小麦-顶芒山羊草2M染色体系的鉴定与评价

为了研究小麦-顶芒山羊草新种质材料的染色体组构成及开发其在育种中的利用价值,本研究利用分子标记和荧光原位杂交对小麦-顶芒山羊草杂交新种质进行鉴定,并通过小麦主要病害生理小种接种和农艺性状调查等方法对鉴定的材料进行综合评价。分子标记和原位杂交结果显示,所鉴定材料分别为小麦-顶芒山羊草2M附加系、2M(2D)代换系、2AS-2ML.2MS易位系和2DS-2ML.2MS易位系;抗病性鉴定结果表明,含2M染色体的材料均高抗小麦条锈病,其小麦亲本则高感条锈病,表明2M染色体上可能含有抗条锈病新基因;农艺性状调查分析结果表明,2M染色质导入小麦,可影响其小穗数、穗粒数和穗粒重等产量性状。因此,在创制和利用抗条锈病的小麦-顶芒山羊草2M染色体小片段易位系时,应加强对上述农艺性状的考察,并利用当前主栽品种进行回交改良。本研究鉴定出的抗条锈病小麦-顶芒山羊草2M染色体系丰富了小麦抗病基因库,为小麦育种提供了新抗源。     

小麦M染色体组的RELP标记

利用２９个小麦ＲＦＬＰ探针与６种限制性酶切的Ｍ染色体组ＤＮＡ进行分子杂交，获得了５５个Ｍ染色体组的ＲＦＬＰ标记，其中１５个与小麦ＡＢＤ染色体组相同，４０个染Ｍ染色体组的特殊标记，在顶荒山羊草和小麦－顶芒山羊草双二倍体以及小麦顶芒山羊草异代换系中稳定遗传     

基于SSR标记的粗山羊草遗传多样性分析

粗山羊草(Aegliops tauschii)是普通小麦遗传改良的重要基因资源.随机选取8份粗山羊草对普通小麦的211对SSR引物进行了引物筛选.选取稳定清晰、有特异带的32对SSR引物对78份粗山羊草进行多样性分析,共扩增出308个等位位点,平均每对SSR引物扩增出8.14个等位位点,其中A组引物、B组引物和D组引物分别扩增出39个、51个和212个等位位点,平均每个组等位位点分别为5.57个、6.38个和12.47个,D组引物检测到遗传变异最丰富,说明粗山羊草D组的同源性与普通小麦的D组同源性最高,同时,能在A、B引物中扩出特异性条带,说明粗山羊草D组与普通小麦的A、B基因组也有一定的同源性.UPGMA聚类结果表明,78份粗山羊草在遗传相似系数0.77时聚为6个主要类群,而且遗传聚类关系与材料的地理来源有一定的相关性.     

小麦远缘杂交种质资源创新

小麦近缘种是改良小麦的一个重要基因库, 具有许多栽培小麦所不具备的优良特性。我们通过远缘杂交、染色体工程的方法创制了一大批不同类型的材料, 经基因组原位杂交GISH、多色FISH 和特异分子标记鉴定, 抗条锈病、白粉病、叶锈病鉴定, 品质、营养性状以及产量性状鉴定, 共选育出10 类可为育种家利用的抗病、优质、富含微量营养元素、氮高效、丰产性状优良的远缘杂交新种质和新不育系种质; 开发了414对黑麦基因组专化的EST 引物, 31 个黑麦染色体(臂)专化的EST 分子标记, 可应用于分子标记辅助育种, 或追踪检测小麦背景中的黑麦染色体或染色体片段; 进行了抗病新基因的遗传分析和分子标记定位工作。利用新种质, 选育出了一批表现突出的抗病、营养高效的小麦-黑麦、小麦-冰草远缘杂交新品系     

簇毛麦5V染色体特异性ISSR标记的建立和应用*

摘要：为建立簇毛麦特定染色体上的特异性ISSR标记，以二倍体簇毛麦、小麦中国春等为材料，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)，而中国春等小麦均未扩出该片段。进而利用UBC848对小麦近缘种偏凸山羊草等进行扩增，发现它们均未扩出该片段。用一套中国春-二倍体簇毛麦附加系CSDA1V~CSDA7V对目标片段进行染色体定位，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、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，发现它们均能扩出目标片段，这表明，在小麦染色体工程育种中， 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。     

