Molecular characterization and genetic variability studies associated with fruit quality of indigenous mango (Mangifera indica L.) cultivars

Start codon-targeted (SCoT) markers were used for characterization and genetic comparison analysis among 20 mango cultivars (15 indigenous and 5 popular) with respect to fruit quality. Out of 80 SCoT markers used, 19 were able to amplify. These primers produced total 117 loci across 20 cultivars, of which 96 (79.57 %) were polymorphic with an average of 5.05 polymorphic fragments per primer. Out of 19, 17 SCoT primers produced 34 cultivar-specific DNA finger prints. These were 25 unique fragments for identification of 15 indigenous cultivars and 9 fragments for the identification of five popular cultivars. The three SCoT primers—40, 45, and 51 are most informative in identifying mango cultivars as they possess the higher primer index values. The 20 mango cultivars were clustered into two major groups based on the SCoT data analysis with UPGMA. Three indigenous cultivars—Khodi, Amrutiyo, and Kaju and one popular—Dasheri out grouped from other 16 cultivars and shared only minimum similarity (11 %). In clustering pattern, indigenous cultivars—Kaju and Amrutiyo grouped together and shared 37 % similarity with higher boot strapping values (63 %). Clustering pattern is corresponding well with their physical and/or biochemical properties of fruits. The results of principal coordinates analysis (PCoA) analysis were comparable to the cluster analysis. The first three most informative PC components explained 56.61 % of the total variation. In PCoA, three indigenous cultivars—Jamrukhiyo, Chappaniyo, and Sopari appears to be distinct from other 12 indigenous, which be different in fruit size, sugars, ascorbic acids, and carotenoids content. Similarly, popular cultivars—Jamadar and Kesar were also discrete from Alphonso, Dasheri, and Neelum in PCoA. The results demonstrate that the SCoT marker system is useful for cultivar identification and genetic diversity analysis of mango cultivars based on their biological traits.     

Analysis of diversity and relationships among mango cultivars using Start Codon Targeted (SCoT) markers

Genetic variation and relationships among 47 mango germplasm and 3 relative species from Guangxi province in China, were analyzed using Start Codon Targeted (SCoT) markers. Using 33 selected SCoT primers 273 bands were generated with an average of 8.27 bands per primer among the 50 accessions, of which 208 (76.19%) were polymorphic. Genetic relationships estimated using the SM similarity coefficient generated values between different pairs of accessions that varied from 0.531 to 0.923 with an average of 0.782. These coefficients were utilized to construct a dendrogram using the UPGMA. All 50 accessions were basically classified into six clusters and correspond well with their recorded pedigrees. The results will provide much more useful information for the management of germplasm and will also be useful to improve the current breeding strategies. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.     

ISSR鉴定亲缘关系非常近的芒果栽培品种

用ISSR技术鉴定7个吕宋芒品种(系)和柳州吕宋芒。从30个引物中筛选出6个多态性好的ISSR引物建立DNA指纹图谱用于区分吕宋芒品种(系)。分析DNA指纹图谱,发现这6个引物中每个引物都能区分吕宋系列品种(系),表明ISSR-PCR技术对芒果品种(系)的鉴定非常有效,能区分亲缘关系很近的品种(系)。基于69条多态性条带的聚类分析结果,发现吕宋芒和其它供试的7个品种(系)同源性低,而这7个品种(系):高州吕宋芒、湛江吕宋芒、田阳香芒、金钱芒、柳州吕宋芒、粤西一号、攀西红吕宋同源性较高,可归为一类。     

桂热杧系列品种（系）的亲缘关系分析

从100条ISSR引物中选取12条多态性好的引物,对广西亚热带作物所选育的16个杧果（Mangifera indica L．）品种（系）及其可能亲本共22个样品的亲缘关系进行了分析。结果表明：这12条引物共扩增出127条DNA谱带,其中多态性条带为85条,条带多态百分率为66．93％。聚类分析显示,所有供试品种（系）之间的亲缘关系都比较近,相似系数在0．81以上;如果以相似系数0．82为标准,可将供试的22个品种（系）分为3大类。利用ISSR技术能较好地区分桂热杧系列品种（系）的亲缘关系。     

Genetic variability and structure of Quercus brantii assessed by ISSR,IRAP and SCoT markers

