藜麦EST-SSR的开发及通用性分析

藜麦因营养均衡受到越来越多的关注,但尚未深入开展其基础研究。开发微卫星序列重复SSR分子标记将为藜麦的遗传分析提供重要资源。本研究利用NCBI数据库中藜麦RNA测序RNA-Seq及表达序列标签EST数据挖掘、验证及评价藜麦EST-SSR,共发现1862个藜麦非单核苷酸EST-SSR。其中,二核苷酸重复最多(38.3%),六核苷酸重复最少(11.7%)。不同重复类型SSR的数量随着核苷酸数目的增加呈下降趋势。在随机选取验证的119个EST-SSR标记中,66 (55.9%)个能够扩增出清晰条带,39个在4份藜麦资源中具有多态性,且其多态性与重复序列长度不具有显著相关性。t测验显示,多态性EST-SSR在藜麦与其他藜科种质间不存在显著差异,说明其具有良好的通用性,可用于藜科物种的遗传关系分析。     

ACGM标记在竹子中的通用性

用39对ACGM引物对竹亚科5个属13个种进行PCR扩增,结果共有34对引物可以在至少一个竹种中获得特异性PCR产物。利用PHILIP软件,得到13个供试竹种材料的聚类图,总体上刚竹属是聚在一起的,而大明竹属的华丝竹和箭竹属的箭竹分别各自成类。其中有差别的是赤竹属的菲白竹和箬竹属的阔叶箬竹与刚竹属发生聚类在一起的情况。从刚竹属内部的聚类结果看,9个种可以大致分为4类,毛环竹、黄槽石绿竹、迟竹和刚竹为一类;毛芽竹和早园竹为一类;花竹和乌哺鸡为一类;红壳竹自成一类。他们之间的亲缘关系可以简单表示为:(((毛环竹 黄槽石绿竹 迟竹 刚竹) (毛芽竹 早园竹)) (花竹 乌哺鸡)) 红壳竹。本研究结果表明ACGM遗传标记可以有效地用于竹子的遗传分类及相关的分子生物学研究。     

Development and characterization of SSR markers in Chinese jujube (Ziziphus jujuba Mill.) and its related species

In order to study the extensively genetic diversities of more than 700 cultivars of Chinese jujube, it is necessary to utilize various informative DNA markers. SSR markers are highly polymorphic, co-dominant, locus-specific markers widely used in genetic studies, but less used in Chinese jujube because of no specific primers available. In this study, we used the approach of selectively amplified microsatellite (SAM) to develop SSR markers for Chinese jujube and its related species. Three cultivars (Dongzao, Dalilongzao and Jinsixiaozao) were selected to perform the approach of SAM with CT repeats. There were totally 25 primers obtained, of which we selected 16 primers available to detect the polymorphism in populations of 24 Chinese jujube cultivars, two wild jujube varieties and two Indian jujube cultivars. Based on these primers, genetic relationships of the 28 samples were constructed in a dendrogram according to the UPGMA cluster analysis. The samples were clustered into three main groups, including Chinese jujube, wild jujube and Indian jujube as expected. The 16 sequence-specific SSR primers could efficiently distinguish all the 24 cultivars of Chinese jujube, except for two cultivars, Jinsixiaozao and its ‘stoneless’ mutant, Wuhexiaozao. As a result, SAM was a very efficient method in targeted developing sequence-specific SSR primers in Chinese jujube. Furthermore, SAMs could also be used as high polymorphic molecular markers independently. The further study would focus on developing other oligonucleotide repeat types and applying more SSRs available in the genetic research of Chinese jujube.     

冰草EST-SSR引物和小麦EST-SSR引物在黑麦属基因组的通用性分析

为研究黑麦属植物的遗传多样性,开发R基因组特有的分子标记并绘制其遗传连锁图谱,选用1 343对冰草EST-SSR引物和786对小麦EST-SSR引物对新疆杂草黑麦和栽培黑麦(共计6份材料)的全基因组进行了PCR扩增,结果显示,有679对冰草EST-SSR引物能够扩出清晰的条带,占引物总数的50.6%;其中有187对引物在6份黑麦材料基因组中扩增产物表现为多态性,占其引物总数的13.9%,平均每对引物扩增条带数为1.1。有364对小麦EST-SSR引物可扩增出清晰的条带,占其引物总数的46.3%;其中有135对引物在6份黑麦材料基因组中扩增产物具有多态性,占其引物总数的17.1%,平均每对引物扩增条带数为2.0。冰草EST-SSR引物在黑麦中的有效扩增效率高于小麦EST-SSR的有效扩增效率,但扩增多态性后者大于前者。两种来源引物扩增强带比率分别为51.8%和32.6%。结果表明小麦和冰草的EST-SSR引物均可用于黑麦基因组分析研究。     

Development of microsatellite markers in cultivated vanilla: Polymorphism and transferability to other vanilla species

The sources of natural vanilla are the cured fruits of two obligatorily hand-pollinated and clonally propagated orchids: ‘Bourbon/Mexican vanilla’ (Vanilla planifolia G. Jackson) and ‘Tahitian vanilla’ (Vanilla tahitensis J.W. Moore). In this paper we describe for the first time the isolation and characterization of 14 microsatellite loci from V. planifolia. These were monomorphic within cultivated accessions, as expected from the probable single clonal origin of this crop and previous genetic studies. These markers were transferable to V. tahitensis and 11 loci were polymorphic between these two closely related species. Furthermore, some of these markers were transferable and polymorphic across 15 other wild American, African and Asian species and revealed consistent relationships between species, together with a strong pattern of Old World versus New World differentiation in the genus. These microsatellites will be very useful for diversity, hybridization and phylogeographic studies in the genus Vanilla.     

楸树SSR标记在滇楸中的可转移性与应用

利用开发的13对楸树SSR引物,对其在近缘物种滇楸中的可转移性与应用进行研究。将13对楸树SSR引物对75份滇楸无性系进行PCR扩增,所使用的引物对滇楸的扩增成功率与引物多态性比例均为100%,表明楸树SSR标记对近缘种滇楸具有较高的可转移性和多态性。共检测到76个多态位点,平均有效等位基因数(Ne)为2.364 4个,平均期望杂合度(He)为0.550 2,Nei’s基因多样性指数(H)为0.013 2～0.833 5,平均值0.446 8,结果显示滇楸无性系中遗传差异处于中间水平,各无性系之间亲缘关系较近。UPGMA聚类将75个无性系划分为5个类群,类群中地域性聚集不明显、亚群地域性聚集较明显。     

茄子SSR标记在番茄上的通用性分析

微卫星标记(SSR)是分子育种中常用的遗传标记之一,但目前报道的番茄SSR标记并不能满足番茄分子育种的需要,有必要发展出更多的SSR标记,近缘物种转移法是一种发展SSR标记简便快速的方法。本研究分析了茄子SSR标记在番茄上的通用性情况,结果表明:300对茄子SSR引物中有111对能在番茄基因组DNA上扩增出产物,97对引物扩增出的带型在茄子、番茄间相似程度高,标记通用率为32.3%;EST-SSR比基因组SSR的通用性更好,前者通用率为36.6%,后者为30.0%。将这些标记转移到番茄上,能节省开发番茄SSR标记的成本。     

IncK plasmid-mediated tetracycline resistance in Edwardsiella ictaluri isolates from diseased freshwater catfish in Vietnam

Eight tetracycline resistant Edwardsiella ictaluri isolates obtained from diseased freshwater catfish (Pangasianodon hypophthalmus) in Vietnam, and showing different resistance phenotypes to other antimicrobial agents, were studied. The tet genes were determined using PCR. Conjugation experiments were performed to assess transferability of the tetracycline resistance determinant and the size and incompatibility group (Inc) of each tet-carrying plasmid were determined. PCR and sequencing were used for characterization of the co-transferred resistance genes. A tetA gene was demonstrated in the E. ictaluri isolates and for all of them, Escherichia coli transconjugants were obtained. All transconjugants contained high-molecular weight tetA-carrying plasmids (~ 140 kb) belonging to the incK group, as was shown with the PCR-based replicon typing method. The strA–strB, dhfr1 and sul 2 genes were detected on the tetA-carrying plasmids of the transconjugants showing resistance to streptomycin, trimethoprim and sulfonamides, respectively. The dhfr1 gene was found to be located in a class 1 integron as determined by PCR and sequencing. Interestingly, the 3′ CS region of class 1 integrons was not detected by PCR. This study shows the presence of incK plasmid-mediated tetracycline resistance among E. ictaluri isolates from diseased freshwater catfish in Vietnam.     

棉花非冗余性EST-SSR新标记的开发及其评价

利用Clustal X等软件对公共数据库现有的393 753条棉花EST序列分析,得到349 815条非冗余EST序列,借助自主开发的SSRmine软件共发掘SSR位点11 372个,分布于10 507条EST中,EST-SSR的频率是3%,平均相隔21 kb出现一个SSR。在2~6 bp的重复基元中,三核苷酸和六核苷酸分别占34.1%、40.6%,二、三、四、五和六核苷酸基序分别以AG/CT、AAG/CTT、AAAT/ATTT、AAAAG/CTTTT和AAAAAG/CTTTTT的类型最多。利用去冗余的且在亚洲棉、陆地棉、海岛棉中没有被开发过的410条EST序列设计开发了200对非冗余性SSR引物,利用自主开发的SSRD软件通过SSR引物序列下载、预处理、Blastn、提取相似性分值≥81%的引物编号、提取引物冗余对、冗余引物写成一行等6个步骤去除来源于自身部分同源序列以及与CMD释放的不同棉种相似性SSR引物,得到了非相似性引物,定名为CRIXXX (CRI即Cotton Research Institute)。并分别选用棉花12个种的代表性材料对其中100对进行引物功效评价,包括多态信息含量(polymorphism information content, PIC)及引物通用性研究。结果显示,从自主开发的100对SSR引物筛选出56对均能在12份材料间扩增出稳定明显的条带, 其中多态性引物35对,多态率占35%。引物的PIC变幅为0.097~0.888,平均为0.482;1对海岛棉EST-SSR引物在12份材料间的通用性为100%,25对亚洲棉引物通用性为81%,74对陆地棉引物通用性为80.1%。