| The cultivated peanut（Arachis hypogaea L.）is a widely grown crop in the world,and its seeds are rich in high-quality oil and protein,which provide humans with abundant nutrients.In China,peanut is an important oil and cash crop,and also the second largest source of domestic vegetable oil.In recent years,edible peanut oil and food have been increasingly consumed globally,therefore,the improvement of pod yield has become the primary goal in peanut breeding.Pod size is one of the major traits determining yield and commodity characteristic of peanut.Therefore,exploring the genetic and molecular mechanisms of pod size regulation has a wide prospect on molecular breeding of peanut.In this study,a RIL population containing 151 lines and an F2 population containing 1020 individuals were constructed by crossing the varieties 79266 with common pod and D893 with super larger pod,and a secondary F2 population containing 1039 individuals was constructed by crossing the LA123 line that selected from the RIL population with 79266.Based on BSA-seq and linkage analysis in F2 and RIL populations,combined with genotype and phenotype analysis of recombinant exchange individuals in secondary F2 population,we fine mapped a QTL for pod size and mapped based cloning of key gene.Transcriptome analyses of shells and seeds were performed to further explore the regulatory mechanisms of pod size.The main research contents are as follows:1.During the rapid expansion stage of peanut pod,the shell expansion rate and seed filling time of D893 were significantly higher than those of 79266.The single pod weight（SPW）,pod length（PL）,pod width（PW）and pod shell thickness（PST）of mature pods of D893 were significantly greater than 79266.The cytological analysis of pod shell and cotyledon in R2～R5stages showed that the duration of mitosis and the number of layers of D893 shell cells were greater than 79266,which were the main reasons for the differences in pod size.2.The BSA-seq was performed using the F2 population,a QTL controlling pod size was located on chromosome A07,named q AHPS07,with a physical interval length of 700 kb.The genetic effects analysis of q AHPS07 using the F2 and RIL populations revealed that the phenotypic variation explained（PVE）of q AHPS07 for SPW,PL,PW and PST reached 38.6%,23.35%,37.48%,and 25.94%,respectively.Based on the genotypic and phenotypic analysis of recombinant exchange individuals in the secondary F2population,the q AHPS07 locus was narrowed down to a 36.46 kb region on chromosome A07（Arahy.07:424670 bp～461129 bp）.3.There are six annotation genes in the interval of q AHPS07（36.46 kb）.The results of genome re-sequencing of 79266 and D893 showed that there were 14 SNPs in the 36.46 kb region,none of which has caused changes in the amino acids.The expression patterns of the six annotation genes were tested in the pod shells and seeds of 79266,D893 and LA123,at the four developmental stages,R2～R5,it is speculated that the RUVBL2 protein coding gene（Arahy.TSR8I7）is candidate gene for q AHPS07.4.Genome re-sequencing results of 79266 and D893 showed that there were 3 SNPs,SNP＿460907（G/A）,SNP＿461023（A/T）and SNP＿461129（C/T）,in the promoter region of AhRUVBL2.Amplified the 5’ends of AhRUVBL2 in 79266 and D893 by 5’RACE and found that these three SNPs did not cause change in the transcription start site（TSS）.The coding sequence（CDs）of AhRUVBL2 were cloned from 79266 and D893,and there was not any difference at the nucleotide level.Subcellular localization of AhRUVBL2 showed that it was located in the nucleus.Overexpression of AhRUVBL2 in Arabidopsis led to larger seeds and plants than the wild type.AhRUVBL2-silenced peanut seedlings represented small leaves and shorter main stems.5.Genome-wide association study and haplotype analysis using the genome re-sequencing data of 119 peanut cultivated accessions from previous studies by our group showed that SNP＿460907,SNP＿461023 and SNP＿4611293 were significantly correlated with pod size.Three haplotypes were identified according to three SNPs in the promoter of AhRUVBL2 among119 peanut accessions,including 78 lines carrying Hap＿GAC,38 lines carrying Hap＿ATT and3 lines carrying Hap＿GAT.Hap＿ATT is the dominant haplotype,and the accessions carrying Hap＿ATT had significantly higher single pod weight,single seed weight,pod width,pod shell thickness,single plant pod weight,and single plant seed weight than those carrying Hap＿GAT.In addition,a KASP marker developed from SNP＿461129 will provide an application basis for future genetic improvement of peanut that needs to be done from the pod size aspect.6.In this study,the pod shells and seeds of 79266 and D893 at the R2～R5 stages were used as materials to perform the transcriptome analysis.A total of 49835 expressed genes were identified in pods,among which 10084 genes were specifically highly expressed in shell,8634genes were specifically highly expressed in seed.A total of 32289 different expression genes（DEGs）were identified from shells and seeds.The expression trend analysis of DEGs showed that compared to 79266,the response to light stimulus and glycolysis are the more dominant biological processes of D893,which may be involved in the regulation of pod size.The genes co-expression with AhRUVBL2 were identified through WGCNA,and genes interacting with AhRUVBL2 protein were predicted by STRING database and gene expression level.Based on the above analysis,a molecular network regulating pod size by AhRUVBL2 has been initially established. |
