Fine Mapping Of Purple Seed Character Of Guizi Wheat No.1

As a special germplasm resource,purple grain wheat has high nutritional value and health benefits due to the high anthocyanin content in the seed coat.In order to clarify the location of the genes controlling the purple grain trait and the content of anthocyanins in the purple grain,a study on the fine localization of the purple grain gene and the content of anthocyanins in the purple grain was carried out in Guizi Wheat No.1,with the aim of laying the foundation for the cloning and genetic transformation of the purple grain gene and germplasm utilization.In this study,whole-genome re-sequencing（WGS）,combined with group segregation analysis（BSA）strategy,was used to detect the whole-genome re-sequencing of DNA from two extreme mixed pools and two parents of purple and white.Based on the resequencing data,517 pairs of InDel markers were successfully developed,and 271single individuals of F₅generation obtained from the cross between Guizi Wheat No.1（purple grain）and Guinong Wheat 30（white grain）were used as the study population to construct a genetic linkage map.The genetic linkage map was constructed,and the fine localization of purple grain gene and anthocyanin content of purple grain were investigated.The main results of this study were as follows.1.A cross was made using the parent,Guizi Wheat No.1（purple grain）,and the parent,Guinong Wheat No.30（white grain）.The hybrid F₁was continuously bagged and selfed to construct a recombinant inbred line（RIL）consisting of 271 single plants as the research population.Thirty plants each with very purple and white grains were selected from the study population by agronomic trait identification and purple grain color shade in the field.DNA was extracted using a DNA genome kit,and the DNA was mixed in equal amounts to construct two extreme mixed pools of purple and white.The whole genome resequencing technique was used to resequence the DNA of the two purple and white mixed pools and the two parents.The sequencing results showed that there were two candidate regions on chromosomes 2A and 7D,and the purple grain gene controlling Guizi Wheat No.1 might exist on chromosomes 2A and7D with physical intervals of（673Mb-698Mb）and（571Mb-601Mb）,respectively.2.517 pairs of InDel markers were successfully developed based on whole genome resequencing data.The 517 pairs of molecular markers were polymorphically identified in the purple and white mixed pool and in both parents,and a total of 39pairs of specific markers were obtained on both chromosomes 2A and 7D,including three pairs of co-dominant markers.The results showed that the two dominant complementary genes controlling the purple grain trait in Guizi Wheat No.1 were located on chromosomes 2A and 7D,respectively.3.PCR amplification using 39 pairs of specific markers in the study population was used to identify two dominant complementary genes of noble purple wheat 1localized on chromosomes 2AL and 7DL.The purple grain gene GZMpp1 of Guizi Wheat No.1 was localized on chromosome 2AL,and the genetic distance between the purple grain gene GZMpp1 and the marker chr2A32 was 1.2 c M;the genetic distance between GZMpp2 and the marker DY-7D6 was 0.5 c M.4.According to the results of the fine gene localization of purple grains in Guizi Wheat No.1,two functional genes related to the anthocyanin synthesis pathway were found in the 2AL chromosome localization interval,and their gene functions were annotated as b HLH protein;five functional genes related to the anthocyanin synthesis pathway were found in the 7DL chromosome localization interval,and their gene functions were annotated as MYB-related transcription factor.The positions of the seven candidate genes overlapped with the resequenced candidate intervals by NCBI,and it was assumed that the target genes controlling the purple grain trait of Guizi Wheat No.1 were within these seven candidate genes.5.Molecular markers chr2A34 and chr7D37 were screened for co-dominant markers by specific marker screening,and PCR amplification was performed in monocultures of 271 using chr2A34 and chr7D37 markers.According to the results of the amplified bands,there were 28 heterozygous plants in the 271 study population;133 plants with the same band type as the parental Guizi Wheat No.1;104 plants with the same band type as the parental Guinong Wheat 30;and 6 plants without band type recorded as deletion.The anthocyanin content was determined by extracting anthocyanins from the seeds of the three genotypes according to the method of Guiping Yang.The results showed that the highest anthocyanin content was 18.14mg/kg when the genotype was dominantly pure（AA）,12.86 mg/kg for heterozygous（Aa）and 7.01 mg/kg for recessive pure genotype（aa）,and the genotype of Guizi Wheat No.1 seeds was gradually changed from heterozygous（Aa）to recessive pure（aa）and dominant pure（AA）through segregation and recombination.The heterozygous（Aa）genotype was gradually changed to recessive pure（aa）and dominant pure（AA）through segregation and recombination.The anthocyanin content decreased sharply when the heterozygous（Aa）genotype was segregated and recombined into recessive pure（aa）,and increased when the heterozygous（Aa）genotype was segregated and recombined into dominant pure（AA）.It was observed that the color of the seeds gradually deepened when the seed genotype changed from recessive pure（aa）to dominant pure.This indicates that the difference of seed coat color,genotype and anthocyanin content of Guizi Wheat No.1 are positively correlated.