非洲黑麦染色体特异性标记的建立与应用

以非洲黑麦、小麦-非洲黑麦双二倍体、安岳排灯麦等为材料筛选100条ISSR引物。其中, 引物UBC815可在非洲黑麦中扩增出1条长561 bp的特异性片段(命名为pSaUBC815₅₆₁), 而小麦对照均未扩出该片段。引物UBC815同样能在黑麦属的瓦维洛夫黑麦(Secale vavilovii Grossh.)、森林黑麦(Secale sylvestre Host.)等5个种扩增出pSaUBC815₅₆₁。根据pSaUBC815₅₆₁设计特异PCR引物U815-F、U815-R, 对小麦族多物种进行扩增, 表明pSaUBC815₅₆₁为黑麦属特有。进而利用一套中国春-Imperial黑麦二体附加系及小麦-黑麦异源材料进行扩增, 结果显示, pSaUBC815₅₆₁分布在黑麦整套染色体上, 并且所有后代材料都能扩增出pSaUBC815₅₆₁, 表明pSaUBC815₅₆₁可作为特异性标记用来检测小麦背景中的黑麦染色质。     

小麦族中含St染色体组物种的特异分子标记的建立

以拟鹅观草(Pseudoroegneria spicata)、偏凸山羊草(Aegilops ventricosa)、二倍体簇毛麦(Dasypyrum villosum)、荆州黑麦(Secale cereale cv. Jingzhou rye)、普通小麦中国春(Chinese Spring)等15个物种为材料, 用200条10碱基随机引物进行RAPD分析, 筛选到拟鹅观草基因组中1个542 bp的特异DNA片段(GenBank登录号为DQ992032), 命名为OPH11542。根据OPH11542设计特异引物, 对小麦族物种进行PCR扩增, 发现拟鹅观草可以扩增出OPH11542以及分子量分别为742 bp (GenBank登录号为DQ992033, 记为OPH11742)和743 bp (GenBank登录号为EF014218, 记为OPH11743)的DNA片段, 而其他材料均未扩增出这3个片段。经序列比对结合多个软件的分析结果认为该3个片段为同一类新重复序列。利用特异引物对15份含St染色体的物种进行扩增, 发现含StY染色体组的物种均能扩增出OPH11742或OPH11743, 而含StH染色体组的物种均能扩增出OPH11542。这表明St染色体组在与其它染色体组组合形成多倍体的过程中往往会出现不同程度的重组或修饰。OPH11542、OPH11742和OPH11743可以作为检测St染色体的分子标记。     

拟鹅观草St基因组ISSR特异标记的建立

以拟鹅观草(Pseudoroegneria spicata)、中间偃麦草(Thinopyrum intermedium)、长穗偃麦草(Th.elonga-tum)、二倍体簇毛麦(Dasypyrum villosum)、澳冰草(Australopyrum retrofractum)等10份小麦族物种为材料,对100条ISSR引物进行分析,结果显示引物811可以在拟鹅观草(GenBank登录号为EU368859)和中间偃麦草中扩增出一条长441 bp的特异DNA片段命名为St441,而其它供试物种均未扩出.经序列比对、软件分析结合原位杂交结果认为St441为一类新的低拷贝重复序列.利用ISSR引物811对10份不同居群的中间偃麦草、20份披碱草属物种、4份小麦-偃麦草部分双二倍体、6份小麦-茸毛偃麦草后代和12份小麦对照进行扩增,结果发现除对照小麦外均能扩增出St441;进而对小麦-中间偃麦草两套附加系进行扩增,将St441初步定位于包括第四同源群在内的8条St染色体上.同时,发现只含有整条St染色体和St染色体片段的材料能扩增出St441,而仅有Js染色体的材料未扩增出St441.因此,该标记St441可以作为检测不同背景下St染色质的分子标记.     

