Rediscovery of a diploid cytotype of Dasypyrum breviaristatum in Morocco

The chromosome numbers of a total of 273 Dasypyrumbreviaristatum plants sampled from 20 Moroccan and one Greek naturalpopulations were examined. 270 plants from all of the populations weretetraploids. Among them, 223 were eu-tetraploids with 28 chromosomes and 47 wereaneuploids with 25, 27 and 29 chromosomes. Besides, three plants were found tobe diploids among 16 plants from a population in the Moyen Atlas Mountains ofMorocco. This is the second report of a diploid cytotype ofD. breviaristatum since the firstcommunication in 1957. Morphologically, the diploids were similar to but smallerthan the tetraploids in plant height, spike length, spikelet number and leafepidermis cell size. However, it was not possible to distinguish between the twocytotypes based on those morphological characteristics alone. A distinctdifference was found between the two cytotypes only in the number of trichomeson the leaf surfaces. The karyotype of the diploid cytotype consisted of a pairof SAT-chromosomes, five pairs of metacentric and a pair of submetacentricchromosomes. Based on the similarity in karyotype and in plant morphologybetween the two cytotypes of D.breviaristatum, we suggest that the diploid cytotype is themost probable candidate for the ancestral form of the tetraploid cytotype.     

Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality

Dasypyrum villosum (L.) Candargy (DV) is adiploid (2n = 14, VV genomes), allogamous grass of theMediterranean region. It may be hybridized with wheatand is thus a gene resource for wheat improvement. Westudied grain protein concentration andSDS-sedimentation (SED) as indicators of end-usequality. The latter is a good predictor of glutenstrength. A-PAGE and SDS-PAGE were used to identifymonomeric and polymeric seed storage proteins,respectively, to relate proteins of DV to those foundin Chinese Spring (CS), Triticum aestivum L.,wheat. Two full-sib lines of DV had high grain protein(19.3 and 20.3%), but one had very low mean SED (69mm) and one had very high (118 mm) based on onegreenhouse and one field test. CS had very low grainprotein (12.0%) and weak gluten (33 mm). Single-DVchromosome addition and substitution lines and twoDV-wheat recombinant lines all had higher grainprotein than CS (range 13.9 to 16.7%). SED valuesshowed a different pattern. CS=4V and CS=6V hadlow SED, 63 and 44 mm, similar to CS, whereas CS=1Vand full sib DV 200 had very strong gluten, 118 mm, asdid substitution lines CS1V (1A) and CS1V (1B), 125and 131 mm, respectively. One hybrid-derived line withDV-wheat 1V recombinant chromosome had SED of 99 mmand one line with a 6V added chromosome had SED of 64mm. The large positive effects of quality in the wheathaving DV chromosome 1V are believed to be due to DValleles at the Glu-V1 and Gli-V1/Glu-V3loci. DV chromosomes 4V and 6V did not contribute toimproved quality probably due to Gli-V2 and Gli-V3 which, as the orthologous loci in wheat, donot enhance wheat quality. Based on the positiveeffects of alleles on DV chromosome 1V in a breadwheat background, we conclude that D. villosumis a source of allelic diversity that can beconsidered for improving end-use quality in breadwheat.     

多年生簇毛麦a-醇溶蛋白基因的分离与序列分析

以多年生簇毛麦(Dasypyrum breviaristatum)基因组DNA为模板, 用小麦种子醇溶蛋白保守引物进行PCR扩增。扩增产物经克隆测序, 获得999~1 018 bp的7个序列(GenBank登录号为EU186102–EU186108), 分别编码283~290个氨基酸。序列比对结果表明, 它们具有a-醇溶蛋白基因的保守特征, 是a-醇溶蛋白基因家系成员。该7个序列与乳糜泻(celiac disease)病人毒性抗原相关序列的比对结果表明, 有5个序列在C末端具有Glia-a-2型抗原序列的特征。根据a-醇溶蛋白基因的编码氨基酸建立系统树, 发现来自多年生簇毛麦的a-醇溶蛋白基因序列单独聚为一类, 不能与普通小麦A、B或D染色体组的相关序列聚在一起。在序列分析的基础上设计了簇毛麦基因组a-醇溶蛋白基因的特异引物AS-V-gli-F和AS-V-gli-R, 通过特异PCR扩增, AS-V-gli-F和AS-V-gli-R仅能对多年生簇毛麦、小麦-多年生簇毛麦部分双二倍体进行特异性扩增, 而不能对小麦和其他小麦族物种进行扩增。因此, AS-V-gli-F和AS-V-gli-R可作为检测多年生簇毛麦a-醇溶蛋白基因向栽培小麦转移的分子标记。     

