兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

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。     

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。     

中国东北落叶松属植物亲缘关系的研究

本文用RAPD方法对中国东北落叶松属植物的亲缘关系进行了研究。从100个引物中筛选出41个引物,检测出120个多态位点。应用UPGMA法计算了种群内和种群间的遗传距离,并构建了系统树。实验结果表明,分布在长白山的落叶松与分布在大、小兴安岭的落叶松之间的差异,在遗传距离上还不能达到种间分化水平。分布在长白山的落叶松应视为兴安落叶松种下的变种,分布在东京城的落叶松可能是兴安落叶松与长白山落叶松的杂交种。     

利用正交设计优化兴安落叶松RAPD-PCR反应体系

以兴安落叶松针叶DNA为模板,对影响落叶松RAPD-PCR 扩增的重要参数进行了优化试验,以期建立兴安落叶松RAPD PCR反应的最佳体系。通过采用正交设计L₁₆(4⁵)对兴安落叶松RAPD-PCR反应的5因素(Taq酶、Mg²⁺、dNTP、模板DNA、引物)在4个水平上进行优化试验,结果表明兴安落叶松最佳的RAPD-PCR的反应体系(20 μL)中含有模板90 ng,0.5 μmol·L^-1的引物,1×反应缓冲液,DNTP各为0.25 mmol·L^-1,1 U的Taq DNA聚合酶,Mg²⁺ 2.5 mmol·L^-1。在此基础上筛选出20个扩增稳定、多态性丰富的RAPD引物,并通过梯度 PCR试验,确定了引物最佳退火温度。     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

与棱果沙棘性别相关的RAPD标记

应用RAPD技术筛选与棱果沙棘性别相关的分子标记,对棱果沙棘雌雄株的基因组DNA进行混合分组分析（BSA）,在194条随机引物中有50条引物能够在雌雄DNA反应池间形成多态性条带,应用这50条引物分别对棱果沙棘雌雄个体（雌雄个体各选取5个）进行RAPD分析,其中引物S10扩增得到1个约为1030 bp的与雌性相关的RAPD标记。该标记的获得进一步表明棱果沙棘雌雄株间存在基因水平的差异,为棱果沙棘的性别研究提供分子依据。进一步利用该雌性特异位点设计出更加稳定的SCAR标记,可望用于棱果沙棘的早期性别的准确鉴别。     

四种落叶松种子球蛋白免疫血清学的初步研究

本文对兴安落叶松、长白落叶松、华北落叶松和新疆落叶松种子的球蛋白进行了免疫血清学研究。通过双扩及免疫电泳分析,结果表明四种落叶松之间的抗原距离为零。这可提示用球蛋白进行免疫血清学方法不能鉴别这四个种。     

利用RAPD法对虹鳟鱼基因组DNA多态性研究初报

本文通过利用RAPD（随机扩增多态DNA）方法,选用20种10bp的随机引物（OPC－01－OPC—20）,用于虹鳟鱼基因组DNA多态性分析,其中发现一种引物（OPC－02）在虹鳟鱼群体中能扩增出多条带,说明该引物能反映其基因组DNA的多态性,可用于检测出不同品系间的差异。     

RAPD法对罗曼蛋鸡基因组DNA多态性分析初报

利用RAPD（随机扩增多态DNA）方法,选用37种10bp的随机引物,对罗曼蛋鸡基因组DNA进行多态性分析,发现其中两种引物（OPS－08,OPY－06）在三代7个品系中都能扩增出多条带并且反映其基因组DNA的多态性,可用于检测出不同品系间的差异,并且这两种引物的碱基序列有80％是对应互补的。     

Seed-specific identification of Larix gmelinii,Larix olgensis,and Larix principis-rupprechtii using sequence-characterised amplified region markers

Larix gmelinii, Larix olgensis, and Larix principis-rupprechtii are the three native and sympatric larch species in North China, and each of these species has a distinctive ecological niche. It is difficult to identify them based only on certain morphological characters, particularly the seed appearance. In this study, the seed endosperms of these three larch species were analysed using the random amplified polymorphic DNA (RAPD) technique to screen for interspecific differences. The following three RAPD markers linked to species-specific segments were observed in the different species: 1475-bp (Larix gmelinii and L. olgensis), 505-bp (Larix principis-rupprechtii), and 1121-bp (Larix gmelinii) markers. The three seed-specific fragments amplified by the RAPD markers were sequenced, and the sequences were used to design and synthesise species-specific SCAR markers. The size of the SCAR fragments was concordant with that of the RAPD species-specific fragments. Therefore, these SCAR markers can be used to identify the seeds of different larch species, thereby providing a new molecular tool for the identification of larch seeds that leads to considerable savings in terms of time and economic resources.     

