广西野生濒危植物掌叶木遗传多样性的ISSR与SRAP分析

采用ISSR和SRAP技术对中国野生濒危植物掌叶木[Handeliodendron bodinieri（Lévl.）Rehd.]的5个野生居群的95份材料进行分析,10条ISSR引物共扩增得到114个条带,其中多态性条带68个,多态性条带百分率（PPB）59.65%,掌叶木在物种水平上的Nei’s基因多样性（H）为0.1817,Shannon’s遗传多样性信息指数（I）为0.2792;观测等位基因数（Na）为1.5965,有效等位基因数（Ne）为1.2984,居群间基因间分化度（Gst）为35.17%。15组SRAP引物共扩增出292个条带,其中多态性条带269个,多态性条带百分比为92.12%,物种水平上,掌叶木的Nei’s基因多样性（H）为0.3171,Shannon’s遗传多样性信息指数（I）为0.4758;观测等位基因数（Na）为1.9212,有效等位基因数（Ne）为1.5380,居群间基因间分化度（Gst）为23.17%。结果显示两种标记均能检测到掌叶木具有较高的遗传多样性,掌叶木不同居群间存在一定的遗传分化和基因流动,但遗传分化主要存在于居群内。     

三叶悬钩子自然居群遗传多样性的ISSR 分析

 利用ISSR分子标记对云南特有植物三叶悬钩子（Rubus delavayi Fanch.）的12个居群共248个个体进行了遗传多样性分析。结果表明：16个ISSR引物共扩增到199个位点,其中185个是多态性位点,占92.96%。三叶悬钩子居群具有很高的遗传多样性水平,在物种水平上平均每个位点的多态位点百分率（PPB）为97.99%,有效等位基因数（A_e）为1.427,Nei’s遗传多样性（H）为0.267,Shannon’s多态信息指数（I）为0.417;在居群水平上PPB为62.10%,A_e为1.289,H为0.177,I为0.275。居群间基因分化系数G_st = 0.3351,与AMOVA分析的居群间遗传变异量占总量的33.03%相近,说明三叶悬钩子居群间存在一定程度的遗传分化。居群间遗传分化占总遗传变异的33.51%,居群内的遗传变异为66.49%,基因流（N_m）为0.9923。通过Mantel检测,居群间的遗传距离与地理距离不存在相关性。UMPGA聚类分析和二维主成分分析（PCA）结果一致。导致居群内高遗传变异水平原因主要是有限的基因流,而居群间较低的遗传多样性水平可能与生态破坏和生物入侵有关。     

野生天门冬遗传多样性的ISSR分析

通过ISSR技术对19个野生天门冬居群共67个个体进行遗传多样性分析.结果表明:用13个随机引物共扩增出125条清晰条带,其中92条具多态性,平均多态性位点比率为73％,建立了不同居群的ISSR标记;Nei's基因多样性指数H=0.19,Shannon,s多样性指数I=0.30,遗传分化指数Gst=0.8206.聚类分析表明,青海、云南和贵州省的5个居群聚为一大支,其它居群为另一大支.野生天门冬种内具有较高的遗传多样性,遗传变异主要存在于居群间;ISSR标记可以作为研究天门冬遗传多样性及居群鉴定的有效标记.     

地木耳遗传多样性的ISSR分析

采用ISSR分子标记技术对7个居群的地木耳进行了遗传多样性研究。结果表明:8条ISSR引物共检测到41条清晰的谱带,其中,多态性条带40条;地木耳在物种水平上遗传多样性较高,其PPB为97.56%、He为0.411 4、I为0.598 1;在居群水平上遗传多样性相对较低,其PPB为43.90%、He为0.165 4、I为0.246 3;居群间遗传变异较高,其中,Gst为0.597 3,PHIst为0.613 4,(Hsp-Hpop)/Hsp为0.588 2;聚类分析发现,以遗传一致度0.72为标准,7个地木耳居群可分为4类。地木耳物种水平遗传多样性大于居群水平,居群间遗传变异大于居群内。     

10 个山茶岛屿天然居群的遗传多样性分析

 采用ISSR 分子标记对舟山群岛、长门岩岛、鹿儿岛、四国岛和五岛的10 个山茶（Camellia japonica）居群的遗传多样性和分化程度进行分析。20 条随机引物扩增出210 个可分析位点,多态性百分比（PPL）为90%。试验结果显示,山茶居群水平的Nei’s 基因多样性指数为0.2732,Shannon 信息多态性指数为0.4003,表明其具有较高的遗传多样性。鹿儿岛、四国岛和五岛的居群相对于舟山群岛和长门岩岛居群而言,遗传多样性水平更高。10 个居群间遗传分化系数Gst = 0.2028;地理距离与遗传距离之间具有显著相关性（r = 0.9081,P < 0.05）,表明岛屿地理隔离对山茶居群间的遗传分化具有重要影响。UPGMA 聚类显示了居群间的亲缘关系,同岛内居群间亲缘关系更近,长门岩岛居群与舟山群岛居群亲缘关系近于和鹿儿岛、四国岛以及五岛居群间的亲缘关系。     

基于ISSR标记的江西野生寒兰居群遗传多样性研究

采用ISSR分子标记对江西省主要山脉12个野生寒兰居群共185个体进行遗传多样性和群体遗传结构研究。结果表明:12条ISSR引物共扩增出123个条带,其中多态性条带97个,多态性条带百分率(PPB)为78.9%。12个寒兰居群在物种水平上的遗传多样性较高,群体总观测等位基因数(N_a)为1.7967,有效等位基因数(N_e)为1.4461,N_ei’s基因多样性(H_e)为0.2649,Shannon’s信息多样性指数(I)为0.3995;在居群水平上遗传多样性较低,PPB平均值为43.97%,N_a平均值为1.4397,N_e平均值为1.2478,H_e平均值为0.1462,I平均值为0.2204;遗传分化系数(G_(ST))为0.4415,居群间基因流(N_m)为0.6325,表明居群内的遗传分化大于居群间的遗传分化;UPGMA聚类结果显示12个寒兰居群可聚为两支,第一支由资溪、武夷山大叶、武夷山小叶、崇义小叶、靖安、宜丰、井冈山、邵武大叶和邵武小叶9个居群组成;第二支由石城、安远和崇义大叶3个居群组成,聚类结果没有呈现出山脉分布的特点;Mantel检验结果表明遗传距离和地理距离之间无显著相关性(r=0.3791)。     

珙桐遗传多样性的AFLP 分析

 采用7对AFLP引物对中国珍稀濒危植物珙桐（Davidia involucrata Baill.）的6个天然居群和2个人工居群的229份材料进行扩增,共检测到100 ~ 500 bp的位点506个,其中多态性位点488个,多态性位点百分率96.44%;各居群多态位点百分率为52.77% ~ 79.25%,平均为67.00%。在物种水平上,珙桐的Nei’s基因多样性H_s = 0.3429,Shannon’s遗传多样性信息指数I = 0.5107。而居群水平上,各居群的Nei’s基因多样性介于0.2229 ~ 0.3389,各居群的平均Nei’s基因多样性H_s = 0.2748,各居群Shannon’s遗传多样性信息指数（I）介于0.3219 ~ 0.4877,平均Shannon’s遗传多样性信息指数I = 0.3995。居群间基因间分化度（G_st）为21.60%,基因流N_m为1.8。说明珙桐的遗传多样性较高,这8个居群间存在一定的遗传分化和基因流动,但遗传分化主要存在于居群内。在珙桐与其变种光叶珙桐之间未找到差异标记。     

基于SSR 标记的四川野生中国樱桃遗传多样性和居群遗传结构分析

 采用SSR 分子标记技术对四川野生中国樱桃5 个居群共133 株的遗传多样性水平及居群的 遗传结构进行了研究。结果显示：10 对SSR 引物共检测到78 个等位基因,平均每位点等位基因7.8 个。 Nei’s 基因多样性指数（H）为0.6112 ~ 0.6689,Shannon’s 信息指数（I）为1.1984 ~ 1.3786。基于分子方 差分析（AMOVA）,92.53%的变异来自居群内,7.47%的遗传变异来自于居群间。居群间遗传距离（GD < 0.2416）、遗传一致度（GI > 0.7854）、遗传分化指数（Fst = 0.0844）以及较强的基因流（Nm = 2.7125）均 表明居群间的遗传分化水平较低,居群内存在显著近交现象（Fis = 0.3986）,且居群在大多数位点上偏离 Hardy-Weinberg 平衡。基于上述结果,分析讨论了居群较高遗传多样性和居群间较低遗传分化形成的可能 原因,并提出野生中国樱桃的保护利用策略。     

新疆野扁桃种质资源遗传多样性的ISSR分析

采用ISSR分子标记技术,对中国新疆分布的野扁桃(Amygdalus ledebouriana Schlecht.)5个居群共120个个体的遗传多样性进行了研究。9条引物共检测到114个位点,其中105个为多态位点。在物种水平上,野扁桃的多态位点百分率为92.1%,Nei's基因多样度(h)为0.2809,Shannon信息指数(I)为0.4327;在居群水平上,多态位点百分率平均为50.7%,Nei's基因多样度(h)平均为0.2062,Shannon信息指数(I)平均为0.2999。基于Nei's遗传多样性分析得出的居群间遗传分化系数Gst=0.2660,表明有26.6%的遗传变异存在于居群之间。居群间的遗传一致度平均为0.8964,估测的居群间基因流(Nm)为1.3794,可以认为5个野扁桃居群间基因流畅通,与Nei's多样性指数揭示的大部分遗传变异存在于居群内是一致的。根据Nei's遗传距离对不同居群进行UPGMA聚类分析显示居群间的遗传距离与居群的地理距离之间没有显著的相关性。基于试验分析结果,野扁桃种质资源保护和利用的策略为在优先保护现有群体的基础上,加强筛选和保存居群内的变异单株。     

贵州砂梨种质资源遗传多样性的ISSR分析

【目的】探明贵州砂梨种质资源遗传多样性及亲缘关系,为其保护与利用提供理论依据。【方法】采用ISSR标记技术,对贵州59份砂梨种质资源的遗传多样性及遗传结构进行分析。【结果】16条ISSR引物共扩增出155个位点,其中126个是多态性位点,多态百分率为81.29%。POPGENE分析结果表明,贵州砂梨种质资源具有一定的遗传变异(在物种水平上Ne=1.418 0,H=0.251 1,I=0.381 6;在区域品种群上Ne=1.300 2,H=0.177 1,I=0.266 7),以黔西南地区和毕节地区遗传多样性最高,且亲缘关系最近。【结论】贵州砂梨种质资源具有丰富的遗传多样性,区域品种群的较高遗传分化可能与地理隔离以及人为干预等因素有关。ISSR标记可有效揭示贵州砂梨种质资源的遗传多样性、分化程度和亲缘关系,研究结果对梨种质资源的保护利用和遗传改良有重要意义。     

Genetic structure of Buxus sinica var. parvifolia, a rare and endangered plant

Buxus sinica var. parvifolia, a rare and endangered tree species in some semitropics alpine areas of China, plays an important role in the maintenance of the landscape and ecosystem. In this study, RAPD and ISSR markers were used to investigate the genetic diversity and structure of five natural populations and one tamed population of B. sinica var. parvifolia. 21 RAPD primers amplified 209 bands with 167 (79.90%) polymorphic and 21 ISSR primers amplified 518 bands with 467 (90.15%) polymorphic. The genetic diversity, estimated by Shannon’ index, was 0.4343 (by RAPDs) and 0.3661 (by ISSRs). Both RAPD and ISSR analyses revealed a high level of genetic diversity in natural populations of B. sinica var. parvifolia. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations. The proportion of variation attributable to within-population differences was very high (69.2% by RAPDs; 84.51% by ISSRs). Moderate differentiation was detected among populations using RAPDs (30.80%), while only a small amount of variation (15.49%) was detected among populations using ISSRs. We suggest that the present genetic structure is due to high levels of environmental variability and gene flow, which still need further study to confirm. Conservation measures are suggested, including in situ and ex situ strategies, based on the observed population genetic information.     

