| Waxy barley is such a kind of barley whose endosperm has low or no amylose. Waxy gene (Wx in short) , a key gene responsible for the barley amylose synthesis and locates in the short arm of chromosome 7H in barley, has a direct impact on the structure, composition and processing quality of starch in barley endosperm. At present, no any identification and related research have been conducted for the waxy barley germplasm and Wx gene in China. Identification of waxy barley and research on genetic diversity of Wx gene not only will find out waxy barley and new Wx genes, providing parents for barley quality breeding, but also will help to understand origin, development, distribution and spread of waxy barley cultivar and alleles, characteristics, distribution and function variation of Wx gene, increasing knowledge on evolution of cultivated barley.In this study, more than 7700 Chinese barley cultivars preserved in National Crop Germplasm Resources Center of China were employed as basic materials. Waxy barley genotypes were screened by the method of half kernel with I2-KI. The geographic distribution of waxy barley was analyzed and the origin of waxy barley cultivars discussed. The individual plant content of amylose in these cultivars was measured by Near infrared reflectance spectroscopy. Wx proteins from cultivars with different amylose content were identified by SDS-PAGE. Genetic diversity of the barley cultivars with waxy genotype was analyzed based on SSR markers. According to the published Wx gene sequence, the contig primers were designed for the whole region of Wx gene with Primer primer5 software. After PCR amplification and gene clone, 30 Chinese normal and waxy barley cultivars, which had high, medium and low amylose content respectively, were sequenced and polymorphism analyzed for Waxy gene with 2 Canadian waxy barley as controls. The main findings were obtained as following:1. 252 cultivars with waxy genotypes were screened from the 7713 domestic barley cultivars in China, accounting for 3.3% of the total identified. But the proportion of waxy genotypes in each cultivar varied much, ranging from 2.7% to 90.0% with an average of 32.8%. The waxy barley mainly distributed in the middle and lower va1ley of Yellow River and Yangtze River geographically in China, with Henan, Jiangsu and Yunnan-Tibet as centers respectively. The three distribution regions is dependent each other in origin, because no notable spreading grade of waxy gene frequency was found among them. Henan province seems to be the earliest place where barley waxy gene mutation took place with the highest rate of waxy barley cultivars and the highest rate of waxy genotypes in waxy barley cultivars in China. The reason that waxy barley cultivars were formed was due to smaller kernel and increasing kernels per spike and enhancing coefficient of propagation and expansion of its proportion in the population when keeping its intrinsic tillering ability and the yield.2. The results of amylose content (AC) measure with NIRS showed that all waxy barley were low AC cultivars and no one no-AC was found. The amylose content ranged from 13.34% to 38.23% among 162 waxy barley cultivars, with an average of 25.23%, a standard deviation of 4.18 and a variation coefficient of 16.56%. The genotypes with AC lower than 15% were found in 28 barley cultivars. Among them 7 plants were found to be lower than 10% in amylose content, which are Jian24 (ZDM09222),Juyemidamai (ZDM08677),Midamai (ZDM00478),Mangdamai (ZDM00693),Sanyuehuangdamai (ZDM09028),Guangmanghuangdamai (ZDM09113) and could be used in barley quality breeding and waxy gene study. Because there was much difference of amylose content between and within cultivars, it should be pay attention not only to AC but also to population purity in identification and cross parent selection of waxy barley.3. There could be four types of biochemical genetics in the low and no amylose barley. Among them two types were related to no AC barley: in one of which waxy gene could transcribe normally, but the enzyme waxy protein produced has no catalytic activity of amylose synthesis. in the another of which waxy gene could not transcribe normally and no waxy protein produced. In the third type, waxy gene could transcribe normally, but catalytic activity of waxy protein was low. In the fourth type waxy gene could only partly transcribe and produce insufficient waxy protein.4. 50 pairs of SSR molecular markers were used for genetic diversity analysis of the 162 barley cultivars with waxy genotypes. In all testing sites, the effective number of alleles was 188.51, with an average of alleles 3.77, accounting for 92.86% of the total gene locus. All these showed that there was a high genetic diversity within the barley containing waxy genotypes of China, which is a rich gene pool for genetic improvement of barley. The SSR analysis for the barley with waxy genotypes from Henan, Jiangsu, Yunnan-Tibet showed that there was no difference in polymorphism information index and the genetic diversity index between the three provinces or districts, which offered further evidence for the independent mutation origin of waxy barley in the three areas.5. There were four kinds of amplification production with primer P1 found: 600bp, 800bp, 1000bp and 800bp+1000bp, in barley till now. Only thee kinds of 800bp, 1000bp and 800bp+1000bp, but no 600bp were found in 501 plants from 162 Chinese barley cultivars, which contain waxy genotype individuals. And the types of 1000bp and 800bp+1000bp occupied overwhelmingly majority and extremely minority, with 85.2% and 0.04% respectively.6. By polymorphism identification of waxy gene nucleotide sequences for the 32 barley cultivars, 169 polymorphism sites were identified, which included 143 SNP and 26 InDel with frequencies of 1/310 and 1/196 respectively. It was found that the intron 1,3,5,8, 2,5, exon 2,5 and 5'-UTR, 3'-UTR were the rich nucleotide variation regions, while nucleotide variation was rare in other regions. At the same time, Wx gene showed the phenomena of linkage disequilibrium (LD) and exon 2 and intron 1 regions were subject to the least selection pressure in its entire gene sequence. 7. Research on Wx gene nucleotide diversity from 30 accessions showed that 5bp,4bp,4bp insertions, appeared in 637 site,757site and 891 site in the leading sequence of Wx gene respectively in all 6 low amylose content barley accessions of China. Meanwhile, there were a lot of SNPs appeared in 5′-UTR and coding regions of the gene, of which 11 SNP in coding regions were non-synonymous. Insertion of 15 bp in 1654 site was found in 23 accessions with medium and high amylose content, but not found in all the 6 low amylose content accessions. This showed that the low amylose content barley in China had some common characteristics in sequence variation of Wx gene and their decrease of amylose content perhaps related to the 15bp deletion when compared with the accessions with medium and high amylose content. |
