Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of a repeat sequence of rye DNA in wheat and related species

Polymerase chain reaction (PCR) and enhanced chemiluminescence (ECL) were used to determine the distribution of the rye-specific sequence contained in the pSc119.1 probe among wheat and related species. A specific pair of primers targeting this rye-specific sequence was used. A 745-bp fragment, the predicted size of pSc119.1, was present inSecale cereale, Triticum aestivum, XTriticosecale, Hordeum vulgare, H. bogdanii, andH. parodii. PCR results were verified by hybridizing the rye-specific probe pSc119.1 to dotblots of DNA from the different species used. Strong hybridization signals detected by ECL were consistent with the PCR results. The results demonstrate the effectiveness of PCR and dot-blot ECL in screening plants for defined DNA sequences, and indicate that pSc119.1 has counterparts with strong homology inT. aestivum, H. vulgare, H. bogdanii, andH. parodii.     

A polymorphic microsatellite in the limit dextrinase gene of barley (Hordeum vulgare L.)

A DNA fragment containing the exons 16, 17 and intron 16 of the limit dextrinase gene was cloned using a 654 bp cDNA as probe. Intron 16 contained a simple sequence repeat (microsatellite). PCR primers were designed to amplify that microsatellite. Using these primers, the limit dextrinase gene was mapped to the short arm of chromosome 1 (7H) using 150 DH lines from the Steptoe × Morex mapping population. This gene co-segregated with the RFLP marker ABC154A. QTLs for malt extract, -amylase activity, diastatic power and fine-coarse difference previously mapped in the North American Barley Genome Mapping Project have been located in this chromosome region. Five limit dextrinase alleles were detected in 31 barley cultivars with a PIC of 0.75. Ten different alleles/genes were identified in 23 uncultivated Hordeum species or subspecies using these microsatellite primers. The primers also amplified one fragment from wheat and two from oat. This microsatellite should be useful for marker-assisted selection for malting quality.     

The use of RAPD markers in Hordeum phylogeny.

The phylogenetic relationships among 39 wild Hordeum species, subspecies, and cultivated barley were investigated using RAPD markers as discriminating characters. Seventy-six RAPD fragments were generated using 12 single decameric primers of arbitrary nucleotide sequences. Amplification reactions resulted in fragments ranging in length between 200 and 2000 bp. Clearly resolved bands were scored for their presence or absence in a binary matrix. Amplified products were treated as independent characters to generate a phenogram using the NTSYS-PC package. Tree topology was generally found to be consistent with those based on morphological treatments. However, a few species like H. erectifolium, H. jubatum and, to a lesser extent, H. bulbosum occupied a position different from previous classifications. The results demonstrated that RAPD technology represents a useful and reliable tool for detecting polymorphism for phylogenetic studies. Key words : RAPD analysis, molecular markers, phylogenetic studies, Hordeum species, barley.     

Genomic fingerprints in the tribe Triticeae produced by PCR using a tRNA consensus primer

Forty-one accessions belonging to ten genera of the tribe Triticeae representing both wild and cultivated species were analyzed by polymerase chain reaction (PCR). Of two consensus tRNA primers tested, one primer revealed characteristic amplification products for all of the species. A total of 35 tRNA markers were scored across all accessions. Five genus-specific and three species-specific markers were obtained. Genomic fingerprints were largely conserved within a genus. The phylogram obtained using parsimony has separated most of the accessions into their prevailing taxonomic species and genus groups. The phylogram showed close association among the three genera Secale, Triticum and Hordeum as expected. The Triticum-Secale relationship was closer than the Triticum-Hordeum or the Secale-Hordeum relationships. The tree also reflected the close associations among the forage grass species belonging to Leymus and Elymus. Thus tDNA-PCR helped to identify species and genera. This revised version was published online in July 2006 with corrections to the Cover Date.     

菠菜CBF转录因子基因片段的克隆及序列分析

根据拟南芥CBF基因序列的保守区设计合成一对特异引物,以菠菜基因组DNA为模板,采用PCR扩增的方法扩出一条DNA特异片段并克隆到pMD18-T载体中。用PCR法和酶切分析法对克隆片段进行鉴定并进一步进行核苷酸序列分析。序列测定该片段长为423bp。OMIGA2.0软件分析结果表明,该片段的推断氨基酸序列与黑麦(AAL35759)、小麦(AAL37944)、拟南芥(AAC78646)、大麦(AAL84170)的同源性分别为33.8%、33.1%、30.8%和30%。     

Genomic organization, evolution, and structural peculiarities of highly repetitive DNA of Hordeum vulgare

A fraction of highly repeated DNA sequences of Hordeum vulgare has been investigated by cloning 19 separate highly repetitive sequences in the plasmid pBR327. Characteristics studied included genus specificity of isolated sequences, their prevalence, and genome organization. Sequences (pHv7161, pHv7191, pHv7179) have been identified that are the most widespread in the H. vulgare genome and have a complicated arrangement. A tandemly arranged sequence, pHv7141, was also identified. The primary structure of a 999 bp long, BamHI fragment of one of the most widespread sequences, pHv7161, as well as the adjacent pHv7302 and pHv7245 sequences was determined. The fragment abounds in inverted repeats, of which two are flanked by direct repeats, and contains short subrepeats, A, B, and C, and a great variety of potential protein-binding sites. A comparison is drawn between the content and genome organization of highly repeated DNA sequences of H. vulgare and those of the wild barley species Hordeum bulbosum, Hordeum jubatum, Hordeum geniculatum, Hordeum brevisubulatum, Hordeum turkestanicum, and Hordeum murinum. According to the above characters (close copy number and genome organization similarity of highly repetitive sequences) the species under discussion have been classified into four groups. This division is in good agreement with other data on interspecific crossing in Hordeum and on chromosome pairing in hybrid meiosis.     

