Seed Size Genes/QTLs Mapping And Functional Analysis In Peanut

Seed size is one of the most important agronomic characters of crops.Larger seeds usually have larger plants,and the seedlings are more competitive for light and nutrients and have higher resistance to stress environments.Therefore,the understanding of the molecular mechanism of seed size can provide important information for crop breeding.In recent years,there are extensive studies on the mechanisms of seed size.With the rapid development of genomics,bioinformatics and other molecular biological technologies,more and more regulatory pathways of agronomic traits in crops have been established.Peanut is a tetraploid legume plant,an important oil crop and widely grown in the world.Howeevr,there are limited studies on the mechanism of seed size and the genetic basis is not clear.The research in this field is far behind that of rice,corn,wheat,soybean and alfalfa,which not only restricts the utilization of peanut germplasms,but also seriously hinders peanut molecular breeding.Therefore,mining and mapping of pod and seedsize genes/QTLs are of great significance.Recently,the whole genomes sequencing of wild and cultivated peanuts have been completed and more and more peanut germplasms have been re-sequenced.With development of large number of SNP and SSR molecular markers,high-density maps have been constructed.These advances promote the molecular analysis of important agronomic characters and speed up the process of peanut molecular breeding.In this study,we used forward and reverse genetics to study seed size genes/QTLs in peanut by mapping and homologous cloning.The main results of this study are as follows,1.In the aspect of positive genetics（1）In the previous study,our research group made an artificial tetraploid of peanut,Ipadur（ID）,by crossing Arachis duranensis and Arachis ipaensis followed by chromosome doubling.The seed of ID was very small,and was similar to that of diploid wild parents.The cultivated species Tifrunner was used as the female parent to cross with ID,and F₁ seeds were obtained.After F₁ selfed,800 individuals of F₂ were obtained,and the seed size characters were segregated obviously.F₂ population was used for seed size genes/QTLs mapping.（2）The length,width,perimeter and area of pods and seeds of F₂ generation plants were measured.The data of 736 pods and 644 seeds were obtained.Segregation analysis of quantitative analysis and statistical analysis showed that pod area,perimeter and length were controlled by two major genes,and there was an additive dominant epistasis relationship between these two genes.There was no major gene controlling seed area,perimeter,length and width.The coefficient of variation of pod length,perimeter,area,length,width,perimeter and area was more than 10%,indicating the existence of hyperparental inheritance.（3）Twenty F₂ plants with the largest and the smallest average area of single seed were selected as large-seeded and small-seeded extreme individuals,respectively.The leaves from the extreme individual plants were collected to make the extreme pool for RNA extraction and BSA（bulked segregant analysis）sequencing.BSA results showed that the differential genes were mainly distributed on chromosome 03,08,09and 12.（4）BSA results were verified using SSR and Indel markers.The results showed that two SSR and two Indel markers verified on A03 chromosome are related with seed size trait.Through QTLs Analysis of these four markers,a QTL related to seed size was obtained,which was located between SSR-03-2 and Indel-03-2,and could explain 11.97%of the phenotypic variation（PVE）.2.In the aspect of reverse genetics（1）Using the genes that control seed size identified in alfalfa and soybean,and the whole genome sequences of peanuts,we found that two BS1 genes in cultivated peanut,located on chromosome 09 and 19,respectively.The sequences of the two genes were cloned and named AhBS1-1 and AhBS1-2.Gene sequences of peanut BS1showed high similarity to the homologous genes in alfalfa and soybean.（2）The expression of AhBS1 in the roots,stems,leaves,flowers,and seeds in different developmental stages of cultivated peanut were detected.The results showed that the expression level of AhBS1 in leaves was much higher（92-153 folds）than that in other tissues.The expression levels of AhBS1 in roots,stems,flowers and seeds were similar.The expression of AhBS1 in seeds of different developmental stages showed a similar trends in big seeded and small seeded peanut,the expression was highest in S1 and lowest in S3.（3）CRISPR/cas9 gene editing vector,RNAi vector,over expression vector were constructed and transformed these vectors into peanut.For the overexpression vector,a number of transgenic plants were obtained identified by PCR amplification.The overexpression vector was also transformed into Arabidopsis.