猕猴桃雄性基因RAPD标记S1032-850的获得及其应用

利用RAPD技术对美味猕猴桃(Actinidia deliciosa var.deliciosa)海瓦德×秦雄201杂交F1代雌雄分离群体进行分析,获得了与雄性基因链锁的RAPD标记.通过对300个随机引物的筛选和研究,得到了与猕猴桃雄性基因链锁的RAPD标记S1032-850,并在杂种后代、中华猕猴桃(A.chinensis var.chinensis)和美味猕猴桃雌雄个体上进行了验证.进一步对猕猴桃雄性RAPD标记S1032-850回收、克隆和测序,获得了该标记的核苷酸特定序列,由867bp的特定碱基序列组成,该序列片段已被GenBank收录,登陆号为AY336259.     

与南瓜矮生基因连锁的分子标记研究

本研究以世界上首次发现的中国南瓜矮生突变体（矮10）为供体亲本，以印度蔓生南瓜（蒙日南瓜）为轮回亲本，经6代回交2代自交选育出S1—S9共9个南瓜矮生近等基因纯合株系，其中S2株系再与蒙日南瓜杂交获得F2代分离群体共306株。利用RAPD技术, 采用近等基因系（Near isogenic lines, NILs）分析法，对上述材料进行分析。在640条随机引物中，发现RAPD标记S1225-548与矮生单基因D呈现连锁关系，遗传距离为2.29cM。并且本研究进一步将此RAPD标记成功转化成更为稳定的SCAR标记SCAR3-398。     

家蚕胚胎温敏性基因的RAPD标记筛选

用家蚕华1、S栏品种及其杂交后代F2分离群体为材料进行胚胎湿敏性基因的RAPD分子标记筛选，经287个随机引物的PCR扩增产物电泳分析，筛选到2个特异性DNA多态片段，一个只出现在温敏性赤蚁蚕品种中。另一个出现在非湿敏黑蚁蚕品种中，扩增条带的分子量约1350bp，命名为OPA－021350，经共分离的检测分析，与黑蚁非温敏性状有较紧密的连锁关系，并以pEGM^R-T Vector为载体，大肠杆菌DH5α为宿主进行了克隆。     

甘蓝型油菜陕2A细胞质雄性不育的遗传及RAPD标记

本研究以甘蓝型油菜陕 2A细胞质雄性不育系、保持系和F2 分离群体为材料 ,对F2 分离群体的遗传分离情况进行分析 ,结果表明 ,可育与不育株花器存在明显差异 ,符合 3∶1的分离比例 ,因而推断细胞质雄性不育恢复基因受 1对显性基因控制。利用分离群体分组分析法(BSA) ,用 1 0 5个随机引物对细胞质雄性不育恢复基因进行RAPD分析 ,发现有 6个随机引物扩增出多态性差异谱带。引物S62 和S74在可育和不育基因池中扩增出单条特异性谱带所代表的DNA序列 ,很可能与不育系的恢复基因连锁。     

与南瓜矮生基因连锁的分子标记

以中国南瓜(Cucurbita moschata Duch)矮生突变体(矮10)为供体亲本,以印度蔓生南瓜(C.maxima Duch)(蒙日南瓜)为轮回亲本,经6代回交和2代自交选育出S1~S9共9个南瓜矮生近等基因纯合株系,其中S2株系再与蒙日南瓜杂交获得F2分离群体共306株。利用RAPD技术,采用近等基因系(near isogenic lines,NILs)分析法,结果表明,在640条随机引物中,发现RAPD标记S1225-548与矮生单基因D呈现连锁关系,遗传距离为2.29cM。RAPD标记成功转化成更为稳定的SCAR标记SCAR3-398。     

稻瘟菌无毒基因簇的RAPD标记及其SCAR转化

为克隆稻瘟菌无毒基因簇Avr-Pi1、Avr-Pi2和Avr-Pi4a,以RAPD方法对稻瘟菌(Magnaporthe grisea）菌株FJ81278ZB15、GUY11及其F1后代群体进行扩增，得到2个与该无毒基因簇连锁的分子标记P1414700和P1389420 。对2个特异片段进行克隆和测序，并根据序列分别设计2对特异引物，对菌株FJ81278ZB15、GUY11及其F1后代群体进行扩增，扩增结果P1414700 成功转化为SCAR标记SC1414，而P1389420未能成功转化。对分子标记SC1414、P1389420和报道的RPF1.2与无毒基因簇Avr-Pi1、Avr-Pi2和Avr-Pi4a遗传连锁关系进行了分析，结果SC1414、RPF1.2、P1389420与Avr-Pi2的遗传距离分别为5.8、2.2 和4.4 cM；与Avr-Pi1的遗传距离分别为15.9、7.9和5.7 cM；与Avr-Pi4a的遗传距离分别为20.7、12.7 和10.5 cM。     

