【作者】 陈锋；

【导师】 何中虎； 夏先春；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2006， 博士

【摘要】 籽粒硬度是最重要的小麦品质性状之一,对磨粉和食品加工品质有重要影响,研究籽粒硬度基因的分子基础对小麦品质改良具有重要意义。本试验选用805份中国当前主栽冬、春麦品种(系)、地方品种和历史品种(共计3个试验)以及596份来自CIMMYT的小麦品种(系)(共计3个试验)材料,采用单籽粒谷物特性测试仪、PCR扩增、酶切、改进的SDS-PAGE和测序技术对其籽粒硬度及其基因型进行了鉴定,并对286份品种(共计2个试验)的磨粉和食品加工品质进行了测试。主要结果如下: 1.我国冬、春小麦主栽品种均以硬质类型为主,由北向南,硬质麦比例逐渐减少。硬质冬麦中共有Pina-D1b、Pinb-D1b和Pinb-D1d两种已知基因型,所占比例分别为12.9%、73.4%和1.6%;而硬质春麦中则发现有Pina-D1b、Pinb-D1b和Pinb-D1c三种已知基因型,所占比例分别为37.9%、51.5%和4.9%。发现了两种Pinb突变新类型:农大3213等13份冬麦和6份春麦品种(系)的Pinb基因的第42氨基酸中有一碱基A缺失,引起了移码突变,导致该基因第60位氨基酸变成了终止密码子,从而致使该类型PINB蛋白缺失,将其命名为Pinb-D1p;京冬11和沧核030的Pinb基因第218位点碱基G突变成了T,导致该基因第44位点色氨酸变成了亮氨酸,将其命名为Pinb-D1q。 2.我国地方品种和历史品种均以硬质类型为主。从地方品种、历史品种到当前主栽品种,软麦(分别为42.7%、45.2%和25.1%)和混合麦(24.3%、13.9%和14.1%)的比例逐渐减少,而硬麦(33.0%、40.9%和60.8%)的比例显著增加。硬质麦中共发现Pina-D1b、Pinb-D1b和Pinb-D1p三种已知基因型。从基因型的变化来看,PINA缺失(43.8%、48.5%和24.2%)和Pinb-D1p(39.7%、14.7%和8.4%)分布频率逐渐减少,而Pinb-D1b(12.3%、36.8%和63.4%)的频率显著增加。在地方品种中发现了三种新突变类型:来自贵州的光头线麦和红麦Pinb基因中第226位点碱基由G变成了C,导致其推断的第47位氨基酸由甘氨酸变成了精氨酸,命名为Pinb-D1t;来自江苏的红和尚Pina基因的第187位点碱基由C变成了T,导致该基因第35位点脯氨酸变成了丝氨酸,命名为Pina-D1m类型;有6个地方品种的Pina基因第212位点碱基由G变成了A,导致其第43位点色氨酸变成了终止密码子,被命名为Pina-D1n类型。另外,有5份地方品种Pina基因第265位点有一碱基C缺失,产生了移码突变,从而导致该基因推断的第93位点氨基酸变成了终止密码子,致使PINA蛋白不能表达,尽管该类型先前已经报道,但命名有误,本文重新命名为Pina-D1l类型。其中,Pina-D1m是目前发现的第一个含有PINA蛋白并且导致普通小麦胚乳质地变硬的突变类型,修订了先前的籽粒硬度分子理论模型。 3.CIMMYT普通小麦以硬质类型为主,硬质麦中只有Pina-D1b和Pinb-D1b两种基因型,Pina-D1b是最常见的基因型,占硬麦总数的86.3%,Pinb-D1b占13.7%。建议CIMMYT多引进一些其它硬度变异类型的小麦种质,如Pinb-D1b类型等,以减少Pina-D1b对品质改良的负面影响。在人工合成小麦与普通小麦杂交的高世代材料中共鉴定出Pina-D1a/Pinb-D1a、Pina-D1j/Pinb-D1i、Pina-D1c/Pinb-D1h、Pina-D1a/Pinb-D1i和Pina-D1a/Pinb-D1j五种基因型,均表现为软质,其中Pina-D1j/Pinb-D1i是最为常见的突变类型。Pina-D1c/Pinb-D1h和更多还原