Persian oak (Quercus brantii Lindl.) is one of the most important woody species of the Zagros forests in Iran. Three molecular marker techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) markers were compared for fingerprinting of 125 individuals of this species collected from different geographical locations of north-west of Iran. A total of 233 bands were amplified by 18 ISSR primers, of which 224 (96.10%) were polymorphic, and 126 polymorphic bands (97.65%) were observed in 129 bands amplified by 10 IRAP primers. Besides, 118 bands were observed for all 10 SCoT primers, of which 113 were polymorphic (95.71%). Average polymorphism information content (PIC) for ISSR, IRAP and SCoT markers was 0.30, 0.32 and 0.38, respectively, and this revealed that SCoT markers were more informative than IRAP and ISSR for the assessment of diversity among individuals. Based on the three different molecular types, cluster analysis revealed that 125 individuals taken for the analysis can be divided into three distinct clusters. The Jaccard's genetic similarity based on the combined data ranged from 0.23 to 0.76. These results suggest that efficiency of SCoT, IRAP and ISSR markers was relatively the same in fingerprinting of individuals. All molecular marker types revealed a low genetic differentiation among populations, indicating the possibility of gene flow between the studied populations. These results have an important implication for Persian oak (Q. brantii) germplasm characterization, improvement, and conservation.     

IGS2-RFLP、SCoT和ISSR在白灵侧耳遗传多样性分析中的比较研究

应用IGS2-RFLP、SCoT和ISSR标记分析新疆5个采集地点的28个白灵侧耳野生样本的遗传多样性,比较这3种分子标记在白灵侧耳种质资源遗传多样性研究中的效用。结果显示,IGS2-RFLP的3个内切酶、5个SCoT引物、5个ISSR引物分别检测到42、59和77条多态性条带,多态性比率分别为91.3%、92.4%和92.8%。3种标记检测出的有效等位基因数(ne)和位点平均的预期杂合度(Hep)没有显著性差异。表明3种标记都适宜作为遗传学标记对白灵侧耳野生种质进行多样性分析。多态性检测效率最高的标记为ISSR,E=15.4,Ai=23.4,其次为IGS2-RFLP,E=14.0,Ai=22.4,SCoT则为最低,E=11.8,Ai=18.2。3种标记中,SCoT和ISSR标记的聚类结果极其相似,且均能较为准确地反映样本的地理来源,虽然二者的聚类图不完全相同。IGS2-RFLP的标记效率较高,准确性和可重复性最好,可用于菌株标准图谱的制作,更适用于品种权保护中的菌株鉴定鉴别;SCoT和ISSR标记的多态性高,信息量大,评价范围广,则更适用于白灵侧耳种质资源的遗传多样性研究。     

Evaluation of genetic diversity and population structure of polygonati rhizoma germplasms: implications for better crop development and conservation of a traditional Chinese medicine

Polygonati rhizoma (PR) is an important and widely used product in Chinese traditional medicine and edible goods. The time-consuming nature of breaking dormancy in both rhizomes and seeds means that improving variety selection is limited to collection, identification, and selection of germplasms. In this study, we used two DNA-based molecular marker techniques—inter simple sequence repeat (ISSR) and start codon targeted (SCoT)—to assess the genetic diversity and population structure among PR source plants collected from 47 different regions and belonging to 12 populations (P1–P12). For molecular markers analysis, 15 ISSR and 10 SCoT markers were tested. Total number of 159 fragments (150–4000 bp) were amplified based on ISSR analysis with a range from 6 to 17 bands, 153 of them were polymorphic, ranging from 97.27 to 100%. For SCoT analysis, 164 polymorphic bands (150–5000 bp) were observed, varying from 14 to 19 bands for each primer. Nei’s genetic diversity analysis showed that the highest value was found in P11 for ISSR and P4 for SCoT markers. The highest Nei’s genetic diversity value was observed in P5 for combined markers and the low in P2. Nei’s dendrogram constructed with combined markers indicated a 75–89% of genetic similarity coefficient among populations. Population structure analysis revealed an optimum number of three groups, the same as their geographical distribution. This knowledge on PR genetic diversity can be used in future breeding programs, genetic improvement, product enhancement, and germplasms conservation.     

