Genetic Identification And Effect Analysis Of Loci For Spikelet Number Per Spike In Common Wheat (Triticum Aestivum L.)

Wheat is one of the most important food crops in the world,providing 20%of the total calories and 25%of the protein for the world’s population.It is also one of the important pillars of food security,and China will face a huge food deficit due to the slow increase of grain yield.Wheat yield is composed of spike number per unit area,grain number per spike and grain weight.Spike morphology directly determines the yield of wheat and is a key agronomic trait.Grain number per spike（GNS）can be further divided into fertile florets and spikelet number per spike（SNS）,and increasing either one can directly increase wheat yield.Given the potential of SNS to increase wheat yield,it is essential to identify,utilize,and pyramid more genes controlling SNS from diverse wheat germplasm resources.Wheat line 20828 has been widely used in wheat breeding given its desirable agronomic traits including high SNS,suitable plant height and a high level of resistance to stripe rust.However,the genetic basis of SNS in 20828 is yet to be revealed.In this study,two recombinant inbred lines（RILs）were constructed by crossing wheat line20828 with wheat variety Chuannong 16 and line SY95-71,and F_3:4and F_2:3families were constructed by crossing 20828 with Shumai51 and Chuanmai60,respectively.In addition,our team previously identified yield-related traits QTL of a Tibetan semi-wild wheat Q1028,and a major SNS QTL,QSns.sau-QZ-3D,was identified on 3DL chromosome arm.Based on this result,QSns.sau-QZ-3D was further analyzed.The following main results were obtained:1.Genetic mapping and validation of loci for SNS in wheat line 20828Two RIL populations were derived from a common parent 20828 crossed with CN16and SY95-71,respectively.The phenotype of SNS and other agronomic traits were measured in multiple environments.Quantitative trait loci（QTL）for SNS were identified using genetic maps constructed with the Wheat55K SNP array and the phenotypes.Among them,QSns.sau-2CN-2D and QSns.sau-2SY-7A were detected in multiple environments and were considered as major and stable QTL for SNS.QSns.sau-2CN-2D was located in a2.1 cM interval on chromosome arm 2DS flanked by markers AX-109836946 and AX-111956072,explaining 10.16-45.68%of phenotypic variation,with LOD values ranging from 3.47 to 38.24.QSns.sau-2SY-7A was located in a 4.75 cM interval on chromosome arm 7AL flanked by markers AX-110518554 and AX-110094527,explaining10.21-40.78%of phenotypic variation,with LOD values ranging from 4.46 to 16.00.The positive alleles of these two major QTL were both from 20828.In addition,QSns.sau-2CN-2D and QSns.sau-2SY-7A were further validated by the tightly linked Kompetitive Allele Specific PCR（KASP）markers in other populations with different genetic backgrounds The significant correlation between SNS and other agronomic traits were detected and discussed.QSns.sau-2CN-2D significantly increased plant height,spike length and grain number per spike,delayed flowering period and decreased thousand-grain weight.QSns.sau-2SY-7A significantly increased spike length,delayed flowering period and decreased thousand-grain weight.The effects of the two QTL in different genetic backgrounds were also evaluated,and the results showed that the combination of QSns.sau-2SY-7A and QSns.sau-2CN-2D had the strongest effect on SNS by up to15.23-24.02%,followed by QSns.sau-2CN-2D and QSns.sau-2SY-7A by up to 4.98-22.52%.Meanwhile,QSns.sau-2SY-7A might be inhibited by QSns.sau-2CN-2D in different genetic backgrounds.Compared with previous studies,we think QSns.sau-2CN-2D is not Rht8 or Ppd-D1,and might be a novel SNS QTL.In addition,we found that QSns.sau-2SY-7A overlapped with loci identified in previous studies and the candidate gene（WAPO1）in this interval had been recently isolated functionally characterized.In this study,WAPO1 was also isolated and sequenced and it was consistent with previous studies.These results indicated that WAPO1 is also the candidate gene for QSns.sau-2SY-7A.2.Molecular markers development,near-isogenic line（NIL）construction and candidate genes screening for SNS QTLTo further study QSns.sau-2CN-2D and QSns.sau-2SY-7A,as well as another major QTL for SNS,QSns.sau-QZ-3D,identified in our previous study from a Tibetan semiwild wheat Q1028,the molecular markers linked to QSns.sau-QZ-3D and QSns.sau-2CN-2D were developed by searching SSR,analyzing Wheat660K SNP array and re-sequencing transcriptome.Considering that QSns.sau-2SY-7A overlapped with loci identified in previous studies and its candidate gene is WAPO1,we only developed NILs of QSns.sau-2SY-7A for validation.For QSns.sau-QZ-3D and QSns.sau-2CN-2D,we developed 168 and 50 markers nearby the interval,respectively.However,as the genetic basis of D genome is narrower than A and B genomes,only 18 markers closely linked with QSns.sau-QZ-3D,and 6 ones closely linked with QSns.sau-2CN-2D were developed.Based on the expression information of genes in the intervals of QSns.sau-QZ-3D and QSns.sau-2CN-2D,the genes specifically expressed in spikes were screened.Based on the annotation information,4 candidate genes were further identified.The allelic variations of4 candidate genes were analyzed.One candidate gene for QSns.sau-QZ-3D and another one for QSns.sau-2CN-2D were finally screened for following studies.In addition,2 pairs of NILs for QSns.sau-QZ-3D were constructed based on heterogeneous inbred family.For QSns.sau-2CN-2D and QSns.sau-2SY-7A,NILs were also constructed by screening the residual heterozygous lines and using heterogeneous inbred family.Finally,we screened 4and 2 pairs of NILs for QSns.sau-2CN-2D and QSns.sau-2SY-7A,respectively,and there were significant differences in SNS among these NILs.3.Dissection of haplotypes,geographical distribution and breeding utilization of WAPO1 associated with spike development in wheatWAPO1,encoding an F-box protein,is an ortholog of rice APO1 in wheat.In this study,haplotypes of WAPO1 were identified and their genetic effects,geographical distribution,and breeding utilization were further analyzed.The SNP（G/T）in F-box region was identified by using the developed KASP marker（KASP-GT-WAPO1）and 115bp insertion/deletion of promoter was identified to analyze the haplotypes by the reported In Del marker in Chinese wheat landraces.In addition to the previously identified three haplotypes(H1;140^G+115^deletion,H2:140^T+115^insertion,H3:140^G+115^insertion),and a newly fourth one(H4:140^T+115^deletion)was identified and proved that H2 and H4 could significantly increase SNS in wheat.Haplotypes H2 and H4 with more spikelets were grouped as A,and the other two haplotypes H1 and H3 were grouped as B.The KASP marker was also used to genotype Sichuan wheat varieties.In addition,667 wheat lines from public database were also used to identify the distribution of the above haplotypes.The results revealed that the group A（with more spikelets）has been widely selected and bred in China and the world.