小麦Wx-A1和Wx-D1位点的PCR分子标记

为了获得筛选低直链淀粉含量的分子标记,根据已知小麦Waxy基因序列设计PCR引物,通过在基因水平和蛋白质水平进行分离群体验证,建立分别专一检测Wx-A1位点和Wx-D1位点的PCR标记MAG264和MAG269。在分离群体中,MAG264标记能清楚地区分Wx-A1位点的三种不同基因型,呈共显性遗传。MAG269标记引物在野生型中扩增片段为1 400 bp,在突变型(Wx-D1bWx-D1b)中的扩增片段为800bp。但在分离群体中没有出现杂合体带型,从而使本应该表现为共显性的标记表现为显性遗传。MAG264和MAG269引物对DNA的扩增稳定性、重复性好。这些结果表明,本研究获得的Waxy基因分子标记可以用于小麦Waxy基因缺失类型的鉴定和糯质专用小麦的分子标记辅助选育。     

糯性小麦研究进展

糯小麦(Waxy wheat)因有高于普通小麦的支链淀粉含量,在食品和工业方面有很大的应用价值和潜力,自发现后就成为各国小麦研究的重点之一.通过多年的努力,人们在糯小麦的遗传机理、育种理论和方法、品质性状及利用等方面的研究都取得了很大的进展,并在1995首次育成了全糯性小麦.糯小麦根据其糯蛋白缺失情况可分为8种类型.控制3个Waxy位点的Wx-A1,Wx-B1和Wx-D1基因分别位于染色体的7AS、4AL和7DS上.3个基因的核苷酸序列长度分别是2 781、2 794、2 862 bp,基因的完全编码区含有11个外显子和10个内含子,在核苷酸序列上三个糯基因彼此之间有惊人的同源性.研究表明,直链淀粉含量和淀粉糊化特性受Wx-B1的影响最大,其次是Wx-D1,Wx-A1蛋白的影响最小.糯小麦的鉴定主要在细胞水平、蛋白质水平、分子水平上进行,I2-KI染色法和SDS-PAGE法是较常见的鉴定小麦糯性的方法.糯小麦野生型与突变型基因组成差异的发现使得分子标记技术在糯小麦的选育中得到了极大的发展和利用.     

部分小麦品种(系)品质相关基因的分子检测

为了了解小麦品种(系)的遗传信息,提高优质小麦育种的效率,利用已有的品质相关基因的分子标记(Dx5、Wx-A1、Wx-B1、Wx-D1、1BL/1RS、PPO18和PPO29)检测了144份黄淮麦区、3份国内其他麦区和8份德国品种(系)的基因等位变异类型。结果表明,5+10亚基类型、Wx-B1b和1BL/1RS易位的出现频率分别为34.8%、2.0%和47.1%,郑麦、新麦、淮麦、烟台以及徐麦等系列小麦品种(系)5+10亚基出现频率较高,而周麦系列中频率较低;未检测到Wx-A1b、Wx-D1b以及双缺失类型的材料;2A染色体上PPO等位基因Ppo-A1a(高PPO)、Ppo-A1b(低PPO)及杂合型基因型出现频率分别为45.8%、49.7%和4.5%,2D染色体上PPO等位基因Ppo-D1a(低PPO)和Ppo-D1b(高PPO)基因型出现频率分别为47.1%和52.9%,两个基因组合类型Ppo-A1b/Ppo-D1b(中等PPO)的出现频率最高,为29.0%,Ppo-A1a/Ppo-D1a(中等PPO)、Ppo-A1a/Ppo-D1b(双高PPO)以及Ppo-A1b/Ppo-D1a(双低PPO)的出现频率依次为23.9%、21.9%和20.6%;聚合多个优质基因的频率很低,同时含有5+10亚基、缺失1个Wx基因、非1BL/1RS易位以及双位点低PPO活性(Ppo-A1b/Ppo-D1a)的材料仅有1份(郑麦366),同时含5+10亚基、非1BL/1RS易位以及Ppo-A1b/Ppo-D1a的材料也仅占5.2%,应加大多个优异基因聚合品种的选育力度。     

