RAPD and ISSR molecular markers in <Emphasis Type="Italic">Olea europaea</Emphasis> L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.     

Collection of Lupinus angustifolius L. Germplasm and Characterisation of Morphological and Molecular Diversity

Lupinus angustifolius L. is a Mediterranean species, domesticated in the 20th century, representing an important grain legume crop in Australia and other countries. This work is focused on the collection of wild germplasm and on the characterisation of morphological and molecular diversity of germplasm accessions. It reports the collection of 81 wild L. angustifolius accessions from the South and Centre of Portugal, available at the ‘Instituto Superior de Agronomia Gene Bank’, with subsequent morphological and molecular characterisation of a selection of these and other accessions. A multivariate analysis of morphological traits on 88 L. angustifolius accessions (including 59 wild Portuguese accessions, 15 cultivars and 14 breeding lines) showed a cline of variation on wild germplasm, with plants from Southern Portugal characterised by earlier flowering, higher vegetative development and larger seeds. AFLP and ISSR molecular markers grouped modern cultivars as sub-clusters within the wider diversity of wild germplasm, revealing the narrow pool of genetic diversity on which domesticated accessions are based. The importance of preserving, characterising and using wild genetic resources for L. angustifolius crop improvement is outlined by the results obtained.     

黑穗醋栗品种亲缘关系的ISSR分析

 采用ISSR标记技术对39个黑穗醋栗品种的亲缘关系进行分析。结果表明：13条ISSR引物扩增的总带数（A）从3到11条不等,平均扩增出6.77条带,平均多态性带百分比（P）为64.37%。聚类分析显示,在相似性系数为0.8496处将39个黑穗醋栗品种分为5组。第Ⅰ组包含8个起源国家的品种,说明世界各国之间基因交流较为频繁。‘Ben Lomond’和‘Kantata’同其他品种的遗传距离较远,两者又有诸多优良农艺性状,可作为黑穗醋栗的优良育种材料。     

基于ISSR标记初选格鲁桑类型桑树核心种质

核心种质可以用最少份数的种质资源代表该物种及生态类型的遗传多样性。以收集自我国黄土高原的73份格鲁桑类型桑树种质资源为材料,利用15个ISSR引物共扩增出129条带,其中多样性条带为115条,多态性比率为89.15%。根据ISSR分子标记聚类结果和树型图,利用逐步聚类随机取样法和属性约简启发式算法对73份种质资源进行核心种质构建研究,2种方法分别初选出21份和16份格鲁桑类型桑树核心种质,核心种质样本的比率分别为28.77%、21.92%。利用统计软件SPSS 13.0,结合分子标记多态性位点数、多态性位点百分率、观测等位基因数、有效等位基因数、Nei's遗传多样性指数和Shannon's信息指数对2组核心种质的遗传多样性进行评价和t检验,结果显示:采用2种方法初选出的21份和16份格鲁桑核心种质都能很好地保存初始群体的遗传多样性和结构,但采用属性约简启发式算法所得16份核心种质的代表性要优于采用逐步聚类随机取样法所得的21份核心种质。最终确定以属性约简启发式算法初选的16份样本作为格鲁桑类型桑树核心种质。     

引物浓度与退火温度对ISSR扩增片段大小的选择性

用ISSR法,在不同浓度与不同退火温度下,对长豇豆中基因组的不同分子量片段进行扩增,结果表明,引物浓度对扩增片段大小具有强烈选择性,对退火温度也具有一定的选择性。     

海南野生荔枝ISSR反应体系优化及应用

为获得最佳野生荔枝ISSR反应体系,用引物(AG)_8YC,采用单因子试验,优化了ISSR反应体系。结果表明,总体系20μL中,含1．0×(1．5 mmol/L Mg~(2 ))Buffer,0．1～0．15mmol/L dNTPs,0．2μmol/L引物,1．0～1．25U Taq酶,70ng DNA模板,不足体积用无菌超纯水补足。并利用该优化后体系对23条ISSR引物进行筛选,对33份野生荔枝样进行ISSR扩增,证实了该体系稳定可靠。     

Inter simple sequence repeat (ISSR) markers as a tool for the assessment of both genetic diversity and gene pool origin in common bean (Phaseolus vulgaris L.)

In this study, we report the use of ISSR to assess genetic diversity and to determine the relationships among ten cultivars of common bean developed in Argentina and three materials from France. ISSR markers resolved two major groups corresponding to the Andean and Mesoamerican gene pools of common bean. We compared the results of previous analysis, performed with RAPD markers (Galván et al., 2001), with the results generated by means of ISSR. It appears that ISSR are better tools than RAPDs to identify beans by gene pool of origin though they did not revealed as many differences between individuals as RAPDs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of a CSR10 (indica) × Taraori Basmati F3 population segregating for salt tolerance using ISSR markers

Segregation for salinity tolerance and ISSR markers based molecular polymorphism were investigated in a F₃ plant population raised via single-seed descent method from a cross between salt-tolerant indica rice variety CSR10 and salt-susceptible premium traditional Basmati rice variety Taraori Basmati HBC19. A total of 130 F₃plants were evaluated individually for salinity tolerance on 1–9 scale on the basis of seedling growth parameters; the average score ranged between 1.7 to 8.3. Frequency distribution curve obtained using the salinity tolerance data of F₃ population and a chi-square analysis, showed a good fit to a normal distribution. Eleven plants each in the category of salt-tolerant and salt-susceptible were selected from the segregating F₃ population for ISSR marker analysis. A total of 149 bands (4–11 bands per primer) ranging from 200 to 3530 bp were scored for the two rice varieties and the selected CSR10 × HBC19 segregating F₃ plants using 26 ISSR primers. Of these, 89 were monomorphic and 60 were polymorphic. Of the 60 polymorphic bands,36 and 20 bands were specific to CSR10 andHBC19 respectively. The remaining four bands were amplified using UBC primers 810,848, 853 and 886 and present in only some of the CSR10 × HBC19 F₃ plants. Notably, ISSR primers with dinucleotide repeat motif and 5'-anchored end amplified more number of bands (7.0 bands/primer) compared to3'-anchored dinucleotide primers (5.4bands/primer), but 3'-anchored dinucleotide primers revealed higher level of polymorphism (2.6 polymorphic bands/primer) compared to 5'-anchoreddinucleotide primers (1.43 polymorphic bands/ primer). While distribution of majority of the polymorphic bands were more or less in the expected ratios in salt-tolerant and/or salt-sensitive F₃segregating plants, but some of the bands amplified using UBC ISSR primers 823, 825,826, 849, 853, 864, 866 and 884 showed highly skewed distribution. Such polymorphic bands stand greater chances of having a linkage with the genes/ QTLs for salinity tolerance and shall be the target for further studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

苦瓜ISSR-CR反应体系的建立

以苦瓜（Momordica charantia L．）为试材，研究了PCR反应体系的主要成分及退火温度、循环次数对苦瓜ISSR扩增结果的影响。结果表明：在20μl的反应体系中，Mg^2＋的用量为1．2—1．6mmol／L，dNTPs浓度为0．2mmol／L，引物的浓度为0．25μmol／L，Taq DNA聚合酶的用量为1u，模板DNA的用量为30～50ng，在46．4—51.7℃的退火温度下35个循环，能得到清晰、多态性高的ISSR带谱。     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution