SDS-PAGE与分子标记相结合分析宁夏小麦HMW-GS组成与变化特点

为了建立准确有效小麦HMW-GS的检测方法,提高优质小麦品种鉴定和筛选效率,以已知HMW-GS组成的16份小麦品种为对照,优化完善SDS-PAGE结合分子标记检测小麦HMW-GS的方法,并对103份宁夏小麦品种进行了验证和分析。结果表明,8.5%的分离胶可以有效区分除了 Glu-B1位点的7^*、7^OE与8^*亚基之外的其他亚基,分辨效果优良;SDS-PAGE结合 Bx7^OE、 Bx7^*/Bx7和 By8基因的分子标记可以准确鉴定小麦HMW-GS。在103份宁夏小麦品种中,发现15种HMW-GS和29种组合类型;首次在该地区小麦品种的 Glu-B1位点检测出了携带7^OE、7^*和8^*亚基的品种;1/17+18/5+10为当地小麦HMW-GS的优势亚基组合类型,占20%。自1970年至2010年,HMW-GS的优质亚基（1、17+18和5+10）的出现频率呈明显增长趋势;宁夏小麦的HMW-GS种类和优质亚基出现频率随品种更换呈增加趋势。综上所述,分离胶浓度为8.5% 的SDS-PAGE和 Bx7^OE、 Bx7^*/Bx7和 By8分子标记的方法可准确有效地检测小麦HMW-GS组成,此方法可用于优质小麦品种的鉴定和筛选。     

花药培养与麦谷蛋白亚基分子标记结合选育小麦新品种的研究

为快速获得携带麦谷蛋白优质亚基基因的小麦新品种,提高小麦的品质育种技术水平,利用引进的矮败材料与和尚头、甘春20号、临麦34号等10个不同品种(系)杂交,并对杂交后代进行了花药培养,获得了115份花培株系;利用PCR对花培后代株系及杂交亲本进行了优质贮藏蛋白亚基分子标记检测,3个HMW-GS为 Bx7、 Bx14、 Dx5,3个LMW-GS为 Glu-A3ac、 Glu-A3d、 Glu-B3b。结果表明,在115份花培材料中, Bx7的出现频率最高,为94.78%,其余依次为 Glu-A3ac、 Dx5、 Bx14、 Glu-A3d和 Glu-B3b;获得了44份聚合4个亚基以上的材料;结合农艺性状鉴定,筛选出了3份综合性状优异的小麦新品系AB158、AB167和AB332。本研究将花培育种技术、分子标记辅助选择技术及矮败小麦育种技术进行了有机结合,其结果可为提升小麦品质育种技术水平提供参考。     

陕西小麦Glu-A3和Glu-B3位点等位变异的检测和分析

低分子量谷蛋白亚基（LMW-GS）与小麦品质密切相关。为了给陕西小麦的品质改良提供参考依据,采用STS分子标记,检测了175份陕西小麦品种（系）Glu-A3和Glu-B3位点的等位变异组成。结果表明,陕西小麦Glu-A3位点存在4种等位变异,即Glu-A3a、Glu-A3b、Glu-A3c和Glu-A3d,分别占12.6%、1.7%、58.3%和27.4%;Glu-B3位点存在8种等位变异,即Glu-B3a、Glu-B3b、Glu-B3d、Glu-B3e、Glu-B3f、Glu-B3g、Glu-B3i和Glu-B3j,分别占4.6%、2.9%、45.7%、0.6%、2.9%、8.5%、4.0%和30.8%。在陕西不同地区小麦之间,两个位点等位变异的种类、组合及其分布比例存在差异,这可能与地区间不同的自然地理环境、饮食习惯、育种目标及亲本选择有关。     

小麦HMW GS检测方法的优化及应用

为了准确快速鉴定小麦品种的HMW-GS组成,以18个已知HMW-GS组成的品种为对照,将4种不同浓度分离胶的SDS-PAGE与分子标记(Bx7、Bx7OE和By 8)相结合,构建一套适于检测HMW-GS组成的方法,并用该方法检测214份陕西小麦品种(系)的HMW-GS组成。4种分离胶浓度中,除了亚基7、7*与7OE和8与8*外,9%的分离胶可以很清楚地分离Glu1-位点的其他常见亚基,结合分子标记(Bx7、Bx7OE和By 8)检测对照品种,结果与已知亚基(基因)一致。用分离胶浓度为9%的SDS-PAGE结合分子标记(Bx7、Bx7OE和By 8)方法对陕西品种(系)检测结果表明,Glu-A1、Glu-B1和Glu-D1分别有3、8和3个亚基种类,共28种亚基组合类型。以1、Null、7+9、7+8、7+8*、14+15、2+12和5+10为主要亚基类型,频率分别为55.6%、41.6%、43.9%、26.2%、10.3%、15.4%、79.0%和12.6%;同时在Glu-B1位点发现7+8*和6+8*亚基。该方法可以快速准确地评价小麦HMW-GS组成,有效区分Glu-B1位点的亚基7与7OE,8与8*,鉴定结果稳定可靠。     

