Genetic Analysis And Candidate Gene Mining Of Pod Size Traits In Peanut

Peanut（Arachis hypogaea L.）is an important oil crop and is widely grown in many countries and regions in the world.As an important reproductive organ,peanut pod is closely related to yield.Therefore,mining the key genes controlling peanut pod size and analyzing the regulatory mechanism can provide a theoretical basis for breeding peanut varieties with high-yield.In this study,a mutant with an extremely small pod（spm）from Yuanza 9102（WT）by ⁶⁰Coγ-radiation mutagenesis was identified.The morphological,anatomical,and physiological analyses for pods in WT and spm at three key pod developmental stages were performed.The F₂ segregated population was developed from a cross between WT and spm.A major QTL controlling pod size was identified by bulked segregation analysis（BSA）.An association analysis was performed using 392 natural population with a wide range of phenotypic variation for pod size to fine-map the major QTL.Then,the haplotype analysis of the candidate gene was conducted to identify a favorable allele.In addition,the transcriptome sequencing of peanut pods was performed at three different developmental stages.Finally,the bioinformatics analysis and transgenic experiments were performed to verify the function of the candidate gene.The main results were as follows:（1）A mutant with an extremely small pod（spm）from Yuanza 9102（WT）by ⁶⁰Coγ-radiation mutagenesis was obtained,and the morphological,anatomical,and physiological analyses for pods in WT and spm at three key pod developmental stages were performed.Firstly,an extremely small pod mutant with stable inheritance was selected from 158 mutants.Secondly,the pod phenotype,cell size and phytohormone content of WT and spm at three different developmental stages（10 days,20 days and 30 days after the peg elongation into the soil,designated as S1,S2,S3）were analyzed.The differences of pod size between WT and spm were seen at S1 and became even more striking at S2 and S3.The pod cell size was significantly smaller in spm than in WT at S1,S2,and S3.These results suggested that reduced cell size may be one of the important contributors for small pod in spm.The contents of indole-3-acetic acid（IAA）,gibberellin（GA）,and brassinosteroid（BR）were also significantly lower in spm pods than those in WT pods at all three stages.（2）The BSA and association analysis were performed to fine-map the candidate genes which control peanut pod size.First,the F₂ segregating population was constructed by the cross between WT and spm,and a major QTL controlling peanut pod size was located in the0～10 Mb on A08 chromosome by BSA-seq analysis.Furthermore,an association analysis was performed to fine-map the major QTL.A total of 100 SNPs,which were significantly associated with pod size,were detected.Out of 100 SNPs,7 SNPs were repeatly detected in three environments.Within 100 kb of either side of the 7 SNPs,a total of 63 genes were detected,and 4 candidate genes may be important candidate genes for pod size.The important candidate genes were Arahy.PSG53D,Arahy.63YJK9,Arahy.WC4CIS and Arahy.TS21P9.The gene Arahy.63YJK9 has a point mutation in the 5’UTR region.Arahy.63YJK9 is homologous gene with At TMT（TONOPLAST MONOSACCHARIDE TRANSPORTER）in Arabidopsis thaliana.Haplotype analysis showed that three haplotypes of Arahy.63YJK9 were detected.The t-test showed that the pod weight,pod length,pod width and pod area of the TTC haplotypes were significantly higher than thoseof the CCT and TTT haplotypes.（3）The comparative analyses for global transcriptome between WT and spm pods were performed.RNA-Seq analyses showed that 1,373,8,053,and 3,358 differently expressed genes（DEGs）were identified at stages S1,S2,and S3,respectively.Functional analyses revealed that a set of DEGs related to plant hormone biosynthesis,plant hormone signal transduction pathways,and cell wall biosynthesis and metabolism were detected.The protein interaction analysis found that the candidate gene Arahy.63YJK9 has an interaction relationship with genes in plant hormone synthesis and signal transduction,as well as sugar and starch metabolism pathways.The expression analysis showed that all genes in the 0～10Mb interval on A08 were not expressed in spm,however the most of the genes were expressed in WT.Through gene expression,sequence alignment and sequential FISH/GISH analysis,we found that there was a deletion of 0～10 Mb at the top of the A08 in spm.Therefore,we speculated that the small pod size of spm is caused by the deletion of Arahy.63YJK9.（4）Transgenic experiment was performed to verify the function of candidate gene Ah TMT4.To further understand the function of Arahy.63YJK9（Ah TMT4）,we performed a genome-wide scan of TMT family genes in peanut and identified 10 family genes.The phylogenetic analysis showed that TMT genes of A.hypogaea,A.duranensis,A.ipaensis,G.max,M.truncatula,P.vulgaris,and B.napus can be divided into 4 subgroups,and Ah TMT4and At TMT1 genes were classified into the same subgroup.At TMT1 gene is a positive regulator of grain size and yield in Arabidopsis.Over-expression of Ah TMT4 in Arabidopsis plants led to increased silique and grain size,suggesting that Ah TMT4 may play an important role in the regulation of peanut pod size.