小叶杨Δ~1-吡咯琳-5-羧酸合成酶(P5CS)基因克隆及在杂种落叶松中的转化

以小叶杨为材料构建了干旱胁迫和正常生长条件下的cDNA文库,以特异性引物从中扩增出一条1 850bp大小的DNA片段,经序列分析证实该片段编码Δ1-吡咯琳-5-羧酸合成酶(P5CS)。将该片段构建入植物表达载体pBI121中,在落叶松杂交育种中利用花粉管通道法将带有该P5CS基因的植物表达质粒转化入杂种落叶松,收获球果取出种子,提取转化种子发芽长出幼芽的DNA,特异性PCR扩增和Southern,Western Blotting检测证实落叶松中已导入P5CS基因。     

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers

In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.     

Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.     

Chloroplast and mitochondrial molecular tests identify European×Japanese larch hybrids

Hybrids between European and Japanese larches combine the properties of both parental species (drought resistance, canker resistance, stem straightness) and exhibit a fast growth rate. They are produced in seed orchards, generally by natural pollination. Seeds are collected and used for afforestation as interspecific hybrids. However, there are no convenient tests to assess the interspecific hybrid proportion. In the present study, we developed diagnostic molecular markers suitable for the individual identification of hybrids, whatever their developmental stage. Our strategy involved testing a combination of maternally inherited markers from the mitochondrial genome (mtDNA) and paternally inherited markers from the chloroplast genome (cpDNA). Hybrids were then identified by the presence of a mitochondrial sequence inherited from one parental species and a chloroplast sequence inherited from the other parental species. To achieve this aim, markers discriminating both parental species were first sought. Amplifications of mitochondrial and chloroplast sequences were performed using specific PCR primers. After testing 33 primer pairs in combination with nine restriction enzymes, we detected one mitochondrial marker, f13 which was amplified in Japanese larch and absent in European larch, and one chloroplast marker, ll-TaqI which showed different restriction patterns depending on the species. A restriction fragment of 601 bp was obtained in Japanese larch while two fragments of 120 bp and 481 bp were observed in European larch. These patterns were found in all 197 individuals tested from the two pure species. These markers were then used for the evaluation of the hybrid proportion in a seed lot produced from seed orchards; this was assessed as between 43% and 53% depending on the parental species. The male and female parental species could be determined for each progeny.Communicated by D.B. Neale     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

芦笋雌性特异性片段的克隆和分析(简报)

芦笋(Asparagus officinalis L.)又名石刁柏、龙须菜,系雌雄异株宿根性植物,是重要的经济作物之一。芦笋的性染色体为一对同形的L5染色体,雌性的性染色体为XX,雄性的性染色体为XY。性别决定的多态性是由位于一对L5性染色体上的一个显性基因M决定的[1-3],雌株基因型为隐性纯合子mm,雄株为显性纯合子MM(又称超雄株)或杂合子Mm。在生产上,由于雄株比雌株高产25%以上[4],并具有极强的抗病性和生命力,故雄株特别是超雄株则倍受生产者的青睐,但芦笋雌雄鉴定只有等到种植的第二年待植株开花时才能进行,这就严重影响了芦笋的有目的种植和经济效…     

Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP

Two high-resolution genotypic techniques (RAPD-PCR and AFLP) were evaluated for their possibility to discriminate the species Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum and to type these taxa at the infra-species level. In total 23 strains of L. plantarum, three strains of L. pentosus, two strains of L. paraplantarum and two related strains for which the species assignment was not clear, were studied. For RAPD-PCR, suitable oligonucleotides and amplification conditions were selected and tested. For AFLP, a double digest of total genomic DNA was used and a subset of restriction fragments was selectively amplified and visualised using different primer combinations. Both methodologies generated, species-specific electrophoretic profiles. Moreover, the presence of distinct subgroups was revealed within the species L. plantarum.