宁夏小麦黄色素含量相关基因的组成与分布

选育低黄色素含量品种是宁夏小麦品质改良的重要目标之一。为阐明宁夏小麦中控制籽粒黄色素含量基因TaZds-A1和TaZds-D1的组成及分布特点,利用其功能标记YP2A-1和YP2D-1对91份小麦品种进行检测与分析。结果表明,在TaZds-A1位点,等位变异TaZds-A1a(与低黄色素含量相关)和TaZdsA1b(与高黄色素含量相关)分别占59.3%和40.7%;在TaZds-D1位点,等位变异TaZds-D1a(与高黄色素含量相关)占95.6%,TaZds-D1b(与低黄色素含量相关)仅占4.4%。宁夏小麦黄色素含量基因位点存在4种等位变异组合类型:以TaZds-A1a/TaZds-D1a(57.1%)组合类型为主,TaZds-A1b/TaZds-D1a(38.5%)组合类型次之,TaZds-A1a/TaZds-D1b和TaZds-A1b/TaZds-D1b组合类型最低(2.2%);不同等位变异组合类型在不同地区间的分布比例也不同。     

小麦糯性基因多重PCR体系的建立

为建立通过一次反应能够同时鉴定3个小麦糯性基因(Wx-A1、Wx-B1和Wx-D1)的多重PCR反应体系,研究了PCR反应组分和循环参数对多重PCR反应结果的影响.结果表明,反应体系中的引物浓度比例和反应的Tm值是多重PCR成功的关键,当引物浓度比例(SSR/MAG267)为1(0.1 μmol/L∶0.1 μmol/L)～1.22(0.11 μmol/L∶0.09 μmol/L)、Tm为58℃时,Wx-A1、Wx-B1、Wx-D1基因的多重PCR结果最好.该多重PCR方法能快速高效地鉴定3个Wx基因,可用于糯麦选育和小麦品质育种的复合分子标记辅助选择.     

小麦Wx-A1和Wx-B1亚基的生化与分子标记检测

为了解小麦品种(系)Wx亚基的缺失类型,为品质育种提供依据,利用SDS-PAGE和STS分子标记技术对99份国内外小麦品种(系)Wx-A1和Wx-B1亚基的缺失型进行检测.结果表明,供试小麦品种(系)中Wx-B1亚基缺失的有19份,未检测出Wx-A1亚基缺失材料.STS标记结果中,Wx-7A位点的显性标记引物在野生型中扩增出一条1 172 bp的特异带,即Wx-A1a基因出现;Wx-7A位点的共显性标记引物在野生型中扩增出一条593 bp的特异带,在缺失类型中扩增出一条574 bp的特异带.Wx-4A位点的显性标记引物在野生型中扩增出一条425 bp的特异带,即Wx-B1a亚基出现,而在缺失该亚基的突变材料中没有扩增出该特异带.     

颗粒结合型淀粉合成酶研究进展

本文综述了小麦胚乳中合成直链淀粉的关键酶——颗粒结合型淀粉合成酶(GBSS)的研究进展，展望了GBSS的研究前景。小麦中的GBSS基因在种子中特异表达；控制3个Waxy，位点的基因Wx—A1、Wx—B1和Wx—D1，分别位于7AS、4AL和7DS上；六倍体小麦和二倍体小麦中3个GBSS的DNA序列从起始密码子到终止密码子，Wx—A1为2781bp，Wx-B1为2794bp，Wx—D1为2862bp。基因的完全编码区含有11个外显子和10个内含子，在核苷酸序列上三个糯基因彼此之间有惊人的同源性；研究表明，直链淀粉含量和淀粉糊化特性受Wx—B1的影响最大，其次是Wx-D1缺失蛋白，Wx-A1缺失蛋白的影响最小。GBSS的鉴定技术包括I2—KI染色、电泳和分子标记技术鉴定。     

