Association Analysis Of Important Traits With SSR Markers In Waheat Landraces Of The Yangtze-River Region And Molecular Cloning Of A Novel High Molecular Weight Glutenin Subunits Gene 1Dy12.6

Wheat is the third largest food crops after maize and rice in the world, which plays an important role in agricultural production. High-yield, good-quality and multi-resistant are always the major breeding goals of wheat genetic improvement. Due to the rapid development of technology, the breeding methods evolved from the traditional cross breeding to the molecular breeding, which makes the breeding efficiency improved significantly. However, the progress of wheat genetic improvement in China slowed down in recent years, the main reason is that the genetic basis of parent material used in breeding program is becoming more and more narrow. As a product of artificial selection,the wheat landrace has an abundant genetic diversity and geographical adaptability.Analysis of genetic diversity, identifying the excellent characteristics and explore elite germplasm resources with novel genes are important for the genetic improvement of wheat, especially for the quality traits and resistance, which can provides the important theoretical significance and practical value.In the present study, seven agronomic traits of 343 wheat landraces of the Yangtze-River region were investigated. Meanwhile, 56 simple sequence repeat markers(SSR) were employed to evaluate the genetic diversity and population structure. Basing on the results, the association analysis between traits and markers had been carried out for exploring the excellent allelic variations, which could provide useful information for quantitative trait locus (QTLs) and marker-assisted selection breeding. A novel y-type high molecular weight glutenin subunits (HMW-GS) had been identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), which was designated as 1Dy12.6 and had been cloned. The main results were presented below:1. Association analysis of important traits with SSR markers in wheat landraces of the Yangtze-River regionThe four yield-related traits and three quality traits were evaluated, such as plant height (PH), grains of main spike (GMS), length of main spike (LMS), thousand-kernel weight (TGW), grain protein content (GPC), sedimentation volume (SED) and kernel hardness (KH). The KH showed the largest variation with the coefficient of variation (CV)of 86.6%, whereas, GPC exhibited the least variation with the CV of 9.4%. A total of 254 alleles were detected by 56 SSR markers, the average alleles per locus were 4.5357 which ranged from 2 to 13. The polymorphism information content (PIC) value ranged from 0.0425 to 0.8957, with an average of 0.5546. There were 13 SSR loci with the PIC value more than 0.7000, which indicated that the selected materials had an abundant genetic diversity. The 343 wheat accessions were divided into two main groups by UPGMA cluster and Structure cluster, which suggested that the population could be used for detection of marker-trait association mapping. The result showed that 24 SSR loci were detected significant associated with the 6 traits except KH at the P<0.01 level. In which,the most (18) SSR loci were associated with GPC, meanwhile, the least (2) SSR loci were associated with TGW. Xwmc609-1DL was significantly (P<0.001) associated with GPC,whose phenotypic variation explaining rate (R2) was the highest as 22.49%. There were 4 markers were associated with more than 3 traits, especially the loci Xwmc594-3AL was associated with 5 traits (GMS, LMS, TGW, GPC and SED). These results might provide useful information for marker assisted selection in wheat breeding program.2. Molecular cloning of a novel HWM-GS gene 1Dy12.6A novel y-type HMW-GS possessed similar electrophoretic mobility to 1Dy12 and with molecular mass of 69,985 Da had been identified by SDS-PAGE and MALDI-TOF-MS, the novel candidate 1Dy subunit was designated as 1Dyl2.6. Since the highly conservative of HMW-GS coding gene, the wheat genomic DNA can be readily employed as a template to amplify the ORF region, the two primers P1 and P2 were employed to amplify 1Dy12.6 gene of 'Huazhong830'. A single amplified fragment about 2000 bp in 'Huazhong830' was obtained, and then purified the target DNA fragments and sequenced. The result showed that the length of 1Dy12.6 nucleotide sequences were 2022 bp, containing 673 amino acid residues. The deduced mature protein molecular mass(70,165 Da) were consistent with the identified by MALDI-TOF-MS (69,985 Da), the error of measurement represented only 0.26%, further confirmed that 1Dy12.6 lacked extensive post-translational modifications. The deduced amino acid sequences of 1Dy12.6 showed typical similar primary structural characteristics for y-type HMW-GSs.Sequences comparison with other 26 cloned HMW-GS gene sequences revealed that gene sequences of 1Dy12.6 was highly homologous to the 1Dy type (from 93.81% to 99.70%).Selecting 1Dyl2 (JF736016) code genes with identity of 97.63% for further comparison study, three SNPs and a 45 bp insertion was found, which is the main reason lead to the difference between the two amino acids. Based on the secondary structure prediction and phylogenetic analysis, it was speculated that the novel 1D12.6 might be a new member of HMW-GS which was strong similarity to 1Dy12.