Using BSA-seq To Preliminarily Locate Nitrogen Utilization Efficiency Related Genes In Brassica Napus L.

Rapeseed is a widely-grown oil crop.Among N fertilizer used in rapeseed production,only 30%-40% were utilized by plant.Unused nitrogen is lost through volatilization,leaching,denitrification,and microbial decomposition,which causes great pollution to the environment and huge economic losses.Revealing the biological mechanism of rape absorption and utilization of nitrogen can provide a theoretical basis for the cultivation of new rapeseed varieties ultimately,the goal of increasing the nitrogen utilization efficiency(NUt E)of rapeseed can be achieved.Therefore,it is very important to increase the NUt E of rapeseed.In this study,a F2 population with 717 rapeseeds crossed between N-efficient and N-inefficient cultivars was employed from the previous screening.The mixed separation analysis technique(BSA)combined with SNPs and In Del markers were used to describe the candidate regions related to NUt E.The main results are as follows:1.At maturity,effective silique number,seeds per silique and thousand grain weight of717 rapeseeds were counted,and nitrogen content of stems,pod husk and seeds were determined,and then NUt E of single rapeseed was calculated.Results indicated that NUt E is a quantitative trait.All rapeseed plants were ranked by NUt E,30 nitrogen efficient genotypes and 30 nitrogen inefficient genotypes were selected to construct high and low NUt E pools for sequencing the mixed pool.2.A total of 112.37 Gbp data was obtained by sequencing.The Clean Reads obtained after filtration were 111.26 Gbp,and Q30 reached 80%.The average sequencing depth for each sample was 32.71 X.The average comparison efficiency between the sample and the reference genome is 99.45%,the average coverage depth is 21.50 X,and the genome coverage is 94.67%.3.Through SNP association analysis,25 regions were finally obtained,with a total length of 6.21 Mb,containing a total of 1,014 genes,of which 104 were non-synonymous SNP sites.The In Del correlation analysis finally yielded 37 regions with a total length of 6.04 Mb and contained a total of 792 genes,of which 18 were mutated at the In Del site.4.Association analysis was performed using the SNP＿ED(Euclidean distance)method and the In Del＿ED(Euclidean distance)method,focusing on genes related to nitrogen metabolism such as Bna A03g20510 D,Bna A03g20500 D,and Bna A03g20780 D,and genes related to carbon metabolism such as Bna A05g00100 D and Bna A05g01340 D.At the same time,the SNP＿ED and In Del＿ED intersection region was screened.Through the above methods,the candidate gene range was reduced.Finally,24 candidate genes were obtained and distributed on chromosomes A03,A05,A07,and C02.These genes are involved in nitrogen metabolism,carbon metabolism,and other metabolic pathways.