新疆小麦品种Wx基因组成及其淀粉糊化特性研究

为了给新疆小麦淀粉品质改良和优质面条小麦新品种选育提供理论依据,利用WxA1、WxB1和WxD1位点的3个STS标记（MAG264、BDFLBRD和MAG269）对250份新疆小麦品种（包括157份冬小麦品种和93份春小麦品种）Wx基因的组成进行了鉴定,同时测试了185份冬、春小麦品种的淀粉糊化特性。结果表明,新疆小麦品种（系）Wx基因组成比较单一,以野生型（WxA1a/WxB1a/WxD1a）为主,占参试品种总数的84.0%,除了在WxB1位点发现亚基缺失的品种（缺失比例为13.6%）外,没有发现其他亚基缺失类型。WxB1蛋白亚基缺失比例在冬、春小麦间差异较大,春小麦品种WxB1亚基缺失比例（21.5%）要高于冬小麦品种（8.9%）;农家品种、引进品种和育成品种中WxB1亚基缺失比例也各不相同,依次为0%、5.6%和22.6%。分析表明,新疆小麦品种淀粉糊化特性变异范围相当广泛,冬、春小麦品种在峰值黏度、稀澥值和反弹值等主要淀粉指标上均达到极显著差异,春小麦品种的淀粉特性优于冬小麦品种,并且WxB1亚基缺失体淀粉糊化特性的平均表现总体优于非缺失体。本研究所用3个分子标记对小麦基因组DNA扩增的稳定性、重复性较好,检测结果准确、可靠,可以用于小麦品种Wx基因组成的鉴定和糯质专用小麦的分子标记辅助选育。     

中国小麦品种高分子量谷蛋白亚基和低分子量谷蛋白亚基组成分析

为给我国优质小麦品种的选育和改良提供科学依据,应用SDS-PAGE方法对我国235份推广品种和高代品系的HMW-GS和LMW-GS组成与分布进行了分析。结果表明,HMW-GS和LMW-GS分别具有39和40种带型组合;5个位点共发现34个等位基因。Glu-A1、Glu-B1和Glu-D1分别有3、8和6个变异位点;亚基1、Null、7＋9、5＋10和2＋12是主要的HMW-GS类型,频率分别为46．2％、46．2％、44．5％、47．0％和48．3％。Glu-A3和Glu-B3位点（本文不涉及Glu-D3）分别具有6和11个变异位点。Glu-A3c、Glu-A3d、Glu-B3e和Glu-B3j是主要的LMW-GS类型,频率分别为55．1％、21．6％、28％和28．8％。本研究还发现,在品种Z75和绵阳96-319的Glu-D1位点上,亚基组合形式为5＋12;在品种周92034的Glu-D1位点,亚基组合形式为2＋10。虽然这两种组合形式在本研究中出现的频率很低,但这两种亚基组合在以前的研究中很少出现。     

新疆小麦品种Glu-A3和Glu-B3位点等位变异的分布

为给新疆小麦品质育种提供理论依据,利用Glu-A3、Glu-B3位点上的17个STS标记检测了185份新疆冬、春小麦品种Glu-A3和Glu-B3位点的等位变异。结果表明,新疆小麦品种以Glu-A3c、Glu-B3a和Glu-B3j亚基为主,其分布频率分别为64.86%、22.70%和17.84%。新疆冬、春小麦品种在Glu-A3位点上均以Glu-A3c亚基为主,分布频率分别为63.30%和67.11%;在Glu-B3位点上,新疆冬、春小麦品种分别以Glu-B3j和Glu-B3a为主,分布频率分别为22.02%和26.32%。新疆冬、春小麦农家品种亚基类型较少,冬小麦农家品种仅有5种类型（以Glu-A3c和Glu-B3i为主）,春小麦农家品种有10种类型（以Glu-A3c和Glu-B3d为主）。引进品种和自育品种亚基类型丰富,冬小麦引进品种以Glu-A3c和Glu-B3i为主,分布频率为12.84%和6.42%;春小麦引进品种以Glu-A3c和Glu-B3j为主,分布频率为17.11%和6.58%。冬小麦自育品种以Glu-A3c和Glu-B3j亚基类型为主,分布频率为45.87%和18.35%;春小麦自育品种以Glu-A3c和Glu-B3a亚基类型为主,分布频率为36.84%和18.42%。     

97份安徽省种植小麦品种HMW-GS组成及品质分析

为探究安徽小麦品种高分子量麦谷蛋白亚基（HMW-GS）组成及品质性状,对97份安徽省主要种植的小麦品种进行了HMW-GS组成及品质性状分析。结果表明,供试材料中共检测出10种HMW-GS亚基类型和19种亚基组合,在 Glu-A1位点检测出3种亚基类型,以1亚基（57.73%）和Null亚基 （40.21%）为主;在 Glu-B1位点共检测出3种亚基类型,以7+8亚基（52.58%）为主;在 Glu-D1位点共鉴定出4种亚基类型,优质亚基5+10占比最大,为45.36%;供试品种亚基组合品质得分在4~10分之间,10分的亚基组合1/7+8/5+10和1/17+18/5+10共19份材料（19.59%）,8分以下的共44份材料（45.36%）。对供试材料HMW-GS与品质性状进行相关分析发现, Glu-1的三个位点与硬度指数、溶剂保持力和吸水率相关达显著或极显著水平,对溶剂保持力影响表现为 Glu-A1> Glu-B1> Glu-D1;不同亚基组合对品质性状的影响存在差异,亚基组合为1/17+18/5+10的品种具有最高的硬度指数、4种溶剂保持力、吸水率、稳定时间和粉质质量指数,具有最低的L^*和W,该类品种有烟农19、泰农19、山农17和糯小麦1012;亚基组合为Null/7+9/4+12的品种具有最低的硬度指数、4种溶剂保持力、吸水率、稳定时间和粉质质量指数,具有最高的弱化度、L^*和W,该类品种有荃麦725、皖麦52和未来0818。     

50份哈萨克斯坦小麦高分子量谷蛋白亚基解析

为发掘优良的小麦种质资源,利用SDS-PAGE技术分析了50份哈萨克斯坦小麦种质高分子量谷蛋白亚基组成及其品质得分。结果表明,50份供试材料中共检测到8种亚基类型,其中在 Glu-A1位点有2种亚基类型（Null,1）, Glu-B1位点有2种亚基类型（7＋8,7＋9）,在 Glu-D1位点有4种亚基类型（5＋10, 2＋10,2＋12,4＋12）。利用PCR分子标记对所得到的高分子量谷蛋白亚基进行验证,结果一致。在50份哈萨克斯坦小麦中,优质亚基1、7＋8、5＋10的出现频率较高,分别为34%、22%、56%。亚基组合Null/7＋9/5＋10在所有亚基组合类型中出现频率最高,为28%,品质得分为7分,而组合1/7＋8/5＋10出现频率仅为8%,品质得分为10分;供试材料品质得分大多为7、8分,平均为7.10分。出现频率较高的优质亚基可以作为改善现有品种亚基组成状况的亲本材料。     

57份陕西新育成小麦品种（系）HMW-GS组成及品质分析

为探究陕西关中地区小麦HMW-GS亚基与品质性状间的关系,采用SDS-PAGE法对57份陕西关中地区小麦品种（系）HMW-GS亚基组成及相关品质性状进行了分析。结果表明,供试品种（系）中共检测出7种HMW-GS亚基类型和8种HMW-GS亚基组合;Glu-A1位点上有3种亚基类型,分别为1、2^*和Null,以1亚基为主（78.95%）;Glu-B1位点上检测到7+8（61.40%）与7+9（38.60%）两个类型;Glu-D1位点上检测到5+10（70.18%）和2+12（29.82%）两个类型。3个HMW-GS基因位点编码亚基共组成8种亚基组合,品质得分6～10分,其中1/7+8/5+10组合品质得分10分,出现频率最高。就HMW-GS不同位点对品质性状效应进行分析发现,Glu-D1位点对b^*值、形成时间、稳定时间、弱化度和粉质质量指数的影响达到极显著水平（P＜0.01）;对面团流变学特性的影响,Glu-D1>Glu-B1。不同类型亚基对小麦品质的效应存在差异,7+8亚基对蛋白质含量、湿面筋含量和容重具有正效应,7+9和5+10亚基对形成时间和稳定时间的影响显著高于其他亚基（P＜0.05）;携带1/7+8/5+10亚基组合小麦的蛋白质、湿面筋含量和容重最高;携带1/7+9/5+10亚基组合具有较高面粉L^*值和面团流变学特性指标值。     

Definition of the low molecular weight glutenin subunit gene family members in a set of standard bread wheat (Triticum aestivum L.) varieties

Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the viscoelastic properties of wheat dough. The LMW-GSs are encoded by multi-gene families at the Glu-A3, Glu-B3 and Glu-D3 loci, with more than 15 genes present in most bread wheat varieties. However, the genic profile associated with different alleles has not been clearly defined. Here, the LMW-GSs in a set of standard varieties were analyzed using molecular markers. In most cases, each Glu-3 allele was represented by a specific haplotype; however, some alleles were undistinguishable. The Glu-A3e and Glu-A3g alleles showed an identical marker haplotype, as did the alleles Glu-B3c and Glu-B3d, and Glu-B3f and Glu-B3ab. In contrast, two haplotypes among varieties designated Glu-D3c were differentiated. The marker profiles present at the Glu-D3 locus exhibited less variation compared to the genes at the Glu-A3 and Glu-B3 loci. Results show the potential of the LMW-GS gene marker system in the characterization of the LMW-GS alleles present in specific bread wheat varieties, and its reconciliation with protein-based nomenclature. This approach will advance the understanding of the contribution of each of the LMW-GS gene alleles in the control of the end-use quality.     

普通小麦Glu-3位点基因单元型的分离和序列分析

为了从分子水平上探讨优质小麦资源中LMW-GS等位基因与小麦品质的关系,以及在改善小麦品质方面的潜在价值,利用小麦Glu-A3和Glu-B3基因的特异引物从强筋型、中筋型和弱筋型小麦共计10份材料中分离出LMW-GS基因后进行序列分析。结果表明,共发现14个新的核苷酸变异类型和4个肽链变异类型。其中,14个新的核苷酸变异类型中,4个为Glu-A3基因变异类型,1个为Glu-B3基因变异类型,9个为Glu-D3基因变异类型。值得注意的是,有2个半胱氨酸数目特殊的亚基类型被发现,一个是来自师栾02-1含有9个半胱氨酸残基的GluA3-18基因,另一个是来自偃展4110含有7个半胱氨酸残基的GluD3-13基因。     

Effects of allelic variation of HMW-GS and LMW-GS on mixograph properties and Chinese noodle and steamed bread qualities in a set of Aroona near-isogenic wheat lines

High-molecular-weight glutenin (HMW-GS) and low-molecular-weight glutenin (LMW-GS) subunits play an important role in determining wheat quality. To clarify the contribution of each subunit/allele to processing quality, 25 near-isogenic lines with different HMW-GS and LMW-GS compositions grown at two locations during the 2010 cropping season were used to investigate the effects of allelic variation on milling parameters, mixograph properties, raw white Chinese noodle (RWCN) and northern style Chinese steamed bread (NSCSB) qualities. The results showed that Glu-B1 and Glu-B3 made a large contribution to determining mixograph properties and processing quality, respectively. Subunit pairs 17 + 18 and 5 + 10, and alleles Glu-A3b, Glu-A3d, Glu-B3g and Glu-D3f made significant contributions to mixograph properties and no significant difference was detected on most parameters of RWCN and NSCSB for the allelic variation of HMW-GS and LMW-GS. The allelic interactions among glutenin loci had significant effects on wheat quality. The line with 1, 17 + 18, 2 + 12, Glu-A3c, Glu-B3b, Glu-D3c associated with superior mixograph properties, the line with 1, 7 + 9, 2 + 12, Glu-A3c, Glu-B3d, Glu-D3c had superior viscoelasticity of RWCN, and the line with 1, 7 + 9, 2 + 12, Glu-A3e, Glu-B3b, Glu-D3c had the highest total score of NSCSB. These results provide useful information for genetic improvement of the qualities of traditional Chinese wheat products.     

Influence of allelic prolamin variation and localities on durum wheat quality

Thirty-seven varieties of a Mediterranean durum wheat collection grown in Tunisia and Spain were analysed for their allelic composition in prolamins, as well as their protein concentration, sodium dodecyl sulphate sedimentation (SDSS) test and mixograph parameters. Genotype was a greater source of variation in all measurements than locality. Uncommon high and low molecular glutenin subunits (HMW-GS and LMW-GS) were found (V and 2•• subunits at Glu-A1, 13 + 16 at Glu-B1, 5* subunit and ax allele at Glu-A3). The rare combinations 2 + 4+14 + 18 and 8 + 9+13 + 16+18 subunits at the Glu-B3 locus were found. Glu-A3ax had a positive influence on SDSS and mixograph parameters. Of all the prolamins, those that have the B-LMW-GS composition aaa (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d gave the best semolina quality. By contrast, semolina quality is poor when this same composition is associated with the Glu-A1c and Glu-B1e and even poorer when associated with the Glu-A1c and Glu-B1f. In addition, the cultivars with B-LMW-GS allelic composition aab (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d, gave high quality, whereas when associated with the Glu-A1c and Glu-B1e or with Glu-A1o and Glu-B1f, the quality was very poor.     