ISSR and SCoT for evaluation of hereditary differences of 29 wild plants in Al Jubail Saudi Arabian

This survey is concerned with the hereditary differences of 29 wild plants collected from fifteen different regions in Al Jubail, Saudi Arabia using two molecular marker systems, viz. inter simple sequence repeat (ISSR) and start codon targeted (SCoT) molecular markers. Ten ISSR and ten SCoT primers amplified a total of 142 and 163 bands with a 87% and 84% polymorphism, respectively. The average number of polymorphic bands for each pair of ISSR and SCoT primers combinations was 12.4 and 13.7, respectively. The highest genetic similarity for ISSR (0.97) and SCoT (0.90) were recognized between Zygophyllum qatarense-22 and Juncus rigidus-23, and between Zygophyllum qatarense-28 and Zygophyllum qatarense-29, whereas the lowest was (0.59) differentiated between Zygophyllum qatarense-6 and Salsola imbricate-18 for ISSR and between Cyperus conglomeratus-7 and Halopeplis perfoliata-14 for SCoT. This considers confirmed the value of molecular techniques such as ISSR and SCoT to assess the hereditary differences among the selected 29 weeds for hereditary preservation and plant enhancement.     

一种新的DNA分子标记技术——起始密码子-微卫星扩增多态性

综合SCoT和ISSR分子标记技术开发了一种既能将标记位点与表达序列紧密联系,又具有相对较高的多态性的新的分子标记技术——起始密码子一微卫星扩增多态性（start codon-simple sequence repeat, SC-SSR）。SC—SSR标记是基于PCR的目的基因标记系统．上游引物用SCoT标记引物,瞄准基因区域,下游引物用ISSR标记引物,上下游引物间可自由组配。引物设计原则同SCoT标记和ISSR标记。使用50℃的退火温度,保证了扩增结果的稳定性。PCR结果采用琼脂糖凝胶电冰和聚丙烯酰胺凝胶电泳检测。SC—SSR分子标记结合了ISSR标记和SCoT标记的优点,具有操作简单、成本低廉、多态性丰富、重复性好、引物设计简单且通用性良好、同时与表达序列紧密连锁等诸多优点,可用于种质资源的鉴定评价、遗传图谱的构建、重要性状基因标记、gDNA与cDNA指纹分析乃至图位克隆等方面。     

Molecular variation and fingerprinting of Leucadendron cultivars (Proteaceae) by ISSR markers

BACKGROUND AND AIMS: There are more than 80 species of Leucadendron and most are used as cut flowers. Currently, more than 100 cultivars are used by industry and many of them are interspecific hybrids. The origin of most cultivars is unclear and their genetic diversity and relationships have not been studied. This investigation was carried out to evaluate the genetic variation and relationships among 30 Leucadendron cultivars. METHODS: ISSR markers were applied to determine the genetic variation and to discriminate Leucadendron cultivars. Sixty-four ISSR primers were screened and 25 primers were selected for their ability to produce clear and reproducible patterns of multiple bands. KEY RESULTS: A total of 584 bands of 305-2400 bp were amplified, of which 97 % were polymorphic. A dendrogram generated using the Unweighted Pair Group Method with Arithmetic Average based on a distance measure of total character difference showed that the Leucadendron cultivars clustered into two main groups. Twenty-four of the 30 cultivars can be unequivocally differentiated, but identical profiles were observed for three cultivar pairs, 'Katie's Blush' and 'Silvan Red', 'Highlights' and 'Maui Sunset', and 'Yellow Crest' and 'Yellow Devil'. CONCLUSIONS: ISSR profiling is a powerful method for the identification and molecular classification of Leucadendron cultivars. A fingerprinting key was generated based on the banding patterns produced using two ISSR primers (UBC856 and UBC857). In addition cultivar-specific ISSR bands were obtained for 17 of the 30 Leucadendron cultivars tested.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markers

ISSR and SSR markers were used to evaluate genetic diversity among 33 Cynodon dactylon accessions and 22 cultivars from four different countries in order to provide information on how to improve the utilization of bermudagrass germplasms. Eighty eight bands were amplified by nine SSR primer combinations and 236 bands were observed from 23 ISSR primers. The results showed that 97.7% of the SSR primers and 86.9% of the ISSR primers were polymorphic. The genetic similarity coefficients (GSC), gene diversity (He) and Shannon index (I) were 0.58–0.97, 0.27 and 0.41, respectively, for ISSR and 0.52–0.97, 0.29, and 0.43 for SSR. The UPGMA analysis clustered the 55 accessions (cultivars) into three groups. The cluster results produced by the ISSR data were close to the SSR data results. Analysis based on the combined ISSR and SSR data was more closely related to the geographical distribution of the tested germplasm.     