野生二粒小麦导入普通小麦抗白粉病基因MlWE27的鉴定和分子标记

由白粉病菌(Blumeria graminis f.sp.tritci)引起的小麦白粉病是严重影响小麦安全生产的主要病害之一.本研究将来自以色列的野生二粒小麦(Triticum dicoccoides)WE27的坏白粉病基因通过杂交和连续回交,导入普通小麦遗传背景中,育成高抗白粉病小麦新品系3D256(其系谱为燕大1817/WE27//农大015/3/941,F6).将3D256和高感小麦白粉病的普通小麦品系薛早配制杂交组合,对其F_1、F_2分离群体和F_3 家系进行白粉病抗性鉴定和遗传分析.结果表明,3D256携带抗白粉病显性单基因,暂命名为MlWE27.利用集群分离分析法(RSA)和分子标记分析,发现3个SSR标记(Xwmc243、Xwmc 154和Xbarc318)、1个EST-SSR标记(Xdp357)、1个AFLP转化的SCAR标记(XCAUG1)和1个RFLP探针转化的STS标记(XWG516-1)与抗白粉病基因MlWE27连锁,在连锁图上的顺序为Xdp357-Mlwe27-XCAUG1-XWG516-1-Xwmc243-Xwmc154-Xbarc318.利用中国春缺体-四体系、双端体系和缺失系将抗白粉病基因MlWE27定位于染色体2B短臂的末端Bin0.84-1.00上.这一普通小麦抗白粉病种质资源的创制及其连锁分子标记的建立为小麦抗病基因分子标记辅助选择、基因积聚和分子育种提供了新的物质基础.     

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。     

小麦背景中披碱草部分染色体的FISH鉴定

为了建立小麦背景中粗穗披碱草(Elymus trachycaulus)和纤毛披碱草(Elymus ciliaris)染色体的跟踪鉴定方法,本研究利用寡聚核苷酸Oligo-pSc119.2-1和Oligo-pTa-535-1为探针的双色荧光原位杂交(FISH)和以(GAA)₈为探针的单色FISH,分别对4个小麦-粗穗披碱草附加系和5个小麦-纤毛披碱草附加系染色体进行分析。结果发现,经与小麦FISH核型比对后,建立了可用于追踪小麦背景中粗穗披碱草1S^t、5H^t、6H^t和7H^t染色体的标准FISH核型;而纤毛披碱草S^c和Y^c染色体FISH信号较弱。FISH检测发现,小麦-粗穗披碱草1S^t附加系自交自发变成了1S^t(1BS·3BL)代换系,且在其染色体组中检测到了一对T1BL·3BS易位染色体,同时发现5AS端部寡聚核苷酸Oligo-pSc119.2-1序列发生了删除。另外,在小麦-粗穗披碱草5H^t附加系中发现2B染色体短臂末端删除现象,形成了2B-del和2BS-4AS·4AL易位染色体,而另一条2B和4A为正常的完整染色体。这种染色体结构重排事件表明,部分小麦-远缘物种附加系细胞学并不稳定,因此,繁种前后应对材料进行单株细胞学鉴定。上述小麦-粗穗披碱草染色体结构变异体的获得,为研究染色体结构重排与基因转录表达和表型变化的关系提供了研究基础。     

DNA分子标记技术及其在小麦育种及遗传研究中的应用

本文介绍了几种常用的DNA分子标记,如RFLP、RAPD、AFLP、SSR、STS、SNP等,并简要综述了分子标记技术在小麦遗传育种研究中的应用现状,包括基因标记与定位、遗传图谱构建、外源染色体鉴定与标记、种质资源鉴定和辅助育种等。     

