Genetic diversity and gene flow estimation in Prosopis cineraria (L.) Druce: A key stone tree species of Indian Thar Desert

Selected amplicon data obtained through our earlier study using ISSR and DAMD markers were utilized for determination of diversity within and among the populations of Prosopis cineraria (L.) accessions collected from different districts of Rajasthan (India). A total of 83 bands were generated from eight ISSR and five DAMD primers of which 79 were found to be polymorphic (95.18%). Nei’s gene diversity (h) ranged between 0.185 and 0.301 with overall diversity of 0.316 while Shannon’s information index (I) values recorded between 0.253 and 0.438 with an average value of 0.243. The gene flow value (1.713) and the diversity among populations (0.226) demonstrated higher genetic variation within the population. It is concluded that P. cineraria is accompanied by high genetic diversity within the population and elevated gene flow showing indications of adaptation to callous and fragile dry conditions of arid environment.     

ISSR primer screening and preliminary evaluation of genetic diversity in wild