基于专化的反转录转座子序列开发鉴定簇毛麦染色质的PCR分子标记

通过克隆簇毛麦的专化序列,开发基于其序列的可用于鉴定簇毛麦染色质的PCR分子标记.用根据抗病基因保守域设计的XLS和A3简并引物进行PCR,检测到1条簇毛麦的特异PCR产物,酶切分析与测序结果表明:这条特异带由4类不同的片段组成,且都与反转录转座子具有同源性.以其中1个片段pHv29为探针进行Southern杂交,发现只有含有簇毛麦染色质的材料产生强的杂交信号,表明pHv29是一个对簇毛麦专化的反转录转座子序列.根据pHv29序列重新设计1对特异引物pHv29-F和pHv29-R,用一系列含有簇毛麦染色质的易位系或添加系的基因组DNA为模板进行PCR扩增,电泳结果表明pHv29433可以作为鉴定簇毛麦的染色质的分子标记.     

7个大穗型小麦-黑麦代换系间杂交后代的形态学与SSR分析

对从小麦-黑麦代换系5R/5A与1R/1D杂交后代中选育的大穗型品系1-5、1-6、1-7、1-8、1-9、1-10、1-11进行形态学与SSR分析。结果表明,7个品系田间表现遗传性稳定,且具有黑麦大穗、抗病等优良性状;利用黑麦特异引物PSC119.1确定7个品系均导入了黑麦染色体片段,引物SCM138扩增结果表明,在1-6、1-7、1-8、1-9、1-10中导入了黑麦染色体5RS片段,引物SCM120、TSM604可以在7个品系中稳定扩增出黑麦5R长臂和1R短臂特异片段。以上结果可为小麦遗传育种与品质改良提供基础材料。     

黑麦1R染色体特异性PCR引物的分子证据

根据黑麦（ＳｅｃａｌｅｃｅｒａｌｅＬ．）与小麦（ＴｒｉｔｉｃｕｍａｅｓｔｗｕｍＬ．）ｒＲＮＡ基因间隔区序列差异,按Ｋｏｅｂｎｅｒ设计的引物序列,合成了黑麦特异引物ＮＯＲ－Ｒ１。运用该引物对不同植物材料进行ＰＣＲ扩增,观察表明,含有黑麦１Ｒ染色体的植物均扩增出黑麦的特异带,但含有其他黑麦染色体的小麦种质,普通小麦品种及其近缘物种长穗偃麦草（Ａｇｒｏｐｙｒｏｎｅｌｏｎｇａｔｕｍ（Ｈｏｓｔ）Ｂｅａｕｖ）     

中国小麦微核心种质资源Psy基因的等位变异

根据已发表的麦族植物体Psy基因序列的保守区设计引物PsyO2,克隆小麦Psy基因（片段）。结果表明,PsyO2引物的扩增产物出现2种带型：196bp和233bp,序列分析表明两条特异条带涵盖了小麦Psy基因第2外显子全部序列,相差的37bp为Psy基因第2内含子中的一段插入序列,可反映不同黄色素含量（YPC）,属小麦风,,基因的等位变异。验证试验表明,248份小麦微核心种质中有153份材料（占样品数的65．7％）扩增出196bp条带,群体内YPC均值7．314mg kg^-1,属高YPC范畴;另有95份材料（占样品总数的38．3％）扩增出233bp条带,群体内YPE均值为5．207mg kg^-1,属低YPC范畴,方差分析表明二者YPC差异达1％极显著水平差异,说明上述37bp的插入序列是导致小麦品种间YPC产生差异的原因之一,因此该引物扩增的Psy基因对小麦YPC具有显著影响,引物PsyO2是对小麦YPC进行分子鉴定的重要标记。     

辣椒胞质雄性不育保持基因的分子标记

利用RAPD标记技术,以辣椒胞质雄性不育系8A及其同型保持系8B为材料,对辣椒胞质雄性不育基因和保持基因进行比较分析.结果表明:引物H7只在保持基因池有1条稳定的特异扩增带,在不育基因池未扩增出此条带,该标记可能与辣椒胞质雄性不育保持基因相连锁,命名为H7-F850.序列分析结果表明,标记H7-F850序列全长857 bp,GenBank登录号为GU208822.1.核酸序列比对结果显示,H7-F850与其具有部分同源性的片段大小均小于340 bp,未发现与其具有较高同源性的DNA序列.该RAPD标记H7-F850已成功转化为SCAR标记SF640.     

