Fine Mapping And Candidate Genes Analysis Of QKnps-4A,A Major Stable QTL For Kernel Number Per Spike In Wheat

Kernel number per spike(KNPS),as one of the three yield components,is an important trait affecting the final yield of wheat.Increasing KNPS is one of the effective ways to increase yielding potential.qKnps-4A,a major stable QTL for KNPS on chromosome 4A has been detected by our research group,previously.To date,no report regarding fine mapping and map-based cloning of this QTL has been documented.In this study,fine mapping and candidate gene analysis of qKnps-4A,as well as its selection and application effects analysis in wheat breeding programs have been performed.The results will provide an important working basis for the gene cloning,functional analysis and molecular breeding application of qKnps-4A,and will be of great significance for the regulation mechanism study and genetic improvement of KNPS.The main results of this study are as follows:1.The recombinant inbred line(RIL)mapping population with 187 lines derived from the cross between Kenong 9204(KN9204)and Jing 411(J411)(denoted as KJ-RILs)was used to remap qKnps-4A based on physical position of SNP markers in the genome assembly of KN9204.The LOD peak position were concentrated in the physical interval of KN4A: 644.91-656.91 Mb on chromosome 4A in 8 datasets.To verify the results of the primary QTL mapping for qKnps-4A,we divided the 187KJ-RILs into 8 groups based on genotypes of the closest linkage markers of qKnps-2A,qKnps-4B and qKnps-7A,three another major stable QTL for KNPS identified in KJ-RILs.These lines within the 8 groups could be regarded as the near-isogenic lines for qKnps-4A(denoted as QTL-NILs).qKnps-4A showed the significant additive effects in 5 of the 8 backgrounds.These 5 sets of NILs were used for primary fine mapping analysis of qKnps-4A based on key recombinant identification and phenotypic evaluation.qKnps-4A was delimited into an interval of approximately 5.71 Mb from KN4A: 651.57 Mb to KN4A: 657.28 Mb,which was consistent with the primary QTL mapping results.2.Using the whole genome re-sequencing data of KN9204 and J411 as well as the genome assembly of KN9204,sequence variation in the target interval of qKnps-4A was analyzed.A total of 501 Insertion/Deletion(In Del)sites(≥5 bp)were found.Based on their flanking sequences,17 In Del markers with good PCR amplification effect and polymorphism were developed to identify the near-isogenic lines(NILs)including homozygotes NIL-KN9204 and NIL-J411,as well as 6 key recombinants(R1-R6).Combined with phenotypic data of the recombinants,qKnps-4A was finally narrowed down to a region between 4A-ID-6 and 4A-ID-7 markers,covering an intervalof 2.19 Mb from KN4A:653.72 Mb to KN4A: 655.91 Mb.This region included14 annotated genes.3.Based on gene functional annotation,transcriptome analysis,quantitative real-time PCR(q RT-PCR)detection and homologous gene analysis,Traes KN4A01HG38570 was preliminarily speculated as the candidate gene of qKnps-4A.Sequence difference analysis showed that,compared with that in KN9204,Traes KN4A01HG38570 in J411 had single nucleotide variations on exon 7(C→G)and intron 7(T→C),resulting in amino acid substitution and alternative splicing,respectively.The temporal and spatial expression characteristics of Traes KN4A01HG38570 in young spike were analyzed by q RT-PCR.The results showed that the expression levels of Traes KN4A01HG38570 in four stages during young spike development of NIL-KN9204 was decreased first and then increased with the increase of spike length.However,the expression levels of Traes KN4A01HG38570 were very low in the first three stages in NIL-J411,then it was sharply increased at the stage of spike length of 4.0 cm and was significantly higher than that in NIL-KN9204.These results suggested that Traes KN4A01HG38570 might negatively regulated the formation of kernel number.4.The genetic mechanism of qKnps-4A affecting KNPS and its genetic effects on yield-related traits were preliminarily analyzed using the paired NILs.The results showed that the excellent alleles from J411 could increase the central floret number per spike(CFNPS)and floret number per spike(FNPS)and thus increase the central kernel number per spike(CKNPS)and the total KNPS.qKnps-4A had no significant effect on the fertility of florets.In addition,genetic effect analysis showed that excellent alleles from J411 at qKnps-4A had a negative effect on thousand kernel weight(TKW),but showed a positive effect in increasing yielding potential at the single plant level.5.The In Del markers closely linked to qKnps-4A were used to screen and classify the genotypes of the natural mapping population comprised by 318 wheat varieties or advanced lines.Results of genetic effects analysis based on the natural mapping population showed that qKnps-4A was closely related to spike length(SL).Although the excellent haplotypeof Hap-J411 could significantly reduce thousand kernel weight(TKW),it had certain positive effects on increasing yielding potential at the single plant level.The results of breeding selection of qKnps-4A haplotype in different regions showed that the excellent haplotype of Hap-J411 accounted for a relatively high proportion in foreign and most of Chinese varieties,indicating that breeders have selected and applied the excellent alleles of qKnps-4A on purpose.A total of 148 varieties with detailed authorized time were used for characterizing the selection effects of qKnps-4A from the time span in breeding programs.The results showed that the proportion of excellent haplotype of Hap-J411 showed a decreased trend with time passage,but it exhibited high selection efficiency(68.18-84.62%)in each decade,indicating that excellent haplotype of Hap-J411 still played an important role in modern breeding.In conclusion,through QTL fine mapping,functional annotation,differential expression analysis of candidate genes and sequence variation analysis,qKnps-4A was finally mapped to the 2.19 Mb interval and Traes KN4A01HG38570 was preliminarily identified as its candidate gene.The spatio-temporal expression characteristics of Traes KN4A01HG38570 during spike development were analyzed,which provided data support for its further functional verification.The genetic effect of qKnps-4A on yield-related traits and its application in wheat breeding were analyzed based on natural population.This study laid a foundation for the cloning of qKnps-4A and the regulation mechanism study of KNPS,and provided important gene and marker resources for the genetic improvement of KNPS in wheat.