Pre-Harvest Sprouting Resistant Germplasms Screening And Excellent Genes Discovery In Wheat(Triticum Aestivum L.)of Henan Province

As the world’s largest producer and consumer of wheat,China has always placed great importance on genetic improvement of wheat varieties in terms of yield and quality.Pre-harvest Sprouting(PHS)can occur in many wheat varieties when there is a certain amount of rainfall before harvesting,which can lead to a decrease in yield and quality,and have adverse effects on agricultural production.Therefore,selecting wheat germplasm resources resistant to PHS and exploring the related genes that confer resistance to PHS are of great significance for breeding wheat varieties that are resilient to disasters,ensuring the potential yield and stable quality of wheat varieties.In order to screen excellent germplasm resources of wheat resistant to PHS in the Huang-Huai wheat region and identify the excellent genes responsible for resistance to PHS,629 wheat varieties from Henan Province were selected as materials,and the resistance index of PHS was evaluated for two consecutive years to identify and select excellent wheat varieties with resistance to PHS.The distribution and effects of known PHS resistance loci on wheat varieties in Henan Province were analyzed.The wheat 660 K chip was used for genome-wide scanning of the materials,and genome-wide association analysis(GWAS)was performed on PHS resistance based on population structure and linkage disequilibrium analysis.Furthermore,the identified PHS resistance loci were analyzed using transcriptome sequencing.The following research results were obtained:1.The range of variation in the whole spike germination rate of 629 wheat varieties for the two years of 2020-2021 and 2021-2022 were 0.51%～99.22% and 0.00%～97.47%,respectively,indicating that the tested material population has rich spike germination phenotype variation.The study found that the average level of PHS resistance in wheat local varieties to modern breeding varieties is proportional to the number of red wheat varieties in that era,but 38 white wheat varieties with excellent PHS resistance were also identified from the 296 white wheat varieties in the tested material.Among them,only one white grain variety reached PHS resistance: Baixiantiao.37 varieties with middle resistance:Qisiniu,Baifumai,Huangguaxian(Yuxian),Yaerzui,Heshangtou(Anyang),Shuanghoumai,Zhuganqing(Tangxian),Baihulutou,Xiaobaimai(Mianchi),Zigancao(Yichuan),Baimai(Xichuan),Baipimai,Baitiaoyu(Mixian),Ermangmai(Biyang1),Baijuan Mamg,Shiganmai,Fenzhixiaomai,Dongmai,Wuzitoumai,Jinsita,Honghulutou,Baigedatou,Tutoumai(Qixian),Sanbaoxian,Bima6 hao,Fulanni,Zhaomai2 hao,Zhengzhou5hao,Xuzhou14,Fengchan1 hao,Fengchan3hao,Yumai13,Yumai33,Jimai20,Gaoyou2018 and Xinmai30.The above varieties provide important germplasm resources for the cultivation of modern wheat varieties with white grains and resistance to sprouting by exploring anti-sprouting genes that are not linked to red seed coat traits.2.The study used molecular markers for 10 known PHS resistance-related loci to test629 wheat varieties in Henan Province.The results showed that the relative PHS index of each locus’ s resistant allele variation was significantly lower than that of the susceptible allele variation,which is consistent with previous research.The study first counted the number of PHS resistant varieties(R1),white-grained PHS-resistant varieties(R2),and red-grained PHS-resistant varieties(R3)with excellent allele variations for each of the 10PHS-related loci.Secondly,the total number of varieties(N1),white-grained varieties(N2),and red-grained varieties(N3)with excellent allele variations were counted,and the proportion of PHS-resistant varieties with excellent allele variations in the total number of varieties was calculated according to the formula Q1=(R1/N1)*100%,Q2=(R2/N2)*100%,and Q3=(R3/N3)*100%.It was found that Tamyb10-B1 b was the allele variation with the largest Q1 and Q3 values among the 10 excellent allele variations,especially with Q3 reaching 97.9%,indicating that this excellent allele variation could explain 97.9% of the phenotypic variation in red-grained varieties.Ta MKK3-Ab was the allele variation with the largest Q2 value among the 10 excellent allele variations,and it could identify 39.7% of the PHS-resistant white-grained varieties containing Ta MKK3-Ab allele variation,indicating