Identification Of Candidate Genes For Hybrid Seed Production Traits And Their Sensitivities To Exogenous GA3 By Genome-Wide Association Studies In Rice(Oryza Sativa L.)

As one of the major food crops in the world,rice is cultivated in 150 million hectares globally every year.In China,the cultivated area of rice is 30 million hectares,of which hybrid rice takes up about 50%.Large area of hybrid rice requires the production of F1seeds every year,and the annual seed production area in China is about 150,000 hectares.In the process of seed production of hybrid rice,gibberellin injection and leaf cutting are needed to improve the outcrossing seed setting rate of the female parent.Excessive spraying of gibberellin will increase the cost and cause environmental pollution,but also cause the occurrence of rice kernel smut aggravation.Cutting leaves not only increases the use of labor,but also damages the plant.Therefore,the discovery of genes for traits conducive to outcrossing seed production can provide gene and carrier information for improving seed production traits,increasing yield of hybrid seed production and realizing lightweight hybrid seed production.In this study,173 rice varieties were selected as a natural population and planted in Jiangpu Experimental Station of Nanjing Agricultural University（E118°50’,N32°02’）and Lujiang Base of Institute of Rice,Anhui Academy of Agricultural Sciences（E117°13’,N31°）respectively in the regular season of 2019.During the 3rd to 4th stage of rice panicle differentiation,the plants were artificially coated with quantitative GA3,and the plants coated with the same volume of water were used as the control.The phenotypic values of the three target traits（Flag leaf angle,Panicle exsertion length and Plant height）were investigated after the three target traits had been established（Full heading stage）.The difference value（DGA）between each trait and the control and the ratio of the difference value to the phenotypic value of the control were calculated,namely the GA3 sensitivity index value（GSI）,which were used as two indexes to measure the sensitivity of the genotype to GA3.Using rice germplasm of 173 the two sensitivity index as a character value,combining from the 173 germplasm whole genome sequencing of single nucleotide polymorphism（SNP）sites data（the reference genome sequence of Japonica rice variety Nipponbare）as a reference genome sequence,genome-wide association analysis,detection and characters of significant correlation of SNP marker loci,Three seed production traits and favourable candidate genes sensitive to exogenous gibberellin were identified.The main results obtained are summarized as follows:1.The character of flag leaf angle.Taking the trait value（）itself（i.e.the trait value of water treatment）as the index,one peak SNP site on chromosome 2,4,9,12 was significantly associated with the variation of sword leaf Angle under E1 environment（Hefei）,one peak SNP site on chromosome 2,3,9 was significantly associated with the variation of sword leaf Angle under E2 environment The SNP locus detected in both environments on chromosome9 was the same locus.A candidate gene locus（LOC＿OS09G13460）was found in the candidate region where the locus was located,and the excellent gene haplotype was LOC＿OS09G13460^GGCC2.The character of flag leaf angle.Effect value treated with GA3（DGA）.In the E1environment（Nanjing）,one peak SNP locus on chromosome 2,4,7,and 8 was significantly associated with the variation of DGA value.In the E2 environment（Hefei）,one peak SNP locus on chromosome 1,7,7,and 8 was significantly associated with the variation of DGA.The SNP locus detected in both environments on chromosome 7,8 was a repeat locus,andthreefavorablecandidategenes（LOC＿OS07G40630,LOC＿OS07G40930,LOC＿OS08G16310）were found in the candidate region of this locus,and the excellent gene haplotypes were LOC＿OS07G40630^TT,LOC＿OS07G40930^TTAA,LOC＿OS08G16310^G)3.The character of flag leaf angle.Taking the value of GA3 sensitive index,namely GSI value（°/°）as the index,the three peak SNPs in E1 environment（Nanjing）were located on chromosome 3,9,and 11,respectively,and the two peak SNPs in E2 environment（Hefei）were located on chromosome 1,9,respectively.Two favorable candidate genes（LOC＿OS01G52050,LOC＿OS03G13820）were found