Gene Mapping Of White Seed Coat Color In Peanut (Arachis Hypogaea L.)

Peanut（Arachis hypogaea L.）is a significant oil and food crop cultivated worldwide,with China being its primary producer.Achieving high yield and quality has always been the primary objective in peanut breeding.Testa color,an important agronomic trait of peanuts,plays a crucial role in determining their quality.The testa of peanuts exhibits rich color variations,including pink,red,white,black,among others.Colorful peanuts are primarily utilized in the development of food and healthcare products,offering greater commercial value compared to ordinary peanuts.White-seeded peanuts are rare,and their existing yield is low,resulting in limited adoption.Currently,the genes responsible for controlling white testa color in peanuts remain unclear.The mapping and identification of genes regulating white testa color in peanuts hold significant importance in developing high-yield,high-quality peanut varieties with white testa.In this study,peanut varieties with white and pink testa were used to construct mapping population,the testa color of F₁and F₂was investigated.By employing Bulk Segregant Analysis（BSA-seq）,we identified molecular markers linked to the white testa phenotype and examined key genes within the candidate interval.These findings serve as a foundation for the future cloning of candidate genes involved in regulating the white seed coat color in peanuts.The main results of our study are as follows:1.Phenotypic analysis of the F₂generation peanuts resulting from crosses between white and pink peanuts revealed distinct patterns of trait separation.The white seed coat color was found to be controlled by different genes.We analyzed two different cross combinations.In combination 1,we used the white peanut FZ001 as the female parent and the pink peanut Jihua 8（JH8）as the male parent.The F₁hybrids displayed the same color as the female parent.Upon selfing the F₁generation,we observed a segregation ratio of 3:1 for white seed coat to pink seed coat in the F₂generation.This ratio aligns with the expected ratio controlled by a single dominant gene,indicating that the white peanut seed coat color is governed by a pair of dominant genes,which we named ah WSC1.In combination 2,the pink-seeded peanut G06G served as the female parent,while the white-seeded peanut Kainongbai was used as the male parent.After selfing the F₁generation,we obtained 202 F₂plants,and the ratio of pink seed coat to intermediate type to white seed coat in the F₂generation was 3:12:1.Based on the segregation ratio observed in the offspring,we inferred that the peanut seed coat color is controlled by two pairs of recessive genes,which we named ah WSC2 and ah WSC3.2.After carefully observing the seed coat color phenotype of the F₂generation peanuts,we meticulously selected 20 white-coated peanuts and 20 pink-coated peanuts to create extreme pools.Through an in-depth analysis using BSA sequencing,we successfully identified the preliminary location of the candidate genes responsible for controlling the white seed coat in FZ001 peanuts within the genomic range of Chr15:143-155 Mb.Similarly,for Kainong white peanuts,the initial discovery of candidate genes associated with seed coat color was within the genomic range of Chr03:23.6-134.8 Mb.3.According to BSA analysis,we employed a total of 65 pairs of In Del molecular markers within the candidate interval of Chr15 in the FZ001×Jihua 8 cross.With meticulous screening,we successfully identified 15 molecular markers that exhibited excellent polymorphism within the progeny population.Within the interval of Chr15:151-153.1 Mb,we located the gene responsible for regulating the color of peanut white seed coat,demonstrating a significant contribution rate of 31.6%and a remarkable LOD value of 41.6%.Moreover,utilizing BSA analysis in the G06G×Kainong White Peanut combination,we skillfully designed 86 pairs of In Del molecular markers along the Chr03 chromosome region.From this set,we identified 16 pairs of In Del molecular markers that displayed polymorphism within the parental varieties.Subsequently,we applied these markers to the F₂population and confirmed the presence of genes controlling the white seed coat color in the genomic regions of Chr03:108.4-110.8 Mb and Chr03:119-120 Mb.These regions exhibited contribution rates of 20.3%and 17.0%respectively,accompanied by LOD values of 11.97 and 8.27.Remarkably,within these intervals,we uncovered a total of 42 and 38 candidate genes,respectively.4.To further elucidate the candidate genes responsible for regulating the color of the peanut white seed coat within the designated interval,we performed the construction and sequencing of transcriptome libraries using RNA-seq technology for both parental and extreme offspring materials.Through meticulous bioinformatics analysis,we successfully identified two genes,namely Arahy.57FMZL and Arahy.DK9749,exhibiting significant variations in expression between the white and pink seed coat varieties within the candidate interval of the FZ001×JH8 combination.These genes encode the transcription factor b HLH68 and MYB-like protein I respectively.Similarly,within the genomic range of 108.4-110.8 Mb for the G06G×KNB peanut combination,we discovered two genes associated with the flavonoid pathway,namely Arahy.ES58RZ and Arahy.SWJY0L,which encode WD40 and b HLH transcription factors respectively.Furthermore,within the range of 119.7-120.6 Mb,we identified the gene Arahy.Q9HDNE,which is closely related to the flavonoid synthesis pathway and encodes the F-box and WD-40 domain protein.