山羊草属异源多倍体物种核rDNA ITS区的进化

本文测定了山羊草属Aegilops 3个组中异源多倍体物种的核rDNA ITS区序列,并用邻接法进行了聚类分析。结果表明,多倍体物种的ITS区序列长度为559∽606bp,其中ITS1、ITS2分别有变异位点51、42个,且存在多态位点。多倍体种均与各自的某一祖先种构成稳定分支,说明在杂交-多倍化后,这些多倍体的ITS区在同步进化的作用下已向着其某一祖先种的ITS区进化。对于sect．Vertebrata的异源多倍体物种来说,其ITS区主要向其祖先种Ae．umbellulata(UU)的ITS区进化,这与山羊草属的细胞遗传学研究结果基本一致。在sect．Cylindropyrum和sect．Polyeides中,Ae．cylindrica(CCDD)朝着Ae．caudata (CC)进化;Ae．ventricosa(DDMvMv)朝着Ae．comosa(MM)进化;Ae．vavilovii(DDMMSS)朝着Ae．crassa (DDMM)进化。     

“缺体回交法”选育普通小麦—山羊草异代换系的研究

利用从兰单体自交分离得到的5个自花结实的4D缺体小麦(映72180、块天选15等)作母本与11个山羊草(Ae.speltoides, Ae.sharonensis等)杂交,再以4D缺体为轮回亲本对杂种进行回交,借助于幼胚培养技术,获得了缺天选15×拟斯卑尔脱山羊草二体异代换系,缺72180×沙融山羊草单体异代换系。代换系生长发育良好,育性基本正常,表明山羊草的4S染色体能够补偿小麦缺失的4D染色体的功能。证明利用“缺体回交法”选育普通小麦—山羊草异代换系是有效的和可行的。     

山羊草属S基因组与小麦属B／G基因组RAPD标记和特异DNA片段克隆及研究

本研究利用132个随机引物,对山羊草属和小麦属11个种的DNA进行扩增。对其中特异RAPD扩增产物进行克隆,然后用其做探针与小麦族23个种属DNA的RAPD扩增产物进行Southern杂交。共得到24个特异克隆：其中小麦族共有特异克隆1个,山羊草属和小麦属共有特异克隆2个,S基因组特异克隆2类7个,B／G基因组特异克隆2类6个,S基因组与B／G基因组共有的特异克隆8个。24个特异克隆中有22个测定了序列,其中15个为Fasta数据库里显示未见报导的序列。用这24个特异DNA克隆制成的探针与相应的23个材料经HindⅢ酶切消化的总DNA进行Southern杂交,发现其中7个可做为基因组特异探针。通过对24个特异DNA克隆分析研究表明：①S基因组是由两个基本不同的类型构成的,即拟斯卑尔脱山羊草为一个类型,其余4个S组山羊草为另一类型;因此建议前基因型符号仍保留为“S”,而其余4个种之间无明显不同,故应把基因组符号统一为“S^1”②S基因组是小麦B／G基因组的供体,而拟斯卑尔脱山羊草可能是最主要的供体,但并不排除其余S基因组的种参与了B／G基因组形成的可能;研究还表明B基因组与S基因组还是有很大区别的,并已找到了B基因组的特异标记。     

拟斯卑尔脱山羊草（Ae．speltoides Tausch）遗传多样性的RAPD分析

用95个引物,在拟斯卑尔脱山羊草15份材料中扩增出416条带,其中无多态性的带为83条,占总数的20％,有多态性的带为333条,占总数的80％。可以看出,拟斯卑尔脱山羊草种内遗传多样性十分丰富。RAPD分析表明,供试的15份材料可以分成4个类群,每个类群内的材料之间差异较明显,而4个类群间差异不是太大,因此拟斯卑尔脱山羊草是由类群内不太一致、类群间差异不十分明显的4个类群构成。类群内差异明显,支持遗传多样性丰富,说明拟斯卑尔脱山羊草是一个古老物种的观点;而类群间差异又不太大,表示该物种在长期进化中,没有分化成新的类型,说明该物种较稳定。本研究结果从分子水平上支持拟斯卑尔脱山羊草是一个比较古老且比较稳定的物种的论断。     

利用改进的Cap-trapper法构建拟斯卑尔脱山羊草(Ae.speltoides Tausch)全长cDNA文库

Captrapper法是目前全长cDNA文库构建的重要方法之一。在引进、消化、吸收的基础上,通过设计引物,同位素检测,对末端转移反应条件控制等方面做了一些切实可行的改进,形成了一套简单易行的技术体系。利用改进的Captrapper方法,成功构建了小麦B基因组的可能供体种拟斯卑尔脱山羊草(Ae.speltoides)的全长cDNA文库。经综合评价,文库的全长比例达到89.6%,重组率为99%,库容量超过3.0×106cfu。     