采用ISSR方法研究簇毛麦属与偃麦草属染色体组的遗传关系

[目的]确证簇毛麦属（Dasypyrum）与偃麦草属（Thinopyrum）物种不同基因组的系统发生关系。[方法]采用ISSR引物对12个小麦族物种扩增后进行了聚类分析。[结果]多年生簇毛麦（D.breviaristatum）、中间偃麦草（Th.intermedium）和二倍体长穗偃麦草（Lo-phopyrum elongatum）亲缘关系最近而聚在一起,而二倍体簇毛麦（D.villosum）与拟鹅观草（Pseduoroengeria spicata）聚在一起。[结论]多年生簇毛麦Vb基因组与偃麦草E基因组关系较近;二倍体簇毛麦和多年生簇毛麦未聚在一起,支持＂多年生簇毛麦不是二倍体簇毛麦加倍产生＂的观点。关键词簇毛麦属;偃麦草属;ISSR;聚类分析;遗传进化 更多还原     

簇毛麦新型低分子量谷蛋白亚基的分子克隆与序列分析

为发掘低分子量谷蛋白亚基（LMWGS）新的变异类型,通过设计引物和特异PCR扩增,从簇毛麦TA2127基因组DNA中得到14个LMWGS基因,GenBank登录号分别为HQ615147～HQ615160。序列分析表明,所有基因具有完整编码区,长度为831 bp和846 bp,可编码277和282个氨基酸残基,推导的氨基酸序列分析显示,14个基因编码的蛋白序列都缺少一段N端保守区,且N端序列是一种新的类型;在14个LMWGS基因中检测到19个SNPs和1个16 bp 的缺失。系统进化分析表明,簇毛麦LMWGS与ω醇溶蛋白和HMWGS亲缘关系相对较远,与普通小麦 Glu3的低分子谷蛋白基因进化关系相对较近。     

基于簇毛麦No.1026转录组的SSR序列分析及其PCR标记开发

【目的】 探究从前苏联引进的簇毛麦No.1026（Dv#4）的EST-SSR序列特征及其在染色体的分布,分析它们在不同簇毛麦间及与小麦间的多态性,为其进一步的研究与利用提供依据。【方法】通过Illumina HiSeq测序获得No.1026植株的转录组序列,利用MISA软件分析转录组SSR序列及特征,采用Primer 3设计SSR引物,随机合成238对引物,对小麦中国春与簇毛麦Dv#4和引自英国剑桥的簇毛麦Dv#2的基因组DNA进行扩增,在琼脂糖凝胶上分离评价扩增产物的多态性,并利用一套小麦-簇毛麦异染色体系进行扩增分析。【结果】 检测了No.1026总长62.76 Mb的转录组序列,发现10 497个SSR位点,它们分布于8 735条Unigene上。在1—6个核苷酸的重复单元中,单、双、三碱基的重复占95.85%,其中三核苷酸串联重复数量最多,占SSR总数的50.33%,而CCG/CGG基序的重复占三核苷酸串联重复的41.66%;单核苷酸重复是第二大类型,出现频率为27.13%,其中A/T重复占单核苷酸重复的74.58%。二核苷酸重复类型的数量位列第三,占SSR总数的18.39%。在238对EST-SSR引物中,88对在中国春与簇毛麦（包括Dv#2和Dv#4）之间的扩增产物显示多态性;8对只在簇毛麦和单一异染色体系扩增;4对可在簇毛麦和多个异染色体系中特异扩增;但多数可在簇毛麦中清晰扩增的引物不能在任何异染色体系中扩增,推测可能与簇毛麦基因组及染色体导入小麦过程中的变异有关;47对（19.74%）在2份不同来源的簇毛麦Dv#2和Dv#4之间的扩增产物呈现多态性。利用1对EST-SSR引物和1个EST-PCR标记检测48个簇毛麦植株,证明多态性的SSR引物可有效用于检测簇毛麦的异质性。【结果】 簇毛麦No.1026（Dv#4）的转录组中存在丰富的SSR序列,其中CCG/CGG、A/T和AG/CT等三核苷酸、单核苷酸和二核苷酸是其最主要的串联重复基序。部分EST-SSR的侧翼保守序列与单一或若干外源染色体特异相关联,据此开发特异的分子标记,可用于跟踪检测小麦背景中特异的簇毛麦染色体或染色体片段。Dv#4与Dv#2间的部分EST-SSR具有多态性,提示2份不同来源簇毛麦的表达序列间存在一定程度的遗传差异,因此,簇毛麦Dv#4值得进一步的发掘研究。     