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

甘肃冬小麦品种(系)面粉品质性状相关基因分析

为了解甘肃省近20年育成的106份冬小麦品种(系)中加工品质性状相关基因分布情况,用22个分子标记对供试材料的HMW-GS、LMW-GS、面粉色泽及籽粒硬度等品质性状相关基因进行了分析。结果发现,供试品种(系)的HMW-GS相关基因中,在Glu-A1位点检测到34份品种(系)含有AxNull,频率为32.08%;在Glu-B1位点检测到Bx7+By8和Bx14+By15共2种基因组合,分别占17.92%和25.47%;在Glu-D1位点检测到11份品种(系)含有Dx5+Dy10,占10.38%。对LMW-GS鉴定结果显示,29份品种(系)含Glu-A3d基因,分布频率为27.36%。HMW-GS和LMW-GS亚基组合中,含有4个、3个和2个位点优质亚基基因组合的品种(系)分别占0.94%、8.49%和3.77%。对面粉色泽相关基因Ppo-A1、Ppo-D1、Psy-A1、Lox-B1和TaPod-A1位点的检测发现,优异等位变异占比分别为39.62%、50.94%、31.13%、30.19%和38.68%。对籽粒硬度相关基因检测发现,在Pina、Pinb和Pinb-2等位变异位点的检测...     

Effect of allelic variation at the Glu-3/Gli-1 loci on breadmaking quality parameters in hexaploid wheat (Triticum aestivum L.)

Low molecular weight glutenin subunits (LMW-GS) encoded by the Glu-3 loci are known to contribute to wheat breadmaking quality. However, the specific effect of individual Glu-3 alleles is not well understood due to their complex protein banding patterns in SDS-PAGE and tight linkage with gliadins at the Gli-1 locus. Using DNA markers and a backcross program, we developed a set of nine near isogenic lines (NILs) including different Glu-A3/Gli-A1 or Glu-B3/Gli-B1 alleles in the genetic background of the Argentine variety ProINTA Imperial. The nine NILs and the control were evaluated in three different field trials in Argentina. Significant genotype-by-environment interactions were detected for most quality parameters indicating that the effects of the Glu-3/Gli-1 alleles are modulated by environmental differences. None of the NILs showed differences in total flour protein content, but relative changes in the abundance of particular classes of proteins cannot be ruled out. On average, the Glu-A3f, Glu-B3b, Glu-B3g and Glu-B3i_Man alleles were associated with the highest values in gluten strength-related parameters, while Glu-A3e, Glu-B3a and Glu-B3i_Chu were consistently associated with weak gluten and low quality values. The value of different Glu-3/Gli-1 allele combinations to improve breadmaking quality is discussed.     

Comparative analyses of LMW glutenin alleles in bread wheat using allele-specific PCR and SDS-PAGE

One hundred and eighty-two bread wheat cultivars developed in India were characterized for low molecular weight (LMW) glutenins using SDS-PAGE and allele-specific polymerase chain reaction (PCR) to assess allelic diversity encoded by Glu-3 loci, as well as their utility for correctly identifying different alleles. SDS-PAGE indicated Glu-A3c is present in 64.6% of the cultivars, Glu-A3b in 13.8%, Glu-A3d in 12.7% and Glu-A3e/f in 8.8%. Seven types of alleles were present at the Glu-B3 locus: Glu-B3b (29.3%), Glu-B3g (27.0%), Glu-B3h (13.8%), Glu-B3i (16.1%), Glu-B3j (12.1%), Glu-B3c (0.6%) and Glu-B3d (1.1%). SDS-PAGE found three types of Glu-D3 alleles: Glu-D3a (30.2%), Glu-D3b (67.1%) and Glu-D3c (2.7%). However, PCR found two different alleles in cultivars classified as carrying Glu-D3a and three alleles in those identified as carrying Glu-D3b cultivars, indicating a more complex nature of the Glu-D3 locus. In conclusion, the data found greater consistency between the SDS-PAGE and PCR amplification patterns of alleles such as Glu-A3c, Glu-A3d, Glu-B3g, Glu-B3h and Glu-B3i, and less consistency between those same patterns in the Glu-A3b, Glu-A3e/f and Glu-B3b alleles. More studies are needed in order to achieve unambiguous identification of the Glu-3 alleles and thereby allow their greater utility in germplasm evaluation and breeding.