QTL Identification And Key Gene Function Analysis For Seed Size In Peanut(Arachis Hypogaea L.)

Seed size is one of the important agronomic traits selected during crop domestication and a key factor affecting crop yield.Improvement in crop yield has become the major goal of agricultural research to fulfil the increasing demand of food due to the rapid growth of global population.Peanut（Arachis hypogaea L.）is an important oil and cash crop in the world.The peanut cultivar is a heterologous tetraploid（AABB）formed by the natural hybridization of A genome and B genome diploid wild peanut plants in a single or in multiple hybridization event.In recent years,the whole genome sequencing of wild ancestral species and cultivated species of peanuts has been successfully completed,which has greatly promoted the understanding of the molecular mechanism of formation of excellent agronomic traits of peanut,but the molecular mechanism determining peanut seed size has not been studied deeply enough yet.In this study,the cultivated species Tifrunner was used as the female parent to cross with ID,an artificial tetraploid of peanut formed by crossing Arachis duranensis and Arachis ipaensis followed by chromosome doubling.The seed size trait was significantly separated in the isolated population.The QTL（quantitative trait locus）for peanut seed size was used on the selected population and the key genes between candidates were analyzed to screen the gene of interest.The main findings of the study are as follows:1.On the basis of the previous research,after continuous inbreeding through years of cultivation of isolated populations,more than 500 F₅generation plants were obtained.The pod and seed size of F₅generation were analyzed and compared with the pod and seed size of F₂generation.The pod and seed size of F₅generation were significantly larger than that of F₂generation.The genetic variation coefficient of pod area,pod and seed weight,seed length and seed weight of F₅generation were more than 30%higher than that of the genetic variation coefficient of F₂generation.2.According to the results of BSA sequencing of F₂parents and extreme material,the chromosome A05 is mainly mapped between 98 Mb-106 Mb;the chromosome A06 is mainly mapped within 104 Mb-107 Mb intervals;the chromosome A07 is mainly mapped within 1Mb-6 Mb intervals.According to the result of the BSA mapping,the associated molecular markers of the candidate area of chromosomes A05,A06 and A07 are identified.6 and 19SSR markers were designed for validation in candidate intervals A06 and A07 respectively,and the results showed no linkage with seed size trait.40 SSR markers and 16 In Del markers were designed in localization interval of chromosome A05,and 14 markers with polymorphism in parents were screened out for subsequent validation.3.On chromosome A05,14 SSR marks and In Del marks showed polymorphism and were integrated into the genetic linkage map by 107 materials from F₂generation.The average distance between each marker was 9.58 c M.A total of 7 QTLs related to seed and pod were identified.The LOD value is between 5.87 to 9.93,phenotypic variation explained（PVE）is between 23.53-32.62%.The mapping of the seed size QTL is located in two intervals of100.67 Mb-102.52 Mb and 105.05 Mb-106.66 Mb.To further narrow the candidate range,markers related to seed and pod size identified in F₂generation were selected and further identified in F₅generation population.A total of 20 QTLs related to pod and seed size were identified from the F₅generation population.The control of the seed size candidate gene is further narrowed within two intervals of 101.57-101.90 Mb and 105.98-106.66 Mb.According to the peanut genome sequence annotation,18 and 39 genes are predicted in both intervals.4.In order to further determine the gene,RNA-Seq is performed on seeds of different developmental periods of parents and offspring materials,respectively.The results showed that the expression of some key genes involved in hormone signal synthesis and transduction,ubiquitin proteasome pathway and MAPK pathway and some transcription factors were significantly changed during the development of large and small seeds.We found that there was a PPR protein family gene Arahy.33UH6E between 101.57-101.90 Mb,and the expression level of Arahy.33UH6E was significantly different between parents and offspring materials,and higher in large seeds.Within 105.98-106.66 Mb,Arahy.05M0GB gene was highly expressed in large seeds,encoding 3-oxo-delta（4,5）-steroid 5-beta-reductase-like protein.The Arahy.V89RV5 gene encodes gibberellin-regulated family proteins and is highly expressed at the mature stage of large seeds.In summary,through phenotypic analysis of F₂and F₅population,BSA and RNA-seq results,the candidate gene controlling the seed size was mapped in two intervals of 101.57-101.90 Mb and 105.98-106.66 Mb of chromosome A05.The function of candidate genes and their expression differences in large and small seeds were analyzed.Preliminarily,three putative genes involved in the peanut seed size were screened.The results of this study lay the foundation for revealing the molecular mechanism of peanut seed size and provide new marker resources for peanut molecular breeding.