小麦tae-MIR156前体基因的克隆及其靶基因TaSPL17多态性分析

Squamosa-promoter binding protein (SBP)-box基因是植物特有的一类转录因子, 广泛参与植物生长发育, 其部分成员受miR156调控。文章克隆了小麦(Triticum aestivum) tae-MIR156前体基因, 转录后能够形成茎环结构。小麦10个SBP-box基因中, 仅TaSPL3和TaSPL17在编码区存在tae-miR156识别位点。SPL17在普通小麦的A基因组供体种乌拉尔图小麦(Triticum urartu, AA) UR209和B基因组供体种拟斯卑尔脱山羊草(Aegilops speltoides, BB) Y2001中均为多拷贝(SPL17-A1、SPL17-A2和SPL17-A3; SPL17-B1、SPL17-B2和SPL17-B3), 在D基因组供体种粗山羊草(Aegilops tauschii, DD) Ae38中仅检测到一种序列(SPL17-D); SPL17-A2与SPL17-B2, SPL17-A3与SPL17-B3、SPL17-D两两之间序列的一致性程度均大于99%, 且与普通小麦(中国春、衡观35和双丰收)的TaSPL17序列具有较高的一致性, 提示它们可能来源于共同的祖先基因, 并且在进化过程中高度保守。靶基因TaSPL17中的tae-miR156识别位点非常保守, 在根据单株穗数和基因型多样性挑选的SubP1和SubP2群体中均未检测到tae-miR156识别位点存在变异碱基。     

山羊草属核型分析及其与小麦属的进化关系

作者研究了山羊草属(Aegilops)中的新疆节节麦(Ae.squarrosa)、拟斯卑尔脱山羊草(Ae.speltoides)、沙融山羊草(Ae.sharonensis)、尾状山羊草(Ae.caudata)、卵圆山羊草(Ae.ovata)、偏凸山车草(Ae.ventricosa),钩状山羊草(Ae.triuncialis)、三芒山羊草(Me.triaristata)、欧山羊草(Ae.biuncialis)、柱穗山羊草(Ae.cylindrica)、可兹山羊草(Ae.kotschyi)和肥厚山羊草(Ae.crassa)的核型和部分材料的Giemsa N-带,结果表明山羊草属的C组核型为:4sm+3st;D组核型为:6m+1sm;S组的核型为:6m+1sm;M组的核型为:4m+1sm+2t。在四倍体、六倍体中,各染色体组保持着相对稳定。山羊草属S、D染色体组的核型与带型表明它们是小麦B、D染色体组的可能供体,C、M染色体组的一部分染色体带型亦与小麦B组带型相似。     

基于ITS序列分析钩苞大丁草九个居群的亲缘关系

该研究对我国西南地区钩苞大丁草(Gerbera delavayi)9个居群rDNA ITS序列进行PCR的扩增和检测序列,并以非洲菊(G.jamesonii)的ITS序列作为外类群,比较了序列之间的差异,同时分析了钩苞大丁草不同居群在地理距离与遗传距离之间的关系,构建了NJ系统发育树。结果表明:(1)钩苞大丁草9个居群的ITS序列全长介于600~700 bp之间,平均长度约为657 bp,其中,ITS1长度为243~246 bp,(G+C)含量为45.67%~46.80%之间,5.8S长度191~193 bp,(G+C)含量为58.60%~58.61%之间,ITS2长度为220~221 bp,(G+C)含量为57.00%~57.45%之间;ITS序列共有22个变异位点,ITS1序列(17个)、5.8S序列(2个)以及ITS2序列(3个)上均有变异。(2)地理距离与遗传距离有正相关(r2=0.652),序列间遗传分化距离为0.001 1~0.024 3,其中普洱居群与其他居群间遗传距离最大。(3)钩苞大丁草9个居群分成三个分支,普洱居群单独成支,丽江和洱源居群聚为一支,富源、武定、德昌、石林、新平和开远6个居群聚为一支。rDNA ITS序列可以用于钩苞大丁草群体遗传研究的分析,该研究结果为其保护性开发提供了参考依据。     

拟斯卑尔脱山羊草基因的克隆与表达研究

Avenin—like基因是近年来发现的一类新基因。根据小麦avenin-like基因的保守序列,设计合成了一对特异性引物,对拟斯卑尔脱山羊草（Aegilops spehoides,ss）的基因组DNA进行avenin-like基因扩增、克隆、序列测定和表达分析,发现了一个新型avenin—like基因。基因长855bp,编码284个氨基酸残基,分子量约为33kD。Souchern blot结果表明其属于多基因家族。RT—PCR证实了avenin-like基因在籽粒胚乳中特异性表达。其对应的氨基酸序列含有18个半胱氨酸残基,可以形成7对分子内二硫键。研究表明Avenin-like蛋白是一类新型的储藏蛋白。这为小麦加工品质的改良提供了理论依据和遗传资源。     

虎耳草属山羊臭组的界定和系统发育: 核糖体DNA ITS序列证据

虎耳草属Saxifraga山羊臭组sect. Ciliatae是该属中最大的一个组, 共有175种, 主要分布在喜马拉雅地区, 我国分布有166种, 占总种数的95%; 其中, 112种为中国特有。约80%的种类分布在我国青藏高原和西南地区, 是中国喜马拉雅植物成分的代表类群。山羊臭组内物种分化十分显著, 分类处理也很困难, 该组是否为单系类群, 组下的系统发育关系也不清楚, 均需进一步验证。本文测定了虎耳草属山羊臭组及其他组33种植物样品的核糖体DNA内转录间隔区ITS序列, 并从GenBank调取虎耳草组sect. Saxifraga等组和近缘属唢呐草属Mitella共22种植物的该序列。ITS分析结果表明: (1)所研究的山羊臭组类群聚为单独一支, 而且与垫状组sect. Porphyrion、虎耳草组、球茎组sect. Mesogyne和仅在欧洲分布的sect. Cymbalaria和sect. Cotylea等8个组聚成的另一分支构成姊妹群; (2)根据形态特征建立的山羊臭组的3个亚组即唐古拉亚组subsect. Hirculoideae、莲座状亚组subsect. Rosulares和具芽亚组subsect. Gemmiparae各自聚为一支, 但是莲座状亚组这一支的支持率较低。同时, 山羊臭组的鞭匐枝亚组subsect. Flagellares和subsect. Hemisphaericae的代表类群单独聚为一支, 位于具芽亚组类群分支内部而不能成立; (3)唐古拉亚组和莲座状亚组又聚为一亚分支与具芽亚组构成姊妹群, 而且具芽亚组最早从山羊臭组这一支中分化出来。我们的研究还发现山羊臭组内种间形态分化较大, 而ITS碱基变异较小, 这可能是山羊臭组类群在青藏高原及毗邻地区的高山环境下物种快速分化的结果。     

The phylogenetic relationships among the possible donors of B-genome of common wheat based on internal transcribed spacer (ITS) sequences

The ITS regions of 5 species in Aegilops sect. Sitopsis, the possible donors of Bgenome of common wheat, were amplified by PCR, cloned and sequenced. The phylogenetic relationships among 5 species in Aegilops sect. Sitopsis were constructed based on ITS1 + ITS2 sequences. The results demonstrated that Ae. speltoides was a distinct species in Aegilops sect. Sitopsis. The average of the pairwise distances between Ae. speltoides and the other four species was three times as high as that among the latter four. Ae. speltoides was the earliest lineage of the section under question. Relationship between Ae. longissima and Ae. sharonensis was the closest in Aegilops sect. Sitopsis. Sequence of ITS regioncould be used as a molecular marker to identify origin of B-genome in polyploid wheats.     

Studies on the origin and evolution of tetraploid wheats based on the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA

In this study, the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA in the tetraploid wheats, Triticum turgidum (AABB) and Triticum timopheevii (AAGG), their possible diploid donors, i.e., Triticum monococcum (AA), Triticum urartu (AA), and five species in Aegilops sect. Sitopsis (SS genome), and a related species Aegilops tauschii were cloned and sequenced. ITS1 and ITS2 regions of 24 clones from the above species were compared. Phylogenetic analysis demonstrated that Aegilops speltoides was distinct from other species in Aegilops sect. Sitopsis and was the most-likely donor of the B and G genomes to tetraploid wheats. Two types of ITS repeats were cloned from Triticum turgidum ssp. dicoccoides, one markedly similar to that from T. monococcum ssp. boeoticum (AA), and the other to that from Ae. speltoides (SS). The former might have resulted from a recent integression event. The results also indicated that T. turgidum and T. timopheevii might have simultaneously originated from a common ancestral tetraploid species or be derived from two hybridization events but within a very short interval time. ITS paralogues in tetraploid wheats have not been uniformly homogenized by concerted evolution, and high heterogeneity has been found among repeats within individuals of tetraploid wheats. In some tetraploid wheats, the observed heterogeneity originated from the same genome (B or G). Three kinds of ITS repeats from the G genome of an individual of T. timopheevii ssp. araraticum were more divergent than that from inter-specific taxa. This study also demonstrated that hybridization and polyploidization might accelerate the evolution rate of ITS repeats in tetraploid wheats.     

Molecular analysis of the phylogenetic relationships among the diploid aegilops species of the section Sitopsis

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of S-genome diploid Aegilops species regarded as potential donors of the B genome of cultivated wheat. In total, 21 DNA specimens from six S-genome diploid species were examined. On a dendrogram, Ae. speltoides and Ae. aucheri formed the most isolated cluster. Among the other species, Ae. searsii was the most distant while Ae. longissima and Ae. sharonensis were the closest species. The maximum difference between individual accessions within one species was approximately the same (0.18-0.22) in Ae. bicornis, Ae. longissima. Ae. sharonensis, and Ae. searsii. The difference between the clusters of questionable species Ae. speltoides and Ae. aucheri corresponded to the intraspecific level; the difference between closely related Ae. longissima and Ae. sharonensis corresponded to the interspecific level. The section Sitopsis of the genus Aegilops includes six diploid species containing the S genome, which is regarded as an ancestor of the B genome of cultivated wheat. The species of the section are thought to be closest to the genus Triticum. Note that the taxonomic status of some forms of the section Sitopsis is questionable. For instance, Ae. speltoides and Ae. aucheri are variously considered as individual species or as a single species, Ae. speltoides. The situation with Ae. longissima and Ae. sharonensis is similar. Thus, although the group includes only diploid species and is well studied morphologically, its phylogeny and taxonomy are still questionable.     

ITS regions in diploids of Aegilops (Poaceae) and their phylogenetic implications

The nucleotide sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA in nine diploid species representing six sections of Aegilops were determined by direct sequencing of PCR-amplified DNA fragments. These sequences were aligned with two ITS sequences of additional species from Genbank. Sequence divergences were estimated using Kimura two-parameter model, and the phylogenetic analyses were performed using the maximum parsimony (MP) and the neighbor-joining (NJ) methods with PAUP and PHYLIP, respectively. The sequence divergences between the diploid species varied from 0.5% to 4.68%. The resulting MP tree and NJ tree showed relatively congruent phylogenetic relationships among these species, except Ae. caudata. Particularly, Ae. speltoides was basal within the two trees. The paraphyletic relationships between Ae. speltoides and two species of Sect. Sitopsis, and between Ae. uniaristata and two species of Sect. Comopyrum were supported strongly. The ITS data suggest that currently recognized sections within Aegilops should be reconsidered.     

Transferability of wheat microsatellites to diploid Aegilops species and determination of chromosomal localizations of microsatellites in the S genome.

Overall, 253 genomic wheat (Triticum aestivum) microsatellite markers were studied for their transferability to the diploid species Aegilops speltoides, Aegilops longissima, and Aegilops searsii, representing the S genome. In total, 88% of all the analyzed primer pairs of markers derived from the B genome of hexaploid wheat amplified DNA fragments in the genomes of the studied species. The transferability of simple sequence repeat (SSR) markers of the T. aestivum A and D genomes totaled 74%. Triticum aestivum-Ae. speltoides, T. aestivum-Ae. longissima, and T. aestivum-Ae. searsii chromosome addition lines allowed us to determine the chromosomal localizations of 103 microsatellite markers in the Aegilops genomes. The majority of them were localized to homoeologous chromosomes in the genome of Aegilops. Several instances of nonhomoeologous localization of T. aestivum SSR markers in the Aegilops genome were considered to be either amplification of other loci or putative translocations. The results of microsatellite analysis were used to study phylogenetic relationships among the 3 species of the Sitopsis section (Ae. speltoides, Ae. longissima, and Ae. searsii) and T. aestivum. The dendrogram obtained generally reflects the current views on phylogenetic relationships among these species.     

Phylogenetic reconstruction of Aegilops section Sitopsis and the evolution of tandem repeats in the diploids and derived wheat polyploids.