小麦-大麦大穗大粒渐渗系WB13的分子细胞学鉴定

小麦新种质WB13是普通小麦(Triticum aestivum L.)品种7182与农家二棱大麦(Hordeum vulgare ssp.distichon Hsü.)杂交后回交多代衍生而来的大穗大粒材料。为了明确WB13的遗传基础,本研究利用形态学、细胞学、分子标记及特异片段回收测序等技术对其进行了鉴定。结果表明,采用小麦不同同源群简单重复序列(simple sequence repeat,SSR)标记对WB13和小麦7182进行遗传背景分析发现,两者遗传相似系数(genetic similarity coefficient,GS)达97.0%,田间表现为大穗、大粒,综合农艺性状较好;根尖细胞染色体数目为2n=42,以大麦基因组DNA为探针进行基因组原位杂交(genomic in situ hybridization,GISH)未出现杂交信号;利用大麦特异序列标签位点(sequence-tagged site,STS)标记对WB13和农家二棱大麦进行扩增,发现ABG054(4H)和ABC305B(7H)两个标记在WB13中扩增出了大麦特征条带(分别记为WS1和WS3),经测序及序列比对,发现其与扩增到的大麦序列分别具有100%和98%的相似性,与EMBL数据库中的序列比对,与两者有相似性的全为大麦的序列。利用大麦4H和7H染色体上的35对SSR引物对WB13及其亲本进行扩增,发现与千粒重相关的标记MGB396(4H)在WB13中扩增出了大麦的特异条带。综合以上结果确定WB13含有大麦4H和7H染色体的遗传物质,为小麦-大麦渐渗系材料,且4H染色体渗入片段中可能携带与千粒重相关的有益基因。本研究确定了WB13为小麦-大麦杂种后代,此材料的育成丰富了小麦-大麦中间材料和大穗大粒材料的种质资源。同时本研究在分子水平上初步揭示了WB13大穗大粒特性的成因,为后续构建遗传分析群体进行相关数量性状(quantitative trait loci,QTL)定位及推动WB13的研究利用积累了基础资料。     

Identification in Aegilops species of resistant sources to Hessian fly (Diptera: Cecidomyiidae) in Morocco

Hessian fly, Mayetiola destructor (Say), is the major insect pest of wheat in Morocco. Host plant resistance has been the most effective and practical method of controlling this pest. When 347 accessions of Aegilops species were screened in the greenhouse for resistance to Hessian fly, several accessions of Ae. geniculata Roth, Ae. triuncialis L., Ae. neglecta Req.ex Bertol., Ae. ventricosa Tausch, Ae. cylindrica Host and Ae. markgrafii (Greuter) Hammer showed resistance reaction. All expressed antibiosis as the mechanism of resistance against first instar Hessian fly larvae. These Aegilops sources of resistance could be exploited for transferring Hessian fly resistance to wheat.     

Genetic diversity of Central Asian and north Caucasian Aegilops species as revealed by RAPD markers

RAPD analysis of 112 accessions of Aegilops tauschii Coss. (genome DD), Ae. cylindrica Host (CCDD), Ae. crassa Boiss. (DDMM), Ae. biuncialis Vis. (UUMM) and Ae. triuncialis L. (UUCC) collected in the Central Asia and north Caucasia was conducted. Aegilops accessions were divided into two major groups, corresponding to the D genome species and the U genome species. These groups were also separated into sub-groups according to species, except for the Ae. tauschii-cylindrica complex of accessions from Central Asia. Aegilops tauschii from north Caucasia was divided into two varietal groups, tauschii and meyeri. The Central Asian accessions of Aegilops species were more diverse than the accessions from north Caucasia. Aegilops tauschii and Ae. cylindrica accessions from north Caucasia were genetically uniform. Associations between altitudal variation of Aegilops species and variability of RAPD markers were not found.     

Variation in zinc efficiency among and within Aegilops species

Fifteen accessions of Aegilops tauschii (DD), 10 of Ae. speltoides (SS) and 8 of the tetraploid Aegilops species sharing the U genome were used to study the influence of varied zinc (Zn) supply on development of Zn-deficiency symptoms, and on shoot dry weight and Zn concentration. Plants were grown in a Zn-deficient calcareous soil under greenhouse conditions with (+Zn = 5 mg kg^—1 soil) and without (—Zn) Zn supply. Four accessions of wild tetraploid wheat, Triticum turgidum var. dicoccoides (BBAA), a group known for its high sensitivity to Zn-deficiency, were used in the experiments for comparison. As expected, the accessions of wild T. turgidum var. dicoccoides showed the highest sensitivity to Zn deficiency, and had more severe leaf symptoms of Zn deficiency (whitish-brown necrotic patches). Among the Aegilops species, leaf symptoms of Zn deficiency were, in general, more distinct in Ae. tauschii (DD) and least in Ae. speltoides (SS). Zinc efficiency, expressed as the percentage of shoot dry weight produced under conditions of Zn deficiency compared to Zn supply, averaged, 15% for T. turgidum, 32% for Ae. tauschii, 52% for Ae. speltoides and 61% for the tetraploid Aegilops species carrying the U genome. Differences in Zn efficiency among and within Aegilops species and T. turgidum were significantly correlated with the Zn amount per shoot, but not with the Zn amount per unit dry weight of shoots. The results show that Aegilops species can be exploited as an important genetic source for Zn efficiency genes, particularly Ae. speltoides var. ligustica (SS) and Ae. triuncialis (UUCC). Transfer of these genes to cultivated modern wheat may bring about a greater variation in Zn efficiency in wheat, and facilitate production of Zn-efficient modern wheat cultivars for Zn-deficient soil conditions.     