甘蓝型油菜矮秆突变体的遗传及其矮秆基因的RAPD标记

甘蓝型油菜(BrassicanapusL.)矮秆突变体DS-1与中双四号配制正反交组合F1、F2、BC1和BC2,遗传分析表明,该突变体的矮秆性状受一对部分显性主效基因和微效基因控制,将这对显性主效基因命名为Ds1。同时随机选取147株F2植株作为Ds1的RAPD标记的筛选群体,最终从1041条随机引物中筛选到1条引物S470,其扩增的多态性片段S470.416与Ds1连锁,遗传距离为8.9cM。     

甘蔗祖亲种RAPD标记的序列特征性片段扩增区域（SCAR）转化分析

采用RAPD标记技术获得了甘蔗(Saccharum)亲本种RSp2、RSp5、RSp6、RSo13和RSp16 5个RAPD标记特异片段.根据这5个特异片段的测序结果,设计了15对特征性片段扩增区域(SCAR)标记引物,并经SCAR标记特异引物筛选,ZT51、ZT52、ZT53、ZT55及ZT56为割手密种特异引物.RSp2这一甘蔗割手密种特异的RAPD标记被成功转化为割手密种(S.spontaneum)特异SCAR标记,记为ZT51-439.在甘蔗种质检测时,能在崖城割手密、印度割手密及部分云南割手密亲本种中特异检测出,并在具有割手密血缘的47份栽培种中均能检测到明显而唯一的439 bp割手密种特异条带.     

毛头鬼伞中一条新28S rRNA的发现及其同源性分析

本研究从担子菌毛头鬼伞（Coprinus comatus）菌丝中分离获得一条新的28S rRNA序列,序列长度为906bp（GenBank accession No.GU568178）。该序列是我们前期在从毛头鬼伞中克隆一种烟草花叶病毒（TMV）的抗性蛋白基因y3时意外获得的一条非目的条带。将此获得的序列通过NCBI的BLAST,以及与其同源序列进行Clustal w和MEGA聚类分析,证实该序列是28S rRNA,同时还发现毛头鬼伞的系统进化关系比较离散。此外,在这一新28S rRNA与TMV的抗性蛋白基因y3之间发现有两个同源区段有可能是PCR扩增y3基因时出现非目的条带的原因。在这两个同源区段中,其一区段与克隆y3基因时所用的PCR引物之一有较高的相似性,另一区段也是一般PCR引物的类似物。本研究中新28S rRNA序列的获得是PCR扩增中出现非目的条带的新例,该序列的发现及聚类分析的结果有助于真菌基因组学研究及真菌生物分子分类系统的建立。     

与大白菜TuMV抗病基因TuRBCS01紧密连锁的分子标记开发

分子标记辅助选择可大大加快育种进程,然而由于大白菜抗病毒病遗传规律的复杂性,目前所定位的基因和筛选的连锁标记远不能满足育种需要,为了更好地对抗病毒病白菜(Brassica campestris ssp.pekinensis)进行分子标记辅助选择,本研究筛选了与大白菜抗芜菁花叶病毒(Turnip mosaic virus,TuMV)抗性基因TuRBCS01紧密连锁的分子标记.本研究在对该基因进行定位的基础上,利用回交1代(backcross1,BC1)分离群体,采用分离群体分组分析法(bulked segregation analysis,BSA)、简单重复序列(simple sequence repeats,SSR)和序列特异引物(sequence specific primer,SSP)标记技术,筛选与该基因紧密连锁的分子标记.通过对13对SSP引物和16对SSR引物的分析表明,有8对引物在两亲本间表现多态性,有5对引物扩增出的标记与基因TuRBCS01紧密连锁或共分离,分别为mBr4072、Bra025493-1、Bra025493-2、Bra025467-4和Bra025467-5.筛选出与大白菜TuMV抗性基因TuRBCS01紧密连锁的分子标记2个,分别为SAAS_mBr4072_240 (1.5 cM)和Bra025493-1(1.0 cM),另有3个标记与基因TuRBCS01共分离,分别为SAAS—Bra025493-2_749、SAAS_ Bra025467-4—780和SAAS—Bra025467-5_ 956.上述标记丰富了大白菜抗TuMV分子标记的数量和种类,有望用于大白菜抗病毒病分子标记辅助选择.     