The evolution of 2 tandemly repeated sequences Spelt1 and Spelt52 was studied in Triticum species representing 2 evolutionary lineages of wheat and in Aegilops sect. Sitopsis, putative donors of their B/G genomes. Using fluorescence in situ hybridization we observed considerable polymorphisms in the hybridization patterns of Spelt1 and Spelt52 repeats between and within Triticum and Aegilops species. Between 2 and 28 subtelomeric sites of Spelt1 probe were detected in Ae. speltoidies, depending on accession. From 8 to 12 Spelt1 subtelomeric sites were observed in species of Timopheevi group (GAt genome), whereas the number of signals in emmer/aestivum accessions was significantly less (from 0 to 6). Hybridization patterns of Spelt52 in Ae. speltoides, Ae. longissima, and Ae. sharonensis were species specific. Subtelomeric sites of Spelt52 repeat were detected only in T. araraticum (T. timopheevii), and their number and chromosomal location varied between accessions. Superimposing copy number data onto our phylogenetic scheme constructed from RAPD data suggests 2 major independent amplifications of Spelt52 and 1 of Spelt1 repeats in Aegilops divergence. It is likely that the Spelt1 amplification took place in the ancient Ae. speltoides before the divergence of polyploid wheats. The Spelt52 repeat was probably amplified in the lineage of Ae. speltoides prior to divergence of the allopolyploid T. timopheevii but after the divergence of T. durum. In a separate amplification event, Spelt52 copy number expanded in the common ancestor of Ae. longissima and Ae. sharonensis.     

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species.

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.     

Repetitive DNas of wild emmer wheat (Triticum dicoccoides) and their relation to S-genome species: molecular cytogenetic analysis.

We have analyzed the chromosomal GISH molecular banding patterns of three populations of the wild allopolyploid wheat Triticum dicoccoides in an attempt to unravel the evolutionary relationships between highly repetitive DNA fractions of T. dicoccoides and proposed diploid progenitors of the B genome. Aegilops speltoides showed almost complete affinity of its repetitive DNA to C-heterochromatin of T. dicoccoides, whereas other S-genome species demonstrated relatedness only to distal heterochromatin. This substantiates the priority of Ae. speltoides as the most similar to the wheat B-genome donor in comparison with other Sitopsis species. Using molecular banding technique with DNA of different Aegilops species as a probe permits tracing of the origin of each heterochromatin cluster. Molecular banding analysis reveals polymorphism between three wild emmer wheat populations. Comparison of molecular banding patterns with chromosomal distribution of the Ty1-copia retrotransposons, which constitute a large share of T. dicoccoides genome, makes it possible to propose that the activity of transposable elements may lie in the background of observed intraspecific polymorphism.     

Genetic Diversity of the Cytoplasm in Triticum and Aegilops. VIII. Fraction I Protein of 39 Cytoplasms

The electrophoretic characteristics of the cytoplasmically controlled large subunit of the Fraction I protein of 36 alloplasmic and three euplasmic control lines are reported. These lines, representing the cytoplasms of 32 Triticum and Aegilops species, had either H- or L-type large subunits in their Fraction I protein; the diploid Triticum and most Aegilops species, including Ae. bicornis and Ae. sharonensis, had the L-type subunits; whereas, all the polyploid Triticum species (emmer, timopheevi, common wheats), Ae. speltoides, Ae. aucheri, and Ae. longissima had H-type subunits. Therefore, section Sitopsis of Aegilops exhibits interspecific heterogeneity. The H-type is believed to have originated in the Sitopsis section from an L-type subunit because of the prevalence of the latter among the diploid species.     

Intra- and interspecific phylogenetic relationships among diploid Triticum-Aegilops species (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences

This study analyzes intra- and interspecific variation in chloroplast DNA (cpDNA) in diploid Triticum-Aegilops species. This analysis focused on DNA sequence variation in noncoding regions of cpDNA, which included base-pair substitutions, insertion/deletions (indels, 50 loci pooled), microsatellites (7 loci pooled), and inversions. Nine of 13 Triticum-Aegilops species were successfully identified and genotyped using these data. Sixty-two haplotypes were detected in 115 accessions of 13 diploid species. Because of the large number of characters examined, novel deep relationships within and among Triticum-Aegilops species could be identified and evaluated. Phylogenetic trees for the genus Triticum-Aegilops were constructed with Hordeum vulgare and Dasypyrum villosum as outgroups, and the results were compared to previous studies. These data support the following inferences: (1) Aegilops species should be included in Triticum; (2) groups D, T, M, N, U, and section Sitopsis (except Ae. speltoides) underwent speciation concurrently, but most diploid species evolved independently; (3) Ae. mutica does not occupy a basal position in Triticum-Aegilops; (4) Ae. speltoides is in a basal position and differs significantly from other Sitopsis species; (5) Ae. caudata is polyphyletic in all trees; (6) the genus Aegilops is paraphyletic with Secale.