Inheritance in hexaploid wheat of genes for hairy auricles and hairy leaf sheath derived from Aegilops tauschii Coss.

Inheritance of genes for hairy auricles and hairy leaf sheath of Ae. tauschii in hexaploid wheat backgrounds (synthetic hexaploid wheat and common wheat varieties) was analyzed. The results indicated that hairy auricles and hairy leaf sheath of Ae. tauschii can be transferred and are expressed in hexaploid wheat. In a synthetic hexaploid wheat ('Ae. tauschii' 188) hairy auricles was proved to be controlled by a single dominant gene derived from Ae. tauschii, which was different from the Pa gene located on chromosome 4BS of common wheat. The hairy leaf sheath phenotype of 'Altar 84/Ae. tauschii 188' was also controlled by a single dominant gene derived from Ae. tauschii, which is obviously different from the Hls gene in T. dicoccoides. We suggest to designate the Ae. tauschii genes for hairy auricles and hairy leaf sheath as Pa2 and Hls2, respectively; such genes could be used as useful genetic markers in common wheat.     

Powdery mildew resistance in Aegilops tauschii coss. and synthetic hexaploid wheats

Summary A collection of 400 Ae. tauschii (syn. Ae. squarrosa) Coss. accessions were screened for powdery mildew resistance based on the response patterns of 13 wheat cultivars/lines possessing major resistance genes to nine differential mildew isolates. 106 accessions showed complete resistance to all isolates, and 174 accessions revealed isolate-specific resistance, among which were 40 accessions exhibiting an identical response pattern as wheat cultivar Ulka/^*8Cc which is known to possess resistance gene Pm2. Expression of both complete and isolate-specific resistance from Ae. tauschii was observed in some synthetic hexaploid wheats derived from four mildew susceptible T. durum Desf. parents, each crossed with five to 38 resistant diploid Ae. tauschii accessions. Synthetic amphiploids involving different combinations of T. durum and Ae. tauschii generally showed a decrease in resistance compared with that expressed by the Ae. tauschii parental lines.     

Biodiversity of Diploid D-Genome Aegilops Tauschii Coss. in Iran Measured Using Microsatellites

Microsatellite markers were used to analyse the biodiversity of 57 accessions of different subspecies and varieties of wild Aegilops tauschii (2n = 2x = 14; D genome) collected across the major areas where it grows in Iran. Levels of diversity were high, with numbers of alleles averaging 7.3 (ranging up to 12) and polymorphism information contents averaging 0.6591. One accession was notably more similar to two of the D genome in hexaploid wheats (Triticum aestivum) used as outgroups. Within the Ae. tauschii accessions, no markers were characteristic for taxa or geographical origin, suggesting high gene flow between the subspecies and varieties, although some groupings, which could be related to geographical origin, were evident. This survey demonstrates the high diversity present in wild goatgrass in Iran, and indicates that there is value in sampling for useful genes for wheat breeding.     

Chromosome N-banding polymorphism in Aegilops geniculata Roth

The chromosome morphology and the variation in the N-band distribution along the chromosomes were studied in 13 accessions of the wild wheat Aegilops geniculata Roth (2n=28, genome formula UUM°M°). The karyotype consisted of four metacentric, five submetacentric, three subtelocentric, and two satellited pairs of chromosomes. The N-banding technique stained differentially all 14 chromosome pairs. The most prominent bands were observed near the centromeres and in the intercalary regions of both arms. Telomeric bands were found in only seven chromosomes. All chromosomes produced a specific banding pattern, permitting their identification. Polymorphism for presence or absence of particular bands was observed among the different accessions. The variable bands were located predominantly at the terminal and subterminal chromosome regions, and were also unevenly distributed over chromosomes. This polymorphism resulted in up to eight distinct banding pattern variants for each of the Ae. geniculata chromosomes. Each accession in this study showed a unique overall karyotype, sharing 0 to 8 chromosomes of identical banding pattern with the other accessions. A generalized idiogram of Aegilops geniculata is presented.     

