 for fiber length?FL?,36 for fiber strength?FS?,50 for fiber uniformity?FU?and 33 for micronaire?MC?.Among the total QTL,32 could be detected over years and 37 could explain more than 10% of the observed PVs.By analyzing the 95% confidence intervals,33 coincidence regions were found.And four chromosome regions with multiple QTLs and eleven valuable QTLs were pointed out that can classify the traits among years,and these mentioned loci could be important resources for superior fiber quality cotton breeding.4.A total of 55 QTL coincidence regions were found through analyzing 95% earliness related QTL confidence intervals.One vital region at 95.61-96.91 cM on D3 containing stable QTLs related with the six traits was screened out.Based on 12 recombinants in the RIL population,the core region was narrowed down between Marker25958 and Marker25963,spanning nearly 2.0 Mbp.Sixty-eight genes were annotated in this region,and only one gene GhEMF2 has known function influencing flowering,which can suppress flowering and delay earliness.The results of qRT-PCR among early-,medium-and late-maturity cultivars from 3-to 6-leaf stages verified the annotation.The expression level of GhEMF2 in early-maturity cultivar CCRI74 was the lowest,and that was the highest in the late-maturity cultivar Bomian 1,and the expression level in medium-maturity cultivar intermediate.In all three cultivars,the expression level of Gh EMF2 decreased during the progression from the three-to the six true-leaf stage.This Gh EMF2 expression trend was contrary to the progress of flower buds differentiation in cotton.We therefore conclude that GhEMF2 in cotton may also have a significant negative influence on flowering and is thus a potential candidate gene for use in further studies.5.One SSR marker,D03₁348,was selected out by marker screening that might be useful in identifying upland cotton earliness.This marker could sufficiently divide the RIL population into two sub-groups with distinguished maturities.Verified by six cultivars with different whole growth periods,D03₁348 could clearly identify each of the six genotypes by early mature genotype or late mature genotype.And the early mature genotype of D03₁348 had a higher percentage in early maturity populations,which reached 80%,demonstrating that this marker can distinguish cultivars with known maturities.To inspect its usefulness in natural populations,we checked the genotypes of 120 cultivars/lines including early-,medium-and late-maturities by marker bands recognition.The 120 cultivars/lines could also be divided into two sub-groups,one with shorter growth period and the other with longer growth period,and the differences of all six observed traits between the two sub-groups were extremely significant.Thus,we have enough confidence to announce that D03₁348 has tremendous potential in identifying cotton earliness,and has great value in marker-assisted selection in early-maturity cotton breeding.