a relatively high efficiency in identifying white-grained PHS-resistant varieties.This result provides a methodological basis for identifying PHS resistance in white-grained varieties.3.The 629 test materials were scanned using the wheat 660 K chip,and 314,548 SNP markers were obtained after excluding redundancy.Based on this,a genome-wide association analysis of PHS resistance in wheat varieties from Henan Province was performed,and 102 quantitative trait nucleotide(QTN)loci were detected.Among them,22 QTNs were detected by multiple environments and methods,with a phenotype variation interpretation rate(R2)ranging from 0.00001% to 38.1121%.AX-111020384 on chromosome 3A and AX-95124645 on chromosome 3D were detected by seven methods in both environments,with maximum R2 values of 12.8% and 38.1%,respectively.The QTN with the highest R2 value,AX-95124645,was identified as the main-effect QTN for PHS.Based on the LD decay distance of the test materials,the main-effect QTL QSS.AX-95124645(chr3D:569.167 Mb ～ 573.551Mb)for PHS was determined.4.The study aimed to identify candidate genes for resistance to PHS in wheat by using high-throughput RNA sequencing technology on three different time points of transcriptome sequencing for three wheat varieties with different levels of resistance.The results showed that there were 153,699 genes obtained after alignment with the reference genome,and 1,460 up-regulated and 1,559 down-regulated differentially expressed genes between two white wheat varieties with different levels of resistance.There were also2,210 up-regulated and 1,703 down-regulated differentially expressed genes between red and white wheat varieties with different levels of resistance.The study found that the color of the seed coat was closely related to the differential expression of resistance genes.Through KEGG pathway enrichment analysis of differentially expressed genes,it was found that the Glycolysis/Gluconeogenesis pathway was significantly enriched in different wheat varieties with different seed colors,indicating that this pathway may provide energy for seed germination.The study identified 34 genes that were differentially expressed in all three time points and may be related to PHS in white wheat varieties.The study also validated the GWAS results and identified 56 candidate genes for QSS.TAF9-3D in three wheat varieties with different levels of resistance.The study further identified nine candidate genes,including Traes CS3D01G466100 and Traes CS3D01G468500,which may be involved in regulating wheat PHS.The study used bioinformatics methods to predict the functions of these two genes and confirmed them as candidate genes for wheat PHS.5.According to the AX-95124645 SNP locus,the KSAP molecular marker QSS.TAF9-3D-TT/CC was developed and validated through sequencing alignment,tested material haplotype typing,and F2 population construction.QSS.TAF9-3D-TT/CC can effectively perform haplotype typing on tested materials and F2 populations,and is an efficient and reliable molecular marker for screening for PHS resistance varieties.There are263 QSS.TAF9-3D-TT haplotypes and 366 QSS.TAF9-3D-CC haplotypes in the tested materials,with QSS.TAF9-3D-TT being the superior haplotype.There are 11 superior haplotypes of QSS.TAF9-3D-TT in white wheat,and all of them have resistance to PHS.This marker can be used for PHS resistance testing in white wheat varieties,and provides an accurate and efficient molecular marker selection method for breeding white wheat varieties with PHS resistance.Based on the study,38 wheat varieties resistant to spike germination and white grain were identified among 629 wheat varieties in Henan Province.Using GWAS,a PHS resistance main effect QTN AX-95124645 located on chromosome 3D was detected,and a KASP molecular marker QSS.TAF9-3D-TT/CC was developed,which can effectively detect PHS resistance materials.Among them,11 white wheat varieties with excellent haplotype QSS.TAF9-3D-TT showed resistance to PHS.By RNA-seq of wheat varieties resistant and susceptible to PHS,differentially expressed genes related to PHS resistance were obtained.Combined with GWAS results,two wheat PHS resistance candidate genes Traes CS3D01G466100 and Traes CS3D01G468500 were identified in differentially expressed genes.The above research results provide important materials and methods for wheat breeding resistant to PHS in the future.