茶树‘黄山种’自然杂交后代遗传多样性分析

 通过ISSR标记结合形态学性状,分析了茶树‘黄山种’自然杂交后代的遗传多样性。所调查形态学性状的平均变异系数为20.5%（12.5% ~ 33.2%）,Shannon-weave指数平均为1.89（1.60 ~ 2.07）。22条ISSR引物在供试样品中共扩增出402条带,其中多态性条带396条,多态性比率为98.5%,引物的多态性信息指数（PIC）平均达0.90,供试样品的基因多样性（H）和Shannon信息指数（I）分别为0.32和0.49。充分表明‘黄山种’通过自然杂交,发生了基因的分离和重组,产生了丰富的遗传变异。Mantel检测基于形态学与基因型估算的遗传距离矩阵间,存在极显著正相关（r = 0.61,P = 0.01）。基于形态学性状和ISSR标记的聚类结果显示,大多数‘黄山种’自然杂交后代的单株可聚成一大类群,只有少数单株遗传距离较远,单独聚为一类。这些单株一芽三叶长、百芽质量较大,可为今后‘黄山种’系统选种提供优异种质材料。     

Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers

The genetic diversity of the genus Dendrobium is not well known and the phylogenetic relationship of Dendrobium species are mainly determined by studies of the comparative vegetative anatomy and plant systematics. In the present study, we used the technique of inter-simple sequence repeats (ISSRs) to evaluate genetic diversity and phylogenetic relationship among 31 Dendrobium species from Yunnan region of China. In total, 2368 bands were amplified by 17 ISSR primers, resulting from 278 ISSR loci with 100% polymorphism at genus level. Thirty-one species were unequivocally distinguished based on ISSR fingerprinting. Species-specific ISSR markers were identified in nine of 31 tested Dendrobium species. Unweighted pair-group mean analysis (UPGMA) showed that 31 Dendrobium species were grouped into six clusters, indicating the genus was polyphyletic with several well-supported lineages. The high polymorphism and reliable amplification across species demonstrated the utility of ISSR marker for species diagnosis and genetic diversity study of the genus Dendrobium.     

Genetic diversity of Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers

Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass. Although it is widely distributed in China, studies on the genetic variation and relationship among the large-scale indigenous bermudagrass were relatively insufficient, especially at molecular level. The purpose of this study was to assess the molecular variation and relationship among one commercial cultivar ‘Tift3’ and 95 wild bermudagrass accessions collected from 11 provinces in China by ISSR marker technique. The results indicated that 29 ISSR primers generated a total of 248 bands among which 242 (97.6%) were polymorphic bands. The genetic similarity coefficients among accessions ranged from 0.51 to 0.97 with an average of 0.74. All accessions could be clustered into 11 groups with UPGMA method. Accessions from the same or adjacent regions generally were clustered into the same group or subgroups. A few accessions, however, were greatly different from the majority of all accessions. The results suggested that ISSR marker is an effective tool for the study of genetic variation in Chinese natural bermudagrass.     