黏类小麦CMS不育基因rfv1的分子细胞遗传学跟踪鉴定及定向转育研究

为了实现黏类小麦雄性不育基因rfv1的定向转育,创制优良的黏类非1BL/1RS小麦雄性不育新保持系,本研究以1BS上带有不育基因rfv1的非1BL/1RS小麦变种SP4、莫迦小麦为供体材料,以杀雄剂途径培育的小麦强优势组合西杂1号和西杂5号杂交小麦新品种的母本西农Fp1和西农Fp2为受体材料,采用专一核置换回交转育方法,同时结合根尖细胞学镜检、复合引物PCR及SDS-PAGE和A-PAGE技术,进行不育基因rfv1定向转入的鉴定,旨在育成既携带rfv1不育基因,又具有西农Fp1和西农Fp2核遗传背景的黏类非1BL/1RS小麦雄性不育新保持系。结果如下：（1）根尖体细胞随体鉴定和复合引物PCR分析表明,该法不仅能准确鉴定出1BL/1RS纯合易位系,还可鉴定出1BL/1RS·1BL/1BSrfv1 易位杂合体,两者结果一致。其中复合引物PCR更适合于回交后代中大量目标植株的筛选,为小麦雄性不育基因rfv1定向转移到1BL/1RS易位系提供了准确的鉴定方法与依据;（2）利用SDS-PAGE技术对供试材料进行高低分子量麦谷蛋白亚基的分析表明,在低分子量谷蛋白D亚基区存在莫迦小麦和SP4的特征带;而SP4的高分子量谷蛋白亚基区的6+8亚基,也可以作为示踪小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系的特征亚基条带;（3）A-PAGE技术对醇溶蛋白的分析表明,在ω-醇溶蛋白区也发现莫迦小麦和SP4不同于西农Fp2的特异蛋白条带,也可作为示踪小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系的特征蛋白条带。本研究成功地将小麦杂种优势利用中的杀雄剂法和三系法相结合,促进了小麦杂种优势利用新技术体系的建立。同时该方法亦可应用于黏类小麦雄性不育恢复基因Rfv1的定向转育,进而可实现小麦由生理型不育向遗传型不育的定向转化,以探索一套杂交小麦强优势组合多途径利用的新方法。     

Durum wheat cultivation associated with Aegilops tauschii in northern Iran

Hexaploid bread wheat (Triticum aestivum L. ssp. aestivum) is assumed to have originated by natural hybridization between cultivated tetraploid Triticum turgidum L. and wild diploid Aegilops tauschii Coss. This scenario is broadly accepted, but very little is known about the ecological aspects of bread wheat evolution. In this study, we examined whether T. turgidum cultivation still is associated with weedy Ae. tauschii in today’s Middle Eastern agroecosystems. We surveyed current distributions of T. turgidum and Ae. tauschii in northern Iran and searched for sites where these two species coexist. Ae. tauschii occurred widely in the study area, whereas cultivated T. turgidum had a narrow distribution range. Traditional durum wheat (T. turgidum ssp. durum (Desf.) Husn.) cultivation associated with weedy Ae. tauschii was observed in the Alamut and Deylaman-Barrehsar districts of the central Alborz Mountain region. The results of our field survey showed that the T. turgidum–Ae. tauschii association hypothesized in the theory of bread wheat evolution still exists in the area where bread wheat probably evolved.     

Differences in seedling growth characteristics among provenances of Aegilops speltoides Tausch

Differences in physiological characteristics were analysed among accessions/provenances of Aegilops speltoides and between these accessions and the Triticum aestivum cultivar Sparta. The seedlings were cultivated for 21 days in Hoagland 3 nutrient solution. At the end of the experiment, shoot and root dry matter was determined and total plant dry mass and shoot to root ratio were calculated. Daily measurements of the leaf length were used for the calculation of the leaf growth rate and phyllochron intervals. Finally, leaf area of individual leaf blades and the nitrogen content of the whole plant were determined. With few exceptions, no statistically significant differences among accessions of Ae. speltoides were found. Compared to these accessions, T. aestivum cv. Sparta produced considerably more dry matter. This was mainly due to larger leaf blade area, while net assimilation rates were similar. It was concluded that the variability among Aegilops provenances is of minor importance and any of the tested accessions could be used as a representative of Aegilops speltoides for further experiments.