宁夏小麦TaNrx-B1和TaFer-A1基因组成及其与相对发芽率的关系

为给抗旱小麦品种选育和评价提供依据,以117份宁夏小麦品种(系)为材料,鉴定种子萌发期相对发芽率,并利用抗旱相关分子标记检测TaNrx-B1和TaFer-A1基因组成,分析两基因组成分布特点,通过表型与基因型比较分析评价2个基因分子标记的有效性,同时筛选抗旱相关基因组成。结果表明,宁夏地区小麦种子萌发期总体平均相对发芽率为73.3%,变异范围为25.8%~98.2%;中等及以上抗旱性等级品种(系)占75.2%,达到供试材料的四分之三。宁夏小麦品种(系)中TaNrx-B1基因存在2种单倍型TaNrx-B1a和TaNrx-B1b,分别占供试材料总数的65.0%和35.0%,平均相对发芽率分别为71.4%和62.6%;TaFer-A1基因也存在两种单倍型TaFer-A1a和TaFer-A1b,分别占总数的74.4%和25.6%,平均相对发芽率分别为67.3%和62.2%。在两个基因的4种单倍型组合中,TaNrx-B1a/TaFer-A1a、TaNrx-B1a/TaFer-A1b、TaNrx-B1b/TaFer-A1a和TaNrx-B1b/TaFer-A1b依次占46.2%、8.5%、15.4%和29.9%,其种子的平均相对发芽率依次为70.7%、68.3%、65.1%和61.8%;相关分析发现,TaNrx-B1a/TaFer-A1a单倍型组合是抗旱类型组合,TaNrx-B1b/TaFer-A1b单倍型组合是非抗旱类型组合。综上所述,宁夏小麦种子萌发期总体抗旱性较强,以TaNrx-B1a/TaFer-A1a组合类型品种(系)的平均相对发芽率最高,所占比例也最多,两个基因的抗旱相关分子标记可用于小麦抗旱性初步鉴定和筛选。     

利用Wx基因的分子标记筛选部分糯小麦的研究

为了筛选出更多用于培育优质面条小麦的Wx基因缺失材料,用分子标记技术对260份中国核心小麦种质资源进行了分析,并用SDS-PAGE进行了验证.筛选出缺失Wx-A1蛋白亚基的品种2份,缺失Wx-B1蛋白亚基的品种14份,得到1份不同于"白火麦"的Wx-D1蛋白亚基缺失的地方品种"秃芒麦".对它们的地区分布分析发现,长江以北各麦区的小麦品种Wx基因缺失多于长江以南各麦区,且分布更为集中.对收集的20对Wx基因的分子标记引物进行了筛选,并对PCR扩增条件和产物分析条件进行优化,得到了3对可以在相同条件下对Wx基因进行筛选的引物,且能用同一电泳条件对产物进行分析,提高了分析效率.     

面条品质性状相关基因在四川小麦中的分布

为给面条小麦亲本材料的选择提供依据,利用多重PCR技术分析了Wx-B1和2个主效PPO位点的等位基因在104份四川主栽小麦和骨干亲本中的分布,并检测了59份新育成品系的淀粉糊化特性.结果表明,控制低PPO活性的等位基因PPO-2Ab和PPO-2Da的频率分别为42.3%和83.6%,Wx-B1蛋白缺失基因Wx-B1b的频率为26.9%,四川小麦淀粉特性整体表现较优良.Wx-B1缺失小麦在峰值粘度、稀懈值、最终粘度和反弹值方面与正常类型间差异显著.绵阳26 、川麦107、川麦43等是面条小麦育种的优良亲本.准确、实用、高效多重PCR技术适合于多位点控制的品质性状的标记辅助选择.     

