Fine Mapping And Candidate Gene Analysis Of The Major QTLs RT.A09 And RT.C08 For Root Formation In Brassica Napus

Root system is the main part of crops to absorb water and nutrients.Root system architecture（RSA）directly affects the lodging resistance of crops and the utilization efficiency of environmental resources,thereby affecting yield.Mapping and cloning major QTLs for root development in rapeseed is of great significance for understanding the mechanism of root development and providing guidance for high yield breeding in rapeseed from the perspective of root morphogenesis.Two major QTL loci RT.A09 and RT.C08,which explained 10.8%and 14.1%of the phenotypic variation in root development,were obtained by QTL scanning of a recombinant inbred line（RIL）population（ZS11×4D122）.On this basis,the further mapping of RT.A09 and RT.C08were completed and the candidate genes were obtained through the screening of remaining heterozygous lines,phenotypic effect identification,development of KASP markers and construction of mapping population.The main results are as follows:1.KASP markup development.In the initial mapping interval,competitive allele-specific PCR（KASP）markers were developed for genotyping based on SNP microarray information and In/Del sequence differences of parental resequencing.Eleven markers were developed In RT.A09 interval:A9-1,A9-2,A9-4,A9-5,A9-57272,A9-57296,A9-57329,A9-57418,A9-57431,A9-57437 and A9-57560 were developed in the RT.C08range:NC8-5,NC8-6,NC8-7,NC8-10,C8-1,C8-2,C8-3,C8-4,C8-24013,and C8-24663.These markers can be used for accurate classification of A（ZS11 genotype）,B（4D122 genotype）and H（heterozygous genotype）materials,and can be used for validation and further/fine mapping of RT.A09 and RT.C08 genetic effects.2.Selection of remaining hybrid plants（RHL）,phenotypic effect verification and population construction.In the RIL population,the target initial positioning interval was complete or partial heterozygous genotype,and the background was basically homozygous genotype.The residual heterozygous lines（RHLs）were used as RT.A09 and RT.C08 for further positioning.RT.A09 selected eight RHLs:R4,R19,R26,R41,R48,R78,R159 and R183.RT.C08 selected seven RHLs:R53,R107,R119,R168,R186,R260 and R261.A total of 71 RHL-F₁ lines,1137 RHL-F₂ and RHL-F₃ lines were included in the RT.A09 near-isogenic line（NIL）population constructed by RHL selfing progeny and its sister lines,and 83 RHL-F₂ and RHL-F₃ lines were included in the RT.C08 NIL population.According to the phenotypic identification of root traits and shoot fresh weight of the QTL loci isolates at the 3 leaf stage under hydroponic conditions,combined with genotype identification,it was found that the RHL-A values of the eight RHLs isolates of RT.A09 were significantly higher than those of RHL-B（P<0.01 or P<0.05）on shoot fresh weight,root fresh weight,total root length,root surface area and root volume,indicating that the synergistic gene of RT.A09 was derived from ZS11,which was consistent with the initial mapping results.Among the seven RHLs isolates of RT.C08,RHL-B of R53,R107 and R260 was significantly higher than that of RHL-A in shoot fresh weight,root fresh weight,total root length,root surface area and root volume（P<0.01 or P<0.05）,and there was no significant difference in other isolates,indicating that the synergistic gene of RT.C08 was derived from 4D122,which was consistent with the initial mapping results.The genetic effects of RT.A09 and RT.C08 on root and biomass were further verified.3.Further positioning of RT.A09 and RT.C08.A total of 1137 NIL lines were screened by using markers A9-1 and A9-57560 on both sides of the initial location interval of RT.A09,and 14 exchange lines were found.Combined with KASP marker typing and progeny test of exchange plants,RT.A09 was finally located between markers A9-2 and A9-57431,with a physical distance of 129 kb（ZS11 genome）.There are still two KASP markers A9-57329 and A9-574418 co-segregated with RT.A09 in this interval.Analysis of 83 RHL-F₂ and RHL-F₃ lines by markers NC8-5 and C8-24663 on both sides of the initial positioning interval of RT.C08,two exchange lines were obtained.Combined with the relationship between phenotype and genotype,RT.C08 was further anchored in about2.24 Mb（Darmor genome）between markers NC8-6 and C8-24663,and three KASP markers NC8-7,C8-1 and C8-24013 were still co-segregated.4.Candidate gene analysis.Using ZS11 as the reference genome,19 annotated genes from Bna A09G0558900ZS to Bna A09G0560700ZS were obtained in the 129kb region of RT.A09.Eight genes that were not expressed or with very low expression levels in roots or leaves were screened by transcriptome database（http://yanglab.hzau.edu.cn/Bn TIR）,and then q RT-PCR analysis was performed on 11 genes that were expressed in roots or leaves.The results showed that the expression levels of three candidate genes in roots and leaves of NIL-A and NIL-B were significantly different.Among them,the expression levels of Bna A09G0559300ZS and Bna A09G0559800ZS in NIL-A were higher than those in NIL-B,and Bna A09G0560100ZS was the opposite.At the same time,comparative sequencing showed that only seven of the 11 genes had SNP or In/Del differences,among which three genes（Bna A09G0559200ZS,Bna A09G0559300ZS and Bna A09G0559600ZS）showed amino acid differences,and Bna A09G0559600ZS showed code-shift mutation.Combined with analysis of expression patterns,q RT-PCR and sequence analysis,Bna A09G0559200ZS,Bna A09G0559300ZS,Bna A09G0559600ZS,Bna A09G0559800ZS and Bna A09G0560100ZS were selected as the key candidate genes for QTL-RT.A09.In RT.C08,a total of 66 candidate genes were screened using parental resequencing data and gene expression profile analysis,of which 12 genes such as Bna C08g21500D（CYCLIN D3）have been reported to regulate root,leaf and/or hypocotyl growth.This study laid a good foundation for molecular marker-assisted selection breeding of rapeseed fine roots and cloning of related important genes.