中国石榴居群遗传结构的ISSR分析

为了更有效地保护、利用石榴种质资源,采用PopGen32软件,利用ISSR标记对中国7个石榴主产区46个石榴品种进行了研究分析。结果表明:7个石榴种群遗传多样性依次为:山东种群>陕西种群>云南种群>河南种群>安徽种群>四川种群>新疆种群;7个种群间的基因分化系数(Gst)为0.1911,表明分布在种群间的遗传变异占总遗传变异的19.11%,种群内遗传变异占81.89%,种群间表现出低水平的遗传分化;群间基因流的估测值(Nm)为2.1169,种群间的遗传相似性(I)变化范围为0.9218～0.9759,表明种群间存在较强的基因流,遗传相似性很高,石榴品种间的遗传多样性能够得以维持。     

Genetic diversity of ash gourd [Benincasa hispida (Thunb.) Cogn.] inbred lines based on RAPD and ISSR markers and their hybrid performance

Ten inbred lines of ash gourd [Benincasa hispida (Thunb.) Cogn.] were crossed to produce 45 F₁ hybrids (without reciprocal) which were evaluated along with the parents for 20 growth- and yield-related traits, in a replicated field trial. High level of heterosis was observed among the hybrids for most of the traits examined, including yield. These inbred lines were analysed by using 42 RAPD primers those produced 282 DNA marker bands. A total of 130 RAPD markers were obtained with a mean of 3.1 per primer, which in combination discriminated all the inbreds from each other. Pair-wise genetic distance measurements ranged from 0.07 to 0.31, suggesting a wide genetic diversity for these inbreds. These inberds were also analysed with five ISSR primers of which four were informative. Twenty-six ISSR marker bands were generated of which 11 were polymorphic with an average of 2.80 per primers. The percentage of polymorphic bands produced were higher in ISSR markers (>80%) than generated through RAPD markers (46%). Although the results indicated significant positive correlations of genetic distance with hybrid performance and heterosis, the RAPD based genetic distance measures and use of limited ISSR markers in this present study could not effectively predict hybrid performance in this crop. The genetic variation among ash gourd inbred lines examined, herein, defined a marker array (combined ISSR and RAPD) for the development of a standard reference for further genetic analyses, and the selection of potential parents for predicting hybrid performance and heterosis.     

Assessment of clonal fidelity of micropropagated gerbera plants by ISSR markers

True-to-type clonal fidelity is one of the most important pre-requisites in micropropagation of crop species. Genetic fidelity of in vitro raised 45 plants of gerbera (Gerbera jamesonii Bolus) derived from three different explants, viz., capitulum, leaf and shoot tips, was assessed by 32 ISSR markers, for their genetic stability. Out of 32 ISSR markers, 15 markers produced clear, distinct and scorable bands with an average of 5.47 bands per marker. The markers designed from AG motif amplified more number of bands. The markers anchored at 3′ ends produced high number of consistent bands than unanchored markers. Fifteen ISSR markers generated a total of 3773 bands, out of which 3770 were monomorphic among all the clones. The Jaccard's similarity coefficient revealed that out of 45 clones derived from different explants, 44 were grouped into a single large cluster alongwith the mother plant with a similarity coefficient value of 1.00, whereas one clone (C38) remained ungrouped. The clones derived from capitulum and shoot tip explants did not show any genetic variation, whereas, one of the leaf-derived clones exhibited some degree of variation.     

滇西北地区5种羊肚菌遗传多样性的ISSR分析

以滇西北地区11个羊肚茵居群56个羊肚菌样品为研究材料,应用ISSR分子标记方法,进行了遗传多样性与亲缘关系分析。结果表明,11个多态性ISSR引物对全部试验样品进行PCR扩增,共获得88条稳定的条带,其中多态性条带70务（占79．55％）。应用软件POPGENE32分析得出11个羊肚菌居群间遗传距离的变化范围在0．1715～0．4853之间,相似系数的变化范围在0．6155～0．8424之间。遗传分化分析表明,61．35％的变异存在于居群间,38．65％的变异存在于居群内。对56个羊肚菌样品进行分子系统聚类分析（UPGMA）将资源分为三大组,对11个羊肚菌居群进行亲缘关系聚类分析将居群分为两大类群,聚类结果与地理距离有明显的相关性。     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.