基于小麦SNP芯片对簇毛麦6V#2和6V#4染色体及其与小麦6A、6D染色体的多态性分析

[目的]利用大数据比较2条不同的簇毛麦6V(#2和#4)染色体及其与小麦6A、6D染色体间DNA水平上的差异,为小麦-簇毛麦靶向易位的精准设计育种提供依据.[方法]以6V#4(6D)异代换系RW15为父本和6V#2(6A)异代换系南87-88为母本进行杂交,获得F2分离群体,利用6V#4S/6V#2S/6AS/6DS/...     

簇毛麦Dvblt101基因及其启动子的克隆与序列分析

BLT101家族是一类高度保守的小分子质膜蛋白,在植物抗寒、耐旱、耐盐、脱落酸(ABA)应答等过程中起重要作用。利用PCR技术从抗寒、耐旱的簇毛麦中克隆了Dvblt101基因及其启动子。结果表明,Dvblt101cDNA全长422 bp,具有完整的开放阅读框(ORF),编码一条54个氨基酸残基的多肽,并具有2个保守的跨膜结构域;Dvblt101基因长为754 bp,含有2个外显子和1个内含子,启动子区域具有2个干旱/冷应答核心元件(DRE/CRT)、2个MYC结合位点和3个ABA应答元件(ABRE),该基因对低温、干旱等逆境及ABA可能存在广泛的应答。本研究为进一步揭示BLT101家族在簇毛麦抗逆过程中的作用机理奠定了基础。     

簇毛麦5V染色体特异性ISSR标记的建立及其对亲缘物种的检测

为建立簇毛麦(Dasypyrum)特定染色体上的特异性ISSR标记,以二倍体簇毛麦(Dasypyrum villosum)和小麦中国春(Triticum aestivum)等为材料,对96条ISSR引物进行PCR筛选,发现引物UBC848可在二倍体簇毛麦中扩出一条长388bp的特异性片段,命名为pDv848/388(GenBank登录号:EF411201),而中国春等小麦均未扩出该片段。利用UBC848对小麦近缘种偏凸山羊草(Aegilops ventricosa)、荆州黑麦(Secale cereale cv.Jingzhou rye)和节节麦(Ae.tauschii)等进行扩增,发现它们均未扩出pDv848/388。进而用一套中国春-二倍体簇毛麦附加系CSDA1V～CSDA7V对pDv848/388进行染色体定位,将目标片段确定在二倍体簇毛麦5V染色体上。进一步用UBC848对多年生簇毛麦(D.breviaristatum)、小麦-簇毛麦双二倍体、部分双二倍体及其衍生后代进行扩增,发现它们均能扩出pDv848/388,表明pDv848/388可作为簇毛麦5V染色体上的一个特异性ISSR标记,用于快速跟踪检测簇毛麦遗传物质向栽培小麦中的导入。