猕猴桃SCoT遗传多样性分析及指纹图谱的构建

利用SCoT标记对32个猕猴桃品种进行了遗传多样性分析。从47个SCoT引物中筛选了11个引物进行PCR扩增,共扩增出185个条带,其中多态性条带180个,多态性比率为97.30%。各引物Nei's基因多样性指数（H）平均为0.238 4,Shannon's信息指数（I）平均为0.377 8。利用UPGMA构建32份猕猴桃种质资源的聚类树状图。在遗传相似系数为0.78处可将32个猕猴桃品种分为5组,聚类结果与形态学分类基本一致。利用4条引物扩增的16个多态性位点构建了32个猕猴桃品种的DNA指纹图谱,可以将32个猕猴桃品种区分并准确鉴定。     

14个蝴蝶兰品种遗传关系的ISSR分析

采用ISSR分子标记技术对14个蝴蝶兰品种进行品种间遗传关系的研究。利用14个筛选的引物共扩增出179条带,其中多态性条带147条,多态性条带比率(PPB)为82%。品种之间的遗传相似系数范围在0.734~0.936间,说明部分蝴蝶兰品种间存在显著的遗传分化。14个引物组合可区分所有14个品种,并且检测到20条品种特异性条带,这些品种特异条带可用来鉴定供试蝴蝶兰中的10个品种。因此,ISSR分子标记能有效地进行蝴蝶兰品种鉴定。UPGMA聚类分析表明,14个品种可聚为2类,聚类情况与花色特征比较一致,但与花色的划分结果不完全相同,这可能是由于品种间杂交引起的。本文也论讨了ISSR分析结果对蝴蝶兰育种的指导意义。     

皂荚种质资源SCoT遗传多样性分析及指纹图谱的构建

采用SCoT标记分析了18个皂荚种质的遗传多样性,并采用UPGMA法对18个皂荚种质进行了聚类分析。在此基础上,通过筛选出的多态性条带构建了18个皂荚种质的SCoT指纹图谱。扩增结果表明:从51个SCoT引物中筛选了15个引物进行PCR扩增,共扩增出226条带,其中多态性条带216条,多态性比率为96.61%。各引物多态性信息含量(PIC)、观测等位基因数(Na)、有效等位基因数(Ne)、Nei’s基因多样性指数(H)和Shannon’s信息指数(I)的平均值分别为0.875 9、1.964 9、1.440 1、0.272 6、0.426 1。18个皂荚种质的遗传相似系数在0.491 4~0.938 1之间,表明供试材料之间具有较丰富的遗传多样性。聚类分析结果表明:在遗传相似系数为0.60处可将18个皂荚种质分为3组,其中野皂荚单独为一组,山皂荚和皂荚-T聚为一组,其它皂荚材料聚为一组。利用3个引物扩增的8个多态性位点构建了18份皂荚种质资源的DNA指纹图谱,可以将其区分并精准鉴定。该研究结果为皂荚种质的鉴定和新品种选育提供了一定的理论依据。     

24个菊芋品种（系）遗传多样性的ISSR标记分析

利用ISSR分子标记技术研究了来源于国内外不同区域的24个菊芋（Helianthus tuberosus Linn．）品种（系）的遗传多样性,并结合块茎特征采用聚类分析法探讨了它们的遗传关系。结果表明：用16条ISSR引物从24个品种（系）的基因组DNA共扩增出242条带,包含228条多态性条带,多态性条带百分率达94．2％,其中7条引物的多态性条带百分率达100．0％。各品种（系）间的遗传距离为0．19～1．01,遗传距离平均值为0．45。聚类分析结果显示：在遗传相似系数0．68处可将24个品种（系）划分为5类,第1类仅包含‘青芋1号’（‘Qingyu No．1’）,第Ⅱ类仅包含W12,第Ⅲ类包含W30、W42、‘青芋2号’（‘Qingyu No．2’）、W09、W18、W26和WSl,第Ⅳ类包含W06和W84,第V类包含‘青芋3号’（‘Qingyu No．3’）、w23、W36、W43、W54、W75、WS0、W62、W64、W79、S150、S138和W66;多数块茎特征相似的品种（系）被聚在一起,但也有部分块茎特征不同的品种（系）被聚在同一类中;部分品种（系）的聚类分析结果与其形态分类结果及地理分布不一致。研究结果表明：供试的菊芋品种（系）具有较高的遗传多样性,存在着较为频繁的基因交流;基于ISSR标记分析能较准确地揭示出菊芋品种（系）间的遗传多样性。     