黑麦1R染色体的显微分离、体外扩增及扩增产物的鉴定

借助Leitz显微操作器,在国产倒置显微镜下(400×)用玻璃针对处于有丝分裂中期的黑麦根尖细胞中的1R染色体成功地进行了分离。分离出来的1R染色体转入0.5 ml的Eppendorf管中,用蛋白酶K处理,把DNA释放出来;经Sau3A酶切,再与人工合成的Sau3A连接头连接;以连接头的一条链的核苷酸顺序片段为引物对DNA酶切片段进行了PCR扩增。琼脂糖凝胶电泳显示扩增产物的长度大约为300~1000 bp。以生物素分别标记的黑麦总体DNA和小麦rDNA为探针进行斑点杂交,结果表明PCR扩增产物确实来源于黑麦的1R染色体DNA。这个方法为构建黑麦1R染色体亚基因组文库和筛选1R染色体特异性探针奠定了基础。     

利用小麦微卫星引物建立簇毛麦染色体组特异性标记

选位于普通小麦1A-7A、1B-7B、1D-7D染色体上的102对微卫星引物对多年生簇毛麦、二倍体簇毛麦、小麦-簇毛麦双二倍体与后代和普通小麦中国春、R25、R111、MY11进行了PCR扩增, 发现引物对Xgwm301可以在含簇毛麦染色体的材料中扩出一条长415 bp的特异片段(命名为Xgwm301/415), 而所有供试小麦均未扩出此片段。进而用一套中国春-二倍体簇毛麦附加系来进行扩增, 发现1V-7V染色体均可以扩出该片段, 说明该片段为簇毛麦1V-7V染色体所共有。因此, Xgwm301/415是簇毛麦染色体组上的一个特异片段, 可以用来快速跟踪检测导入到普通小麦背景中的簇毛麦染色体。     

几个中国大麦属物种叶绿体psbL和psbJ基因序列分析

对8份来源于中国和2份来自国外的不同大麦属物种或亚种叶绿体基因组psbL～psbJ区进行了序列测定.结果表明,10份大麦属材料的psbL～psbJ区序列长度均为365bp,且无变异位点.同时,将大麦属psbL～psbJ区序列与已报道的黑麦(Secale cereale L.)、小麦(Triticum aestivum L.)、水稻(Oryza sativa L.)和玉米(Zea mays L.)的相同序列比较,发现所有物种的psbL基因序列长度均为117bp,且无核酸变异位点;psbJ基因序列长度均为123bp,共检测到6个核酸变异位点.除水稻外,所有物种psbL和psbJ基因间区长度均为125bp,水稻psbL和psbJ基因间区为126bp.在psbL和psbJ基因间区,除水稻具有1个插入位点外,还发现8个核酸变异位点.psbL～psbJ区揭示的大麦属物种、黑麦、小麦、水稻和玉米的遗传分化距离变化范围为0.0055～0.0426.以玉米为外类群,采用邻接法进行系统发育关系分析发现,同属于小麦族的大麦属植物、黑麦和小麦间亲缘关系较近,而水稻与玉米则相对较近.     

Features of crossability, haploidy and polyembryony in hybrid combinations between common barley Hordeum vulgare L. (2n = 14) and wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between common barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters--frequency of set grains and frequency of grains with embryos--was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1 R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare x T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between common barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

中间偃麦草染色体2Ai-2特异PCR新标记的建立和St基因组特异序列的克隆

中间偃麦草麦、小麦和小麦－中间偃麦草2Ai－2附加系Z1、Z2、X6,代换系ZD28等进行RAPD分析,从320个RAPD引物中,鉴定出2Ai－2染色体特异的2个RAPD标记OPO05650和OPMO414000。利用这2个特异OPO05和OPM04,PCR扩增普通小麦CS（ABD）及其近缘植物中间偃麦草（E1E2St）、拟鹅冠草（St）,长穗偃麦草（E）、簇毛麦（V）、黑麦（R）、大麦（H）粗山羊草（D）等基因组DNA。结果表明,OPO05650和OPO41400均是2Ai－2染色体上St基因组区域的特异标记。将上棕2个特异片段分离回收、克隆、测序,根据测序结果重新设计、合成特异引物,成功地转换RAPD标记为SCAR（sequence characterizked amplifed region）标记SC－05和SC－M4。利用SCAR标记对不同材料进行分析的结果表明,凡含有2Ai－2染色体的抗黄矮病材料及拟鹅冠草均产生一条扩增带,不含2Ai－2染色体的材料,包括小麦、长穗麦草、簇毛麦、黑麦、在麦、粗山羊草以有含有其他他中间偃麦草染色休的附加系,均没有扩增产物,说明上棕2个SCAR标记是中间偃麦草2Ai－2染色体的特异性PCR标记,且是2Ai－2染色体上St基因组区域的特异性标记。克隆与鉴定中间偃麦草的2个SCAR扩增片段TiSCO5和TiSCM4。结果表明,克隆的中间偃麦草TiSCO5和TiSCM4特异片段,分别是St基因组特异性的寡拷贝序列有多拷贝重复序列,为St基因组遗传研究的新探针。     