大豆疫霉菌ITS分子检测程序的建立及其应用

依据GenBank中登录的大豆疫霉菌(Phytophthora sojae)、近缘种及相似种rDNA的ITS区序列差异,进行多重比较后设计合成一对大豆疫霉菌特异引物,并在PCR反应体系和扩增条件优化的基础上,对包括大豆疫霉菌在内的共140个菌株进行PCR检测。结果表明,电泳后只有大豆疫霉菌扩增出一条288bp的特异性条带。运用设计的大豆疫霉菌专用引物(专利申请号200610089105.4)及建立的检测程序对大豆疫霉菌纯培养游动孢子、接种于土壤中的游动孢子和卵孢子以及接种发病的大豆染病组织进行了检测应用,结果显示该检测程序对接种于土壤中的大豆疫霉菌游动孢子和卵孢子的检测理论精度分别达0.3和0.06个孢子,对染病组织检测也表现出了较高的灵敏度。     

落叶松种间及无性系间ISSR鉴别技术的研究

采用ISSR分子标记技术对兴安、长白和日本落叶松种间以及不同无性系进行了鉴别。从49条引物中筛选出13条ISSR引物可以对落叶松种间和无性系间进行鉴别,特异条带个体的百分率为100%,该项技术为落叶松新品种以及良种的准确鉴别提供了新的途径和手段：其中5条引物在日本落叶松、兴安落叶松和长白落叶松不同位置扩增出特异谱带,作为种的鉴定的标准,有9条引物可以对落叶松种内不同无性系分别扩增出特异片段,进行无性系的鉴别。     

AFLP 引物组合数量对准确研究竹子系统关系的影响

利用AFLP技术对26个竹子种类进行了多样性分析, 以探索引物组合数量对准确研究竹子类群系统关系的影响。实验共随机选取10对AFLP引物, 并对所得10组AFLP标记数据随机组合后进行Nei氏遗传距离/UPGMA聚类分析。每对AFLP引物 扩增数据为一组, 随着用于聚类统计的AFLP标记数据随机组合数量的增加, 26个竹子种类的聚类关系趋向一致。这提示我们,在系统学研究中, 足够数量的引物组合是获得供试材料间准确聚类关系的基础, 应采用对各AFLP引物组合数据随机累加后进行聚类分析的方法, 以聚类关系为标准来确定用于分析供试品种的最少引物组合数量。     

商陆高亲和性K＋转运体基因PaHAK1的克隆与表达分析

为了研究高钾植物商陆高亲和性K＋吸收机制,本试验选用野生商陆为材料,以拟南芥、冰叶日中花、小麦、大麦和水稻等多种植物的HAK家族基因的保守序列,设计简并引物,获得了981bp的PaHAK1的基因片段。然后应用RACE技术克隆到PaHAK1基因序列,其cDNA全长为2337bp,编码771个氨基酸。该编码蛋白质分子量为86．66kDa,等电点为7．54。蛋白质疏水性和拓扑结构分析显示该氨基酸序列共有12-13个跨膜区。该cDNA序列与冰叶日中花HAK1基因序列相似性高达88％。Real-TimePCR分析表明PaHAK1主要在商陆根中表达,低钾状态可以诱导PaHAK1的高表达。     

Molecular markers for the identification of the ectomycorrhizal fungus Tuber borchii

Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle.     

Species relationships between antifungal chitinase and nuclear rDNA (internal transcribed spacer) sequences in the genus Hordeum.

The sequences of the chitinase gene (Chi-26) and the internal transcribed spacer of 18S - 5.8S - 26S rDNA (ITS1) were determined to analyze the phylogenetic relationships among species representing the four basic genomes of the genus Hordeum. Grouping analysis based on data for Chi-26 gene sequences placed Hordeum secalinum (H genome) near the Hordeum murinum complex (Xu genome), and Hordeum bulbosum distant from the other species that carried the I genome. ITS sequence data showed the expected grouping based on the genome classification of the species studied. Different sequences of ITS were detected even in the genomes of the diploid species. The results are interpreted in terms of defective or unfinished concerted evolution processes in each taxon.     

Molecular Characterization and Phylogenetic Analysis of Hoplolaimus stephanus

Three Hoplolaimus stephanus populations were characterized morphologically, both by morphometrics and by SEM observations. These populations were used to develop a rapid and accurate molecular identification method for the species, which is useful because of the high level of morphological similarity between H. stephanus and H. galeatus. Species-specific primers for H. stephanus, amplifying a distinct fragment (260 bp) of the nuclear ribosomal internal transcribed spacer1 (ITS1), can be used in multiplex PCR along with previously developed primers for other common Hoplolaimus species. We also infer phylogenetic relationships among H. stephanus, the closely-related H. galeatus, and several other Hoplolaimus species, using sequences of the actin gene, ITS1 and LSUD.     

中国红树科7种红树植物遗传多样性分析

以红树、红海榄、秋茄、角果木、木榄、海莲、尖瓣海莲等7种红树科植物为材料,采用改进的CTAB法获得了纯度较高、得率高、片段完整的基因组DNA。通过筛选出的15个有效引物进行RAPD分析,探讨了7种树植物间的亲缘关系。15个有效引物共扩增出617条DNA带,其中多态性条带415条,占总扩增条带的67.26%。利用Nei指数法得出7个分类群间的遗传一致度和遗传距离,并运用UPGMA法进行聚类分析。7个分类群分为A、B两个大组,平均遗传距离为0.41。将得出的7个分类群的DNA分子分类系统图,与传统的分类进行比较,发现结果相符。同时获得一个OPG05－900的差异片段可作为区分海莲和尖瓣海莲的分子标记。     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。