The Genetic Diversity Of α-amylase Gene In China Barley Germplasm And Association With α-amylase Activity

The important task of crop germplasm research is to meet requirement of production breeding.Therefore it is necessary to identify accurately the phenotypic and evaluate the genetic diversity ingermplasm resources. Characterization of diversified materials with molecular markers offers aneffective measure to conduct marker-trait association analysis for biological and agronomic interest,could be used for marker-assisted selection in barley improvement.In malting barley, high α-amylase activity is desired because this enzyme plays a major role inmalting and subsequent processes. In order to utilize the valuable genetic diversity in barley landracesgermplasm,257landraces were being genotyped using41SSR markers distribution in sevenchromosomes of barley, and phenotypic data were got through analysis for α-amylase activity by DNSmethod. Population structure and genome wide linkage disequilibrium (LD) were investigated, andassociation mapping was conducted between genotypes and phenotypes based on linkage disequilibrium(LD). The diversity of functional genes Amy32b and Amy6-4coding for the α-amylase, wasinvestigated by resequencing, the main finding were as following:1. α–amylase activity in germination seed was identified in257barley landrace of China. Resultsshowed that there was extensive variation in α–amylase activity in barley landrace, with about four-folddifference among accessions assayed. Falling number detected in dry seed did not associate withα–amylase activity in germination seed, suggesting that α–amylase activity in dry seed could not beused for determining degree of α–amylase activity.2. A total of709alleles were identified at41SSR loci in257landraces of China. The number ofalleles per locus ranged from5to44, with an average of17. The polymorphism information contentvalue for SSRs ranged from0.23(Bmag0385) to0.94(Bmac0032).99unique alleles were observed in257barley landrace.3. Cluster analysis depicted that nine clusters were identified based on Nei's genetic distance in257accessions.(labeled Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,Ⅶ,Ⅷand Ⅸ). Cluster I consists of17accessions with spring,six-rowed and covered as the the main features. There were45accessions in Cluster Ⅱ, mainly basedon characteristic of semi-winter, six-rowed and hull. Cluster Ⅲ and Cluster Ⅳ have the same main traitswith spring, six-rowed and nud or hulless. Cluster Ⅴconsisted of27accessions, with spring,two-rowed and hull. Cluster Ⅵ and Cluster Ⅶ consisted of18and33accessions respectively, based oncharacteristics of semi-winter or winter via spring, six-rowed and hulless. Cluster Ⅷ consisted of45accessons based on winter, six-rowed, covered characteristic. Finally in Cluster Ⅸ there were fouraccessions all from Jiangxi province. Cluster analysis showed that germplasm classifications coincidedwith geographic origin and genetic relationships4. Association analysis results showed five SSR markers significantly associated with α–amylaseactivity. Two SSR located in the chromosome7(Bmac0273) and5(scssr09041), could explain almostthe same phenotypic variation (9%). The other three SSR makers in2H (EBmac0415),7H (Bmag03850) and3H (EBmac0708), explained phenotypic variation of9.7,15and8%, respectively.Allele effects were estimated in comparison to the null allele (missing plus rare alleles) for eachSSR locus. At Bmag0385-7H48accessions carrying allele A215produced the highest α-amylaseactivity, and Bmac0273-7H carrying A141produced relatively high positive effect on α-amylase activity,Especially for the locus Bmag0385, tightly linked with α-amylase gene HvAmy2(1.7cM), its alleleA215can be used as a marker for Amy2gene, and in marker-assisted selection for malting barleybreeding.5. Amy6-4gene expressed in the germination seed, coding for barley isozymes with high pI.58barley genotypes were targeted to amplify and sequence for the Amy6-4gene to detect sequencepolymorphisms. The result showed that there were four polymorphic site in the coding region of thegene. Two of them were nonsynonymous polymorphism, involved in amino acid change (Asn/Ser andGly/Ala, respectively). Three polymorphism site (SNP) located in the3'UTR.6. A total of seven single nucleotide polymorphisms (SNPs) were detected which defined fivehaplotypes.The most frequent haplotype was H3with51%frequency, followed by H1with about40%frequency, and the remaining three types of haplotype frequencies were only about10%.The probability for intragenic LD siginificant assessed for all58germplasm between all sevenintrachromosomal SNPs of amy6-4, only SNP3showed no LD to all other SNPs, the extent LD for SNPwith siginificant LD corresponding r2value was beween0.8-1. Associations were carried out firstly byconsidering each individual SNP, and secondly by combining all seven SNPs in haplotypes,the resultshowed that no SNPs or haplotypes were associated with α-amylase activity, but haplotype one wasassociated with the trait for covered or naked seed. The sequence variation for Amy6-4in the barleyaccessions showed neutral selection, which did not impact on selection in the evolutionary process.7Only one single nucleotide polymorphism (G/A) was found located in2269bp in295accessionsof China of Amy32b through sequencing analysis. Two genetype can be indentified among295accessions (Amy_A and Amy_G). Results showed the frequency of allele A was much higher than alleleG, but allele G only occur in naked barley. In terms of geographical distribution, the highest frequencyof allele G taken place in Tibet wild barley, followed by Qinghai-Tibetan cultivated, then by SouthwestNorthwest and The middle and low valley of Yellow River.8. The single nucleotide polymorphism (cSNP) and insertion/deletion variation were found in thecoding region of Amy32b and stop codon formation, respectively. Although the cSNP resulted in aminoacid substitution did not associated with α-amylase activity. But the stop codon formation induced fromthe indel developed the truncated cDNA and further produced no function α-amylase.