Distribution and biodiversity of soybean rhizobia in the soils of Shennongjia forest reserve,China

Soil samples were collected at an altitude of 500, 1,060, 1,500, 1,950, 2,400 and 3,100 m, respectively, from Shennongjia, a forest reserve in Hubei province (central China). Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. By using a plant trap method, a total of 25 soybean rhizobia were isolated from the soil above an altitude of 1,500 m and all identified to be Sinorhizobium fredii. Their genetic biodiversity was characterized by 16S–23S rDNA internally transcribed spacer (ITS) region polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and random amplification DNA (RAPD) analysis. All the tested strains produced a 2.1 kb 16S–23S rDNA ITS fragment. After digestion with three restriction endonucleases (HaeIII, MspI and CfoI), respectively, great variations in 16S–23S rDNA ITS PCR-RFLP patterns were observed. The tested strains could be differentiated into 11 ITS genotypes. The genotypes of rhizobia were not related to geographical location. Twelve primers were applied to RAPD analysis and a dendrogram was obtained, showing that all the strains (including reference strain S. fredii USDA205) were divided into two diverging groups. Moreover, each group could be further divided into two subgroups. Both RAPD and 16S–23S rDNA ITS PCR-RFLP analysis indicated that a high degree of genetic diversity existed among S. fredii strains isolated from Shennongjia virgin soils. Since Shennongjia is an unexploited forest region in central China and the gene centre of soybean is located in China, the symbiotic genes harboured by these strains may be of great importance and the rich diversity of these strains might contribute to the adaptation of soybean to an alpine environment.     

Microbial Diversity and Bioactive Substances in Disease Suppressive Composts from India

The present study aimed to investigate microbial communities in seven Indian composts and their potential for biocontrol of Fusarium oxysporum f. sp. lycopersici. In addition, identification of bioactive substances in disease suppressive composts was also attempted. Composts were chosen based on disease suppressiveness and subjected to molecular microbial analyses. Total genomic DNA from the composts was extracted and amplified with polymerase chain reaction using primers targeting the 18S rRNA and 16S rRNA genes of fungi and bacteria, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprinting and DNA sequencing were used to identify the fungal and bacterial targets. Phylogenetic analysis of the fungal 18S rRNA ITS gene sequences showed that phylum Ascomycota was dominant in all composts, while in the bacterial 16S rRNA gene sequences, the phylum Proteobacteria was dominant. Some fungi in disease suppressive composts grouped phylogenetically close to F. oxysporum. Bacterial sequences with close similarity (>95% identity) with Actinobacterium showed a strong presence only in disease suppressive composts. Disease suppressive composts formed a separate group in the cluster analysis of 18S rRNA ITS and 16S rRNA gene sequences. Gas chromatography-time of flight-mass spectrometry was performed with compost extracts to determine if bioactive substances were present in disease suppressive composts. The analysis of compost organic matter showed a negative association of disease suppressiveness with phloroglucinol, sitosterol, and monoenoic fatty acid, while cholesterol and certain organic acids were positively associated with suppressiveness.     

旱稻(O.sativa)、高粱(S.bicolor)属间远缘杂交种质创新研究

以旱稻基因型远FH 2-1〔旱稻65(O.sativa)/长芒稗(E.caudata)〕为母本、高粱基因型沈农133(S.bicolor)为父本,进行属间杂交旱稻新种质创造研究,分别在F1、F2获得双亲没有的紫色柱头、紫色芒、紫色护颖、紫色颖尖及糯质胚乳5种变异性状。上述性状均能稳定遗传,通过系谱法选育,已获得带有上述新性状的纯合高代品系,部分品系已直接用于旱稻抗逆、高产、优质新品种选育。     

Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship

Fungi belonging to the Cordyceps species have long been used as food and herbal medicines in Asia and are especially popular as commercially available powdered supplements. Despite this acceptance and use, little is known of the phylogenetic relationships of the genus. Presently, the neighbor-joining method based on the ITS1, 5.8S rRNA, and ITS2 regions was used to construct a phylogenetic tree of 17 Cordyceps isolates. Five major groups were evident. Cordyceps sinensis was less closely related to 15 Cordyceps species but shared a closer relationship with Cordyceps agriota. PCR-single-stranded conformational polymorphism was applied to differentiate seven Cordyceps isolates: five were different from those used to construct the phylogenetic tree, based on differences in the internal spacer 2 (ITS2). The length of ITS2, amplified by primers 5.8SR and ITS4, vary between 334 and 400 bp. This segment could be used for intraspecies classification or detection of mutations and represents potential novel means of identification of this fungal genus in herbal medicines and in quality control applications in the fermentation industry.     

小麦抗秆锈突变系龙辐03D51的筛选及其抗病性的遗传分析与RAPD标记

以龙6239幼胚为外植体,利用辐射诱变和组织培养相结合的方法,选育出突变系龙辐03D51,经秆锈接种鉴定,发现其对优势小种21C3CPH免疫,而亲本龙6239对21C3CPH高度感染。遗传分析表明,抗病性由显性单基因控制。该突变系还具有其亲本的优质、高产特点,已成为优异的后备品系。在RAPD检测中,所用的60个随机引物中有3个引物在龙辐03D51和其亲本龙6239中具有多态性,引物E07、E11、E17在龙辐03D51中分别扩增出380bp、700bp和600bp的特异带,初步认为这些谱带可能与秆锈抗性有关。     

Marker-assisted sampling of the cultivated Andean potato Solanum phureja collection using RAPD markers

The potato crop originated in the Andean highlands where numerous farmer's varieties and non-cultivated wild species exist. An Andean potato collection is held in trust at the International Potato Center (CIP) to preserve the biodiversity of this crop and ensure the supply of germplasm for potato improvement worldwide. A core collection representing the biodiversity of the Andean potato germplasm is under construction using morphological, molecular, and geographic data. One of the eight cultivated potato species, Solanum phureja, has been genotyped using the RAPD technique. A protocol suitable for large germplasm collection genotyping has been developed to process numerous samples at reasonable costs. From 106 RAPD primers evaluated, we have selected 12 primers yielding 102 polymorphic markers, which unambiguously discriminated all 128 accessions but 2 that are possible duplicates. The S. phureja germplasm collected throughout the Andean countries appears to have a homogeneous genetic constitution. There was no clear geographic pattern as indicated by cluster analysis of the RAPD data. A sub-group of 20 accessions has been identified on the basis of the marker data and selected to maximize molecular (RAPD) variance and polymorphism. The probability of capturing equal amounts of marker polymorphism in this sub-group of 20 accessions by random sampling is less than 40%. This set accessions represents our first group of accessions that may constitute a core of the S. phureja collection. This tentative core will be challenged for diversity content by alternate markers and agronomic traits. Hence, the methodology for sampling less than 10% of the base collection, proposed for core collections by Brown (1989), can be based on molecular marker data provided cost-efficient fingerprints are developed.     

Comparison of commonly used primer sets for evaluating arbuscular mycorrhizal fungal communities: Is there a universal solution?

Different primer systems have been developed to characterize arbuscular mycorrhizal fungal (AMF) communities; however, a direct comparison of their specificity, potential to describe diversity and representation of different phylogenetic lineages is lacking. Using seven root samples, we compared four routinely used AMF-specific primer systems for nuclear ribosomal DNA covering i) the partial small subunit (SSU), ii) the partial large subunit (LSU), iii) the partial SSU and internal transcribed spacer (ITS; “Redecker”) and iv) the partial SSU–ITS–partial LSU region (“Krüger”). In addition, a new primer combination v) covering the ITS2 region (ITS2) was included in the comparison. The “Krüger” primers tended to yield the highest AMF diversity and showed a significantly higher Shannon diversity index than the SSU primers. We found a strong bias towards the Glomeraceae in the LSU and SSU primer systems and differences in the composition of AMF communities based on the “Redecker” primer system. Our results confirm the crucial role of the choice of target rRNA marker region for analysing AMF communities. We also provide evidence that nested-PCR based data can be interpreted semi-quantitatively and that the extent of observed AMF community overdominance largely depends on the choice of primer.