【作者】 朱彩梅；

【导师】 张京；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2009， 博士

【摘要】 糯大麦是指籽粒胚乳中直链淀粉含量很低或者为零的大麦。Waxy基因(简写为Wx)是控制大麦籽粒直链淀粉合成的关键基因,位于大麦7H染色体的短臂上,其编码酶蛋白的功能和特性直接影响大麦胚乳的淀粉结构、组成和加工品质。目前我国尚未对保存的大麦种质资源进行糯性鉴定及相关研究。开展糯大麦种质资源鉴定,研究我国糯大麦及其Wx基因的遗传多样性,不仅可以发现新的糯大麦种质,挖掘新的糯性淀粉等位基因,从而在亲本材料和糯性淀粉功能基因方面为我国大麦品质育种奠定基础,而且可以深入了解我国糯大麦品种的形成、衍化、分布和传播,以及Wx基因的等位变异类型、特点和分布及功能特点等,为栽培大麦的进化研究积累知识。本研究以我国作物种质资源库中保存的7700多份国内农家大麦品种为基础材料,采用半籽粒碘-碘化钾染色法进行表型鉴定,筛选出含糯性基因型的大麦品种,分析了它们的地理分布,讨论了糯大麦品种的形成;采用近红外法,分单株测定了这些品种的直链淀粉含量;通过SDS-PAGE电泳,对不同直链淀粉含量大麦品种的Wx蛋白进行了检测;采用SSR分子标记,对筛选的含糯性基因型的大麦品种进行了遗传多样性分析;根据公布的大麦Wx基因序列,利用Primer primer 5软件,在该基因的各个不同区域设计重叠引物,以2个国外糯大麦品种为对照,对30份高、中、低不同直链淀粉含量的中国大麦进行了Wx基因的PCR扩增、克隆、测序和等位变异分析。取得的主要研究结果如下:1.从7713份国内大麦种质资源中筛选出252份含糯性基因型的大麦品种,占鉴定品种总数的3.3%。不同品种糯性基因型个体所占比例差异很大,变异幅度为2.7%～90.0%,平均为32.8%。中国的糯大麦品种主要分布在黄河中下游、长江中下游、云贵及青藏高原地区,分别以河南、江苏和滇藏为中心。3个分布区之间,没有出现糯性基因频率从一个地区向另一个地区扩散传播的明显梯度变化,具有独立的突变起源。从糯大麦品种所占比例及其纯合程度来看,河南似乎是我国最早出现糯性基因突变和糯大麦品种的地区。糯大麦品种是糯性突变体在保持原有的分蘖力和不降低产量的情况下,由于籽粒变小、每穗粒数增加、提高了繁殖系数、逐代扩大其在群体中的比例而形成的。2.利用近红外反射光谱(NIRS)法,对含糯性基因型品种的直链淀粉含量测定结果表明,在中国大麦中鉴定发现的糯大麦种质均属低直糯大麦,尚无一份是无直糯大麦材料。162份含糯性基因型品种的直链淀粉含量变异范围为13.34～38.23%,平均值为25.23%,标准差为4.18,变异系数为16.56%。在28个品种中检测到直链淀粉含量小于15%的单株,其中在6个品种中鉴定出7份单株材料,其直链淀粉含量低于10%,可以在我国糯大麦育种和遗传等基础理论研究中优先利用,分别是鉴24(ZDM09222)、钜野米大麦(ZDM08677)、米大麦(ZDM00478)、芒大麦(ZDM00693)、三月黄大麦(ZDM09028)、光芒黄大麦(ZDM09113)。由于大麦品种间和品种内单株之间的直链淀粉含量存在很大差异,因此进行糯大麦种质资源鉴定筛选时,应在品种群体测定的基础上,进一步开展单株纯系繁殖鉴定;在杂交育种中选择糯大麦亲本时,不但要考虑备选品种的直链淀粉含量,还要考虑其个体基因型的遗传一致性。3.在低直糯大麦和无直糯大麦两种类型中,可能存在四种生化遗传变异类型。其中有两种与无直糯大麦有关,一种是Wx基因可以正常转录,但转录产生的Wx蛋白无活性,不能催化直链淀粉合成;另一种是Wx基因不能正常转录,既无Wx蛋白产生,也无直链淀粉合成。另外两种与低直糯大麦有关,一种是Wx基因可以正常转录,但转录产生的Wx蛋白活性有限,催化直链淀粉合成的功能降低;另一种是Wx基因不完全正常转录,产生的Wx蛋白数量有限,影响直链淀粉合成。4.利用50对SSR分子标记对162份含糯性基因型的中国大麦品种进行遗传多样性分析,在所有检测位点中,有效等位基因数为188.51,平均有效等位基因数为3.77,占总等位基因数的92.86%。表明中国糯大麦具有较高的遗传多样性,是进行大麦遗传改良的丰富基因库。对河南、江苏、云南和西藏的含糯性基因型大麦品种进行的SSR分析发现,其多态性信息指数、遗传多样性指数差别不大,进一步为中国不同地区的糯大麦基因可能起源于独立的自然突变提供了佐证。5.迄今利用Wx基因的STS引物P1,在大麦中共扩增出600bp、800bp和1000bp及800bp与1000bp杂合等4种带型。本研究在162个中国大麦品种的501个单株中,只检测到800bp、1000bp两种单一带型和这两条带同时出现的杂合带型,没有发现具有600bp带型的品种或单株,并且以1000bp的带型居多,杂合带型非常罕见,3种基因型分别占14.4%、85.2%和0.04%。6.在对32个大麦品种的核苷酸序列多态性鉴定中,共检测到了169个多态性位点,平均每26个bp检测到一个多态性位点。在所有检测到的多态性位点中,包括143个SNP和26个InDel,二者的频率分别为1/310和1/169。Wx基因的内含子1、3、5、8区,外显子2、5和5′-UTR及3′-UTR区域为变异富集区,其它区域变异较小。同时,Wx基因表现出连锁不平衡现象,外显子2和内含子1区域所承受的选择压力较小。7.中国6份低直链淀粉含量的大麦材料,均在其Wx基因前导序列区第637碱基处插入了5bp、757碱基处插入了4bp、891碱基处插入了4bp的片段。此外,还在这些低直链淀粉材料Wx基因的5′-UTR以及编码区,发现均有大量SNP出现,其中在编码区有11处是非同义突变。在23份中、高直链淀粉含量材料Wx基因的第1654碱基处,同时发现有15bp的碱基插入片段,但在6份低直链淀粉含量的材料中均没有出现该15bp的插入片段,表明中国低直链淀粉大麦材料存在共同的等位变异特征,与中高直淀粉含量材料相比,其直链淀粉含量降低可能与该片段的缺失有关。更多还原

【Abstract】 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.更多还原