【Abstract】 Kernel hardness is one of the most important characters with a profound influence on milling performance and end-use quality of common wheat (Triticum asetivum L.). Understanding molecular characterization of puroindoline genes controlling kernel hardness would be beneficial for improving wheat quality. Materials used in this study are consisted of 805 Chinese cultivars, including current winter and spring wheat cultivars, landraces and historical cultivars, and 596 CIMMYT cultivars and advanced lines. All of the surveyed materials were used to measure kernel hardness by Single Kernel Characterization System (SKCS) or Partical Size Index (PSI) and detect the variation of puroindoline alleles with STS (sequence tagged site) marker, site-specific cleavage with the restriction enzyme PvuII and modified SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) of Triton X-114 soluble protein, 10% glycerol substituting of water for resolving gels and PDA (piperiazine diacrylamide) substituting of N, N’-methylene bisacrylamide for gels, respectively. In addition, 286 genotypes were used to measure milling and processing quality. The main results obtained in this study are summarized below.1. Hard wheat was dominant in Chinese current winter and spring wheats and its percentage was gradually decreased from north to sourth. Pinb-Dlb, Pina-D1b and Pinb-D1d of known alleles were present in hard winter wheat and Pinb-Dlb, Pina-Dlb and Pinb-D1c of known alleles were present in hard spring wheat. Pinb-Dlb was the most prevalent hard genotype in Chinese current winter and spring wheats with percentages of 76.5% and 51.5%, respectively. Pina-Dlb with percentage of 37.9% in Chinese spring wheat was higher than that with percentage of 16.0% in Chinese winter wheat. Pinb-Dld was identified in two Chinese winter wheat cultivars, while Pinb-Dlc was detected in five Chinese spring wheats. Two novel puroindoline alleles were identified in Chinese winter wheat cultivars, which were designated as: i) Pinb-Dlp with characterization of a single nucleotide (A) deletion corresponding to position 42 in the amino acid sequence of puroindoline b, resulting in a Lysine (K) to Asparagine (N) change and leading to a shift in the open reading frame (ORF) and ii) Pinb-D1q with characterization of a base G to T substitution at position 218th nucleotide in the coding sequence of the Pinb gene, resulting in a tryptophan to leucine change (TGG to TTG) at position 44 in the deduced amino acid sequence of puroindoline b.2. Hard wheat was dominant in Chinese landraces and historical cultivars. Pinb-Dlb, Pina-Dlb and Pinb-Dlp of known alleles were present in hard wheat. From Chinese landraces, historical cultivars to current wheat cultivars, the percentages of soft (42.7%, 45.2% and 25.1%) and mixed (24.3%, 13.9% and 12.4%) wheats were gradually reduced, whereas the percentage of hard wheat (33.0%, 40.9% and 62.5%) was significantly increased. PINA null (43.8%, 48.5% and 13.4%) and Pinb-Dlp (39.7%, 14.7% and 8.4%) were gradually reduced while percentage of Pinb-Dlb (12.3% and 36.8% and 76.5%) was significantly increased. Three novel puroindoline alleles were identified in Chinese landrance cultivars, which were designated as: i) Pinb-D1t with characterization of a base G to C substitution at the 226thnucleotide in the coding sequence of the Pinb gene, resulting in a glycine (GGC) to arginine (CGC) substitution at position 47 in the deduced amino acid sequence of puroindoline b, ii) Pina-DH with characterization of a base G to A change at position 212, resulting in tryptophan-43 to a ’stop’ codon, and iii) Pina-Dlm with characterization of a base C to T substitution at the 187th nucleotide in the coding sequence of the Pina gene, which results in a proline (CCG) to serine (TCG) substitution at position 35 in the deduced amino acid sequence of puroindoline a. In addition, the Pina-Dlc allele, characterizing as a base C deletion at position 265, was renamed as Pina-DH due to conflicting with a previous report after negotiation with Drs Mclntosh and Morris. Pina-Dlm allele reported in this study is the first amino acid subsitution in PINA of bread wheat that is known to result in hard endosperm, which modifies the molecular genetic model of grain hardness.3. In CIMMYT wheat cultivars and advanced lines, hard wheat was the most dominant phenotype. Pina-Dlb and Pinb-Dlb were present in hard wheat. Pina-Dlb was the most popular genotype in CIMMYT hard wheats with a percentage of 86.3% and all other hard wheats possessed Pinb-Dlb genotype. Pina-Dlb genotype had inferior milling and processing quality than that of Pinb-Dlb. Therefore, it was recommended that other types of puroindoline alleles should be introduced in CIMMYT wheat breeding program to reduce the negetive influence of the Pina-Dlb genotype on milling and processing quality. In addition, Pina-Dla/Pinb-Dla, Pina-Dlj/Pinb-Dli, Pina-Dlc/Pinb-Dlh, Pina-Dla/Pinb-DH and Pina-Dla/Pinb-Dlj were present in genotypes with puroindoline alleles from various Aegilops tauschii. They were still soft wheats and Pina-Dlj/Pinb-Dli was the most popular genotype among them. Means of SKCS hardness with Pina-Dlc/Pinb-Dlh and Pina-Dla/Pinb-DH were significantly higher than those of Pina-Dlj/Pinb-Dli and Pina-Dla/Pinb-Dla. It is very useful to improve the efficiency of wheat breeding that new puroindoline alleles have recently been introduced through the extensive use of synthetic hexaploid wheat.4. Kernel hardness have significantly positive correlations with flour particle size (r=0.91), water solvent retention capacity (r=0.73), sodium carbonate solvent retention capacity (r=0.59) and farinograph water absorption (r=0.80), and significantly negetive correlations with flour colour L* (r=-0.77). Means of flour ash content and farinograph water absorption of lines with Pinb-Dlb were significantly lower than those of Pina-Dlb, while means of milling yield, volume and total score of steamed bread, firmness and total score of noodle of Pinb-Dlb genotypes were significantly higher than those of Pina-Dlb, suggesting that Pinb-Dlb genotype has slightly superior milling quality and processing qualities of steamed bread and noodle to Pina-Dlb genotype.This is the first extensive survey of puroindoline alleles in Chinese and CIMMYT wheats. It also indicates that variations of puroindoline locus have dramatic effect on milling and processing quality, and suggest that increasing Pinb-Dlb genotype in CIMMYT and Chinese wheat breeding, especially in spring wheat region, will be crucial for improvement of wheat quality in China and CIMMYT.更多还原