Mapping Of Key Genes Associated With Variegated Testa Of Peanut (Arachis Hypogaea L.) And Development Of Molecular Markers

Peanut is an important economic crop and oilseed crop in China.China’s peanut annual planting area is about 70 million mu,and the output is about 17 million tons.Peanut seed coat color is an important agronomic trait of peanuts.Most of the peanuts are with pink or red seed testa,there are few peanut varieties are with variegated testa,however,the yield of them is lower than normal peanuts.The coloration depth of peanut testa is closely related to anthocyanin accumulation,and the variegated region of peanut seed coat is enriched with more anthocyanins.Anthocyanins have various biological functions,such as inhibiting the accumulation of free radicals and antioxidant.In recent years,the market demand for variegated peanuts is in increasing.However,the yield of existing variegated peanut varieties is low.Therefore,high-yield varigated peanuts is the fundamental way to meet market demand.The study focuses on understanding the genetics of variegated peanuts and trying to identify key candidate genes and molecular markers.The results could accelerate molecular breeding and the improvement of peanut germplasm resources for variegated peanuts.In this study,three segregated populations were constructed through crossing between Qicaihuasheng（variegated peanuts）and other three normal peanut varieties.Through forward and reverse genetic analysis,marker development,and QTL localization for peanut color spots.By comparing transcriptomics methods,the gene regulatory network related to variegated peanut was studied,which laid a foundation for the next cloning of genes and molecular breeding that regulated peanut color spots.The following are the main results of this study:1.Using variegated peanut“Qicaihuasheng”as male parent and cross with"Georgia-06G","Tifrunner"and"Jihua 17".All seeds harvested from the mother plants after hybridization had the same color as the mother and did not have varigated spot.For each combination,two pairs of SSR primers were used to identify the true F₁ plant,and the seeds（F₂ seeds）harvested from the F₁plants had varigated spots,but compared with"Qicaihuasheng",the colored spots were linear and the varigated spot area was smaller.The varigated spots of seeds harvested from F₂ plants were separated and divided into three types:obvious varigated spots,intermediate type and no varigated spots.According to the chi-square test,the varigated spots and no varigated spots traits conform to a 9:7 separation ratio,indicating that the spotted trait may be controlled by bigenic dominant inheritance.2.Based on statistical results of F₂ seeds,30 F₂ plants with obviously variegated testa and30 F₂ plants with normal pink testa were selected to construct the extremely variegated testa and pink DNA pools,respectively.Through BSA-seq analysis,the variegated traits were mapped on chromosome 10 and chromosome 06:24.5-73.6 Mb.To further narrow the candidate interval,a total of 59 In Del molecular markers were developed.Among them,seven markers showed polymorphism between parental lines,and further tested in the 374 F₂ individuals.Through constructing the genetic linkage map and QTL analysis,the gene controlling variegated testa was further mapped in a 0.56 c M genetic region,corrospoding to the physical region on chromosome06:24.7-49.9 Mb,which explain 14.11%of phenotypic variation with a LOD of 6.92.3.To further study the molecular mechanism of gene expression regulation of variegated testa.The transcriptome libraries of Qicaihuasheng for whole testa,Georgia-06G for whole testa,Qicaihuasheng’s testa from non-variegation region and Qicaihuasheng’s testa from variegation region were constructed and analyzed using RNA-seq.In total,26 differentially expressed genes were identified within the candidate interval.Among them,the expression of 15 genes in the Qicaihuasheng whole testa was higher than that of Georgia-06G whole testa.The expression level of nine genes in the Qicaihuasheng’s testa from variegation was higher than that in the Qicaihuasheng’s testa from non-variegation.In addition,we found a gene encoding the WD40protein,Arahy.22I0LH.1.In summary,one of the candidate region controlling varigated was mapped in a 24.7-49.9 Mb interval of chromosome 6 and several molecular markers were developed.The present study provided a reference for further studying the molecular mechanism of peanut colored seed coat and development of new spreading peanut varieties.