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the Individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aeativum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S.cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattem of Secale vavilovli was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.     

Effect of rye Secale cereale L. chromosomes 1R and 3R on polyembryony expression in hybrid combinations between (Hordeum vulgare L.)-Triticum aestivum L. alloplasmic recombinant lines and wheat T. aestivum L.-rye S. cereale L. substitution lines

The effect of rye chromosomes on polyembryony was studied for reciprocal hybrid combinations between (Hordeum vulgare L.)-Triticum aestivum L. alloplasmic recombinant lines and five wheat T. aestivum L. (cultivar Saratovskaya 29)-rye Secale cereale L. (cultivar Onokhoiskaya) substitution lines: IR(1D), 2R(2D), 3R(3B), 5R(5A), and 6R(6A), and for direct hybrid combinations between the [H. marinum ssp. gussoneanum (H. geniculatum All.)]-T. aestivum alloplasmic recombinant line and the wheat-rye substitution lines 1R (1A), 1R (1D), and 3R(3B). Chromosomes 1R and 3R of rye cultivar Onokhoiskaya proved to affect the expression of polyembryony in the hybrid combinations that involved the alloplasmic recombinant lines of common wheat as maternal genotypes. Based on this finding, polyembryony was regarded as a phenotypic expression of nuclear-cytoplasmic interactions where an important role is played by rye chromosomes 1R and 3R and the H. vulgare cytoplasm. Consideration is given to the association between the effect of rye chromosomes 1R and 3R on polyembryony in the [(Hordeum)-T. aestivum x wheat-rye substitution lines] hybrid combinations and their stimulating effect on the development on angrogenic embryoids in isolated anther cultures of the wheat-rye substitution lines.     

Isoenzymes of aspartate aminotransferase in perennial and annual rye and their hybrids.

Aspartate aminotransferase patterns were screened in a collection of rye genotypes that included 24 accessions of wild perennial rye (Secale montanum Guss.), 6 accessions of cultivated perennial Derzhavin and Tsitsin rye (Secale cereale x S. montanum), 15 accessions of winter and spring rye cultivars (S. cereale L.), and 9 accessions of perennial and annual rye genotypes bred from S. montanum ssp. kuprijanovii, Derzhavin rye, and winter rye for their resistance to fungal diseases. Aspartate aminotransferase is coded for by four loci. The data fit the model where AAT 1/4 is coded by Aat 1 and Aat 4, two duplicate loci, with null and two active alleles for each locus, alleles 1 and 3 for locus Aat 1 and alleles 2 and 4 for locus Aat 4; dimeric AAT 1/4 enzyme molecules are the products of both intralocus and interloci complementation. Allele 1 of Aat 1 was the most prominent in the isoenzyme patterns of the rye species. Alleles null and 2 of Aat 4 were twice as frequent in the perennial rye accessions, including Derzhavin and Tsitsin rye, than in winter and spring rye. In contrast, allele 4 of Aat 4 was characteristic of S. cereale. Within the screened collection, locus Aat 2 was monomorphic. Among three alleles of Aat 3, allele 2 dominated isoenzyme profiles of both rye species, whereas the other two alleles were species-specific: allele 1 was characteristic of S. montanum and allele 3 was found only in S. cereale. Key words : rye, Secale cereale, Secale derzhavinii, Secale montanum, aspartate aminotransferase, isoenzymes, perennial habit, polymorphism.     

Respiration and Involvement of an Alternative Pathway as Related to Age and Phenological Strategy of the Leaf

Russian Journal of Plant Physiology - Plants of spring wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) pursuing different phenological strategies were studied. Respiratory activity,...