in the candidate region of this locus,and the excellent gene haplotypes were LOC＿OS01G52050^CTGG,LOC＿OS03G13820^C.4.The character of panicle exsertion length.In the group treated with water,one peak SNP in E1 environment（Nanjing）was located on chromosome 9,and the two peak SNPs in E2environment（Hefei）were located on chromosome 1,4,respectively The locus detected on chromosome 9.May have a factor pleiotropy,and a favorable candidate gene（LOC＿OS09G12240）was found in the candidate region of this locus,and the excellent gene haplotype was LOC＿OS09G12240^GA.5.The character of panicle exsertion length.Based on the effect value（DGA,）of GA3treatment,the three peak SNPs in E1 environment（Nanjing）were located on chromosome1,6,and 11,respectively,and the four peak SNPs in E2 environment（Hefei）were located on chromosome 1,4,6,and 11,respectively.Among them,the locus on chromosome 1 of the two environments was the same locus.A favorable candidate gene locus（LOC＿OS01G68260）was found in the candidate interval where the locus was located,and the excellent gene haplotype was LOC＿OS01G68260^CA.10.6.The character of panicle exsertion length.Taking the GSI value（cm/cm）as the index,the two peak SNPs in E1 environment（Nanjing）were located on chromosome 8,10,respectively,and the two peak SNPs in E2 environment（Hefei）were located on chromosome3,12,respectively.Twofavorablecandidategenes（LOC＿OS03G15770,LOC＿OS08G42620）were found in the candidate region of the loci,and the excellent gene haplotypes were LOC＿Os03g15770AGATCG,LOC＿Os08g42620TTGCTG。7.The character of plant height.Taking phenotypic value（CM）as the index,the three peak SNPs in E1 environment（Nanjing）were located on chromosome 2,3,and 9,respectively,and the three peak SNPs in E2 environment（Hefei）were located on chromosome 6,9,and10,respectively.A total of 6 peak SNP loci significantly associated with plant height variation were detected on chromosome 2,3,6,9,10 under the two environments.Four favorable candidate genes（LOC＿OS02G47090,LOC＿OS02G47110,LOC＿OS09G13610,LOC＿OS09G13460）were found in the candidate region of the loci,and the excellent gene haplotypeswere LOC＿Os02g47090A,LOC＿Os02g47110A,LOC＿Os09g13610CCCCAACACCCTC,LOC＿Os09g13460GGCC。8.The character of plant height.Taking the effect value（DGA）of GA3 treatment as the index,the three peak SNPs in E1 environment（Nanjing）were located on chromosome 2,3and 9,respectively,and the two peak SNPs in E2 environment（Hefei）were located on chromosome 6 and 9,respectively.Two favorable candidate genes（LOC＿OS09G13610,LOC＿OS09G13460）were found in the candidate region of chromosome 9,and the excellent gene haplotypes were LOC＿Os09g13610CCCCAACACCCTC,LOC＿Os09g13460^GGCC.9.The character of plant height.Taking GSI value（cm/cm）as the index,the two peak SNPs in E1 environment（Nanjing）were located on chromosome 1,3,respectively,and the two peak SNPs in E2 environment（Hefei）were located on chromosome 1,2,respectively.Three favorable candidate genes（LOC＿OS01G65010,LOC＿OS01G65580,LOC＿OS01G65650）were found in the candidate region of the loci,and the excellent gene haplotype was the haplotype LOC＿Os01g65580GAGGC,LOC＿Os01g65650AGA,LOC＿Os01g65010GCAGCGAAAAGATGCAC.10.To sum up,for the flag leaf angle character,a favorable candidate gene(LOC＿OS09G13460^GGCC)was discovered to increase the trait value.Five candidate genes(LO C＿OS07G40630^TT,LOC＿OS07G40930^TTAA,LOC＿OS01G52050^CTGG,LOC＿OS03G13820^C,LOC＿Os08g16310G)increased the sensitivity of flag leaf angle to GA treatment.For the panicle exsertion length trait,One favorable candidate gene(LOC＿OS09G12240^GA)w as found to increase the trait value,and four favorable candidate genes(LOC＿OS09G12240GA LOC＿OS01G68260^CALOC＿OS03G15770^AGATCGand LOC＿OS08G42620^{TTGC TG})to increase the sensitivity of ear extraction length to GA treatment.For the plant height trait,finding two favorable candidate genes(LOC＿Os09g13610^{CCCCAACACCCTCG},L OC＿Os09g13460^GGCC)increase character value,seven favorable candidate genes(LO C＿Os01g65010GCAGCGAAAAGATGCAC,LOC＿Os01g65580GAGGC,LOC＿Os01g65650AGA,LOC＿O s02g47090A,LOC＿Os02g47110A、LOC＿Os09g13610CCCCAACACCCTCG and LOC＿Os09g13460^GGCC)to increase the sensitivity of plant height to GA treatment.These results provid ed genetic information and germplasm resources for hybrid rice light seed productio n.