Effect of the three waxy null alleles on enzymes associated to wheat starch granules using proteomic approach

In bread wheat (Triticum aestivum L.), waxy genes are present at three loci (Wx-A1, Wx-B1 and Wx-D1) and are responsible for amylose synthesis in the grain. Near isogenic lines (NILs) of the agronomic cultivar Tremie with either one, two or three waxy null alleles were used to study the genome response to these null alleles through proteomic analysis of starch granule associated proteins of mature grain. Among the 352 spots that were revealed, 86 varied significantly between NILs. The main differences were observed for the granule bound starch synthase (GBSS), product of the waxy genes. A strong relation (R² = 0.94) between the amount of GBSS per gram of starch granule and the amylose quantity in the grain was revealed through quantification of the GBSS in each NIL. The analysis of GBSS quantity for each NIL invalidated the additive hypothesis for the expression of this enzyme in (Wx-A1a Wx-D1a Wx-B1b) (Wx-D1a Wx-B1a Wx-A1b) and the normal form of Tremie. In addition, four proteins identified as inactive GBSS were increased (+144.9%) in the triple null isogenic form compared to the normal form of Tremie. The quantitative variations of the proteins present in starch granules support the idea of regulatory mechanisms between genomes.     

Effects of two novel Wx-A1 alleles of common wheat (Triticum aestivum L.) on amylose and starch properties

Common wheat (Triticum aestivum L.) has three Wx proteins (Wx-A1, Wx-B1, and Wx-D1), which are granule-bound starch synthases I and are responsible for the amylose synthesis of flour starch. The effects of two novel Wx-A1 proteins identified by gel electrophoresis on amylose content and starch properties were analyzed. The variant Wx-A1 protein coded by the Wx-A1i allele was present in smaller amounts and produced less amylose (7.3%) compared to standard lines with the Wx-A1a allele (21.5%). Wx-A1i generated altered starch properties; greater swelling power (SP), glucoamylase digestibility, starch paste clarity, and gelatinization enthalpy in differential scanning calorimetry. The starch from Wx-A1i also showed an altered pasting profile on a Rapid Visco-Analyzer, greater peak viscosity, smaller final viscosity, and lower pasting temperature. The Wx-A1i allele is a novel genetic resource for reducing amylose content in wheat. The other Wx-A1 protein coded by the Wx-A1j allele showed a more basic isoelectric point compared to Wx-A1a on an electrophoretic gel. The amylose content of Wx-A1j did not differ from standard Wx-A1a. Starch SP, paste clarity, and glucoamylase digestibility also suggested that Wx-A1j produced amylose as much as Wx-A1a.     

青海高原春小麦糯性新品系的筛选及其直链淀粉含量

为了培育适合青海高原种植的春小麦糯性新品系,利用DNA分子标记技术检测了80株青春533×糯麦1号F2代材料的Wx基因缺失情况,同时测定了不同Wx基因缺失类型F2代材料的小麦籽粒直链淀粉含量。结果表明,在80株F2代分离群体材料中,单缺失WxA1或WxB1或WxD1基因的材料分别为8份、11份、9份;同时缺失WxA1和WxD1基因的材料为10份,同时缺失WxB1和WxD1基因的材料为12份,同时缺失WxA1和WxB1基因的材料为9份;WxA1、WxB1和WxD1基因全部缺失的材料为8份。Wx基因的缺失数量与直链淀粉含量的高低有密切关系,缺失1个Wx基因（部分糯小麦）,直链淀粉含量比对照（青春533）降低5.28%~11.35%;缺失2个Wx基因（部分糯小麦）,直链淀粉含量比对照降低10.73%~19.81%;缺失3个基因（糯小麦）的直链淀粉含量为0.97%,比对照降低30.53%。高原春小麦与糯性小麦杂交的F2代直链淀粉含量下降幅度要比前人所研究的材料下降幅度大,并且WxB1单缺失引起直链淀粉含量下降（11.35%）比WxA1和WxD1双缺失所引起的下降幅度要大（10.73%）。     