茄子耐盐种质资源遗传多样性的SRAP和ISSR分析

利用SRAP和ISSR分子标记,研究了14份耐盐茄子种质资源的遗传多样性,结果表明,2种标记均能揭示材料间较高的遗传多样性,其中ISSR标记多态性略高于SRAP标记。在SRAP分析中,每对引物组合可扩增出8-15条DNA片段,平均为12．12条：26对SRAP引物组合共扩增出315条DNA片段,其中263条具有多态性,多态性比率为83．49％;材料间遗传相似系数变化范围为0．212～0．923,平均值为0．755。在ISSR分析中,每个引物可获得5～16条DNA片段,平均为10．87条;15个ISSR引物共扩增出163条DNA片段,其中141条具有多态性,多态性比率为86．50％;材料间遗传相似系数变幅为0．333-0．957,平均值为0．736。聚类分析表明,2种标记都能将供试材料完全区分开来,聚类结果具有一定的相似性,但也存在明显差异。Mantel相关分析表明,SRAP分析与ISSR分析的相关性达到极显著性水平（r=0．904,P〈0．01）。     

用ISSR标记技术分析山药品种遗传多样性

利用ISSR标记技术对28个山药品种的遗传多样性进行分析。结果表明,从44条ISSR引物中可筛选出7条能够扩增出清晰、稳定条带的引物;这7条ISSR引物对28个山药品种扩增条带间存在较大差异,多态性条带比率为83．01％,Shannon多样性指数为0．3191;构建的分子树状图将28个山药品种划分为4组：第一组含有日本白、花山药和日本园3个品种;第二组为小叶山药;第三组为嵩野1号;其余23个品种归入第四组。而且主成分分析结果支持上述的聚类分析结果。这为利用ISSR标记技术鉴定山药品种,为有效地利用山药种质资源提供了依据。     

Genetic diversity and population structure detection in sponge gourd ( Luffa cylindrica ) using ISSR,SCoT and morphological markers

Investigation of genetic diversity is essential for the selection of parents for crop breeding and conservation of genetic resources. To estimate the genetic variability and population structure in the midst of 45 accessions of sponge gourd brought together from different geographical areas of India, morphological traits and two molecular markers, ISSR and SCoT markers were compared. Principal components analysis of 20 morphological traits showed 72.70% variability and significant positive correlations between fruit traits. All three marker techniques clustered all accessions into two groups with few outgroups. High level of polymorphism was observed among ISSR (74.6%) and SCoT (71.5%) primers. The Bayesian model revealed the hidden grouping and showed admixture type of population. The diversity pattern is influenced by genetic marker used, as different molecular markers have different polymorphism evaluation efficiency. This study can be helpful in amplifying the genetic base and selection of specific traits for breeding. Thus, ISSR and SCoT markers are potential marker for identification in sponge gourd and provide valuable data on its genetic correlation and structure.     

Genetic diversity in some grape varieties revealed by SCoT analyses

Start codon targeted (SCoT) polymorphic markers were used to assess genetic relationships among 64 grape varieties. Seventeen informative primers were selected from 36 SCoT primers based on their ability to produce clear and repeatable polymorphic and unambiguous bands among the varieties. A total of 131 bands were produced; 93.1% of them were polymorphic; the average polymorphism information content was 0.82. Cluster analysis of SCoT markers through the unweighted pair-group method of arithmetic averages analysis and principal coordinate analysis were largely consistent. The partition of clusters in the dendrogram and PCoA plot was similar and some degree of grouping by types of grape and taxonomic status of the varieties was revealed. Four main groups were found after cluster analysis, i.e. table grape of Vitis vinifera; table grape of Euro-America hybrid; wine grape of V. vinifera and wild Vitis species. The results showed that the wild Vitis species originated from America and China could be clearly differentiated. The results also indicated that SCoT markers are informative and could be used to detect polymorphism for grape varieties.