Genome-wide Association Study Identifies Significant Genetic Loci For Root System Architecture Traits And Yield-related Traits In Response To Low Phosphorus Stress In Brassica Napus

Phosphorus（P）is an essential macronutrient for plant growth and development.However,phosphate in the soil is easily fixed by the metal cations to form insoluble compounds,which are difficult to be directly absorbed and used by plants.P deficiency reduced the plant growth,plant height,branch number,and finally decreased the seed yield of B.napus.Therefore,revealing the genetic mechanism of B.napus tolerant to suboptimal P conditions,identify P-efficient candidate genes,and to breed P-efficient varieties are important ways to sort out the"phosphorus crisis".In this study,403 varieties among an association panel of B.napus collected from all over the world were used conducted the field experiments to investigate the seed yield,plant height,and branch number at low（LP）and sufficient（SP）P conditions.There were 84 ancient B.napus varieties and 184 devired B.napus varieties in the association panel of B.napus.Selective sweep analysis between the ancient and devired B.napus varieties were performed,and candidate genes related to P uptake,transport and utilization within the selected region were identified.In addition,the SNP based on whole-genome resequencing were used to conduct the genome-wide association study（GWAS）of root system architecture（RSA）traits by“pouch and wick”system at LP and seed yield and yield-related traits by field trials in this study,and identify potential P-efficient candidate genes.The main results are as follows.1.Ninety-three selected genes in breeding were identified to be associated with P uptake,transport and utilization in Brassica napus by selective sweepCompared with derived B.napus,ancient B.napus had more developed root system architecture and root-secreted APase activity at the seedling stage and more branch number,higher plant height and greater seed yield at maturity stage at LP.Selective sweeps between ancient and derived B.napus were analyzed to identify the genes related to P uptake,transport and utilization in B.napus breeding by F_ST,ROD,and XP-CLR method,and1280 putative selective signals were detected.There were 93 genes related to P uptake,transport and utilization in the selected region,and of which 28 were phosphate transporter family genes and 27 were purple acid phosphatase family genes.GO analysis showed that the most significant GO terms enriched with genes in the selective sweep regions included“acid phosphatase activity（GO:0003993）”,“primary root development（GO:0080022）”,“root cap development（GO:0048829）”,“transporter activity（GO:0005215）”,“nutrient reservoir activity（GO:0045735）”,“sucrose transport（GO:0015770）”,“lateral root formation（GO:0010311）”,“phosphate ion homeostasis（GO:0055062）”,and“cellular response to auxin stimulus（GO:0071365）”.These indicated that GWAS of RSA traits and yield-related traits under contrasting P supplies is very important for the breeding of super P–efficient Brassica napus varieties.2.Genome wide association study ide ntified three candidate genes significantly associated with root system architecture at low phosphorus supplyA total of 403 B.napus varieties have been used to investigate the root dry weight,lateral root length,total root length,lateral root density,lateral root number and mean lateral root length at low P by‘pouch and wick’system in a previous study,and 370 varieties of them have been conducted whole-genome resequencing.Based on the SNPs developed by whole-genome resequencing,GWAS was performed for the above traits and 52significant SNPs were identified.Among them,chr C03＿3535483 was identified by lateral root length and total root length,and chr C07＿42348561 was identified by lateral root number and root dry weight.Two candidate genes（Bna C03g07130D and Bna C07g43230D）were identified for them and they were significantly induced by P deficiency by quantitative real-time polymerase chain reaction（q RT-PCR）.In addition,one SNP cluster significantly associated with lateral root density was detected on A06chromosome,and Bna A06g29270D,a gene whose espression was significantly inhibited by P deficiency,was identified at 54 kb downstream from the lead SNP of significant chr A06＿20030601.Candidate gene association analysis and haplotype analysis were performed on the above candidate genes,and three root favourable haplotypes（Bna A06g29270Hap1,Bna C03g07130Hap1 and Bna C07g43230Hap1）were identified.B.napus varieties containing root favourable haplotypes have more developed root systems than other varieties in the association panel.3.Genome wide association study identified six candidate genes significantly associated with yield and yield related traits at low phosphorus supplyField trials were conducted to investigate the seed yield（SY）,plant height（PH）,branch number（BN）and seed weight（SW）of an association panel of B.napus at LP and SP.Based on the SNPs developed by whole-genome resequencing,GWAS was performed for the above traits.A total of 1773,686,770 and 667 SNPs significantly associated with SY,PH,BN and SW were identified at SP,respectively.A total of 476,131,34 and 420 SNPs significantly associated with SY,PH,BN and SW were identified at LP,respectively.In addition,466 SNPs significantly associated with PHr and 33 SNPs significantly associated with BNr were identified.Among significant SNPs associated with seed yield and yield-related traits at LP,chr C07＿39807169 and chr C09＿14194798 were identified simultaneously for SY＿LP in two trials,and two candidate genes（Bna C07.ARF9 and Bna C09.PHT1;2）significantly induced by P deficiency were identified by combined GWAS with q RT-PCR.There were significant differences in the gene expression level of Bna C07.ARF9 and Bna C09.PHT1;2 between P-efficient and-inefficient varieties at LP.SY＿LP had a significant positive correlation with the gene expression level of both Bna C07.ARF9 and Bna C09.PHT1;2.Bna C07.ARF9 and Bna A01.PHR1 could directly bind the promoters of Bna A01.PHR1 and Bna C09.PHT1;2,respectively.In addition,there were significant positive correlation in the relative expression level between Bna C07.ARF9 and Bna A01.PHR1 at LP in P-efficient and-inefficient varieties.There was a significant positive correlation in the relative expression level between Bna A01.PHR1 and Bna C09.PHT1;2 in P-efficient and-inefficient varieties at LP（Fig.4d）.The above results indicate that the function of Bna C07.ARF9 in P efficiency could be attributed to regulate Bna C09.PHT1;2 by regulating Bna A01.PHR1.In addition,chr A10＿821671 was jointly detected by PH＿LP and PHr in two trials,and located in the coding region of the gene Bna A10g09290D.chr C08＿27999846 was jointly detected by PHr in two trials,and located in the coding region of the gene Bna C08g26640D.In addition,the SNP chr A02＿23899686 located on chromosome A02 was significantly associated with PH＿LP and co-localized with the previously reported PH＿LP QTL（PH＿LP＿A02a）.It was located in the coding region of the gene Bna A02g33340D.Candidate gene association study and haplotype analysis were performed on the above candidate genes,and three P-efficienthaplotype（Bna A10g09290Hap1,Bna C08g26640Hap1and Bna A02g33340Hap1）were identified.B.napus varieties containing favourable haplotypes have higher PH＿LP and PHr than other varieties in the association panel.In summary,ancient B.napus varieties had higher tolerance ability to low P than derived B.napus varieties,and 93 selected genes in breeding related to P uptake,transport and utilization in B.napus were identified by selective sweep analysis.In addition,eight P-efficient genes were identified by GWAS of RSA related traits at seedling stage,yield and yield related traits at low P supply,and six P-efficient haplotypes were identified by candidate gene association analysis and haplotype analysis.