俄引及黑龙江春小麦黄色素含量相关基因分析

为了将俄引小麦资源的优良基因快速用于我国春小麦品种的改良,利用与黄色素含量相关的八氢番茄红素合酶(Psy)基因STS分子标记,对250份俄罗斯引进和125份黑龙江主栽过的春小麦品种进行了研究分析,利用7A和7B染色体上的标记进行鉴定。结果显示,俄引小麦有242个品种(96.8%)扩增出高黄色素含量基因Psy-A1a;8个品种(3.2%)扩增出低黄色素含量基因Psy-A1b;黑龙江小麦有73个品种(58.4%)扩增出Psy-A1a基因,52个品种(41.6%)扩增出Psy-A1b基因。俄引小麦有187个品种(74.8%)扩增出高黄色素含量的Psy-B1a等位基因,63个品种(25.2%)扩增出低黄色素含量的Psy-B1b等位基因,15个品种(6.0%)扩增出高黄色素含量的Psy-B1c等位基因,40个品种(16.0%)扩增出Psy-B1d等位基因;而黑龙江小麦有40个品种(32.0%)扩增出Psy-B1a等位基因,85个品种(68.0%)扩增出Psy-B1b等位基因,5个品种(4.0%)扩增出Psy-B1c等位基因,24个品种(19.2%)扩增出Psy-B1d等位基因,所有供试小麦品种中都不存在Psy-B1e等位基因。2个位点不同等位基因组合共有12种,俄罗斯小麦品种中存在10种组合,黑龙江小麦品种中存在11种。其中,就双低含量等位基因组合Psy-A1b/Psy-B1b而言,黑龙江小麦品种较俄罗斯高28.4个百分点。     

Waxy endosperm accompanies increased fat and saccharide contents in bread wheat (Triticum aestivum L.) grain

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.     

Molecular characterization of two novel null waxy alleles in Mexican bread wheat landraces

Starch is composed of two glucose polymers: amylose, formed by long linear chains, and amylopectin, which is larger and highly branched. The ratio between the two polymers (usually 22–35% amylose, 68–75% amylopectin) affects the starch properties, thereby determining different aspects of wheat quality. Waxy protein, which is the sole enzyme responsible for amylose synthesis, showed polymorphism in a collection of one hundred and three Mexican landraces, including null alleles at Wx-A1 and Wx-B1 loci, respectively. Molecular characterization of these alleles showed that the null Wx-A1 allele presented in only one accession was novel (Wx-A1o) and involved a deletion spanning the three and a half last exons of the gene. Some of the accessions lacking the Wx-B1 protein contained the common allele Wx-B1b (12.6%) (deletion of approximately 67 kb), while four others (3.8%) possessed the novel Wx-B1l, characterized by the deletion of one cytosine in the second exon of the gene and leading to a change in the ORF. This novel allele is particularly interesting because the absence of the Wx-B1 protein in this case was not associated with the lack of the other genes included in the approximately 67 kb region lost with the common null allele Wx-B1b.     

25份澳大利亚小麦种质的性状表现及利用

为给黄淮南片冬麦区小麦育种提供信息,对25份澳大利亚小麦种质于2016-2018年度在新乡进行了部分农艺性状调查,对其品质性状及优质亚基等进行了分析。结果表明,25份澳大利亚小麦种质多表现为春性,综合农艺性状较差;千粒重低于黄淮麦区高产对照品种周麦18,变化范围为31.4 g～43.1 g;湿面筋含量、面筋指数、稳定时间、弱化度的变化范围分别为28.0%～39.1%、55.2%～96.5%、4.0 min～25.0 min、5%～100%。从澳大利亚小麦种质中,筛选出了具有湿面筋含量高、面筋指数高、稳定时间长、面粉粉色白等优良品质特性的材料,鉴定出了含5+10亚基、7+8~*亚基(7超量表达亚基)、17+18亚基、13+16亚基、低PPO活性基因及 Wx-B1基因缺失等优异品质材料,筛选出了含有 Rht-D1b矮秆基因以及 Lr34和 Lr46抗叶锈基因的材料。利用澳大利亚小麦种质为改良亲本,与黄淮麦区表现突出的小麦品种配制三交或四交组合,按常规系谱方法对优良性状进行定向选择,培育出了综合农艺性状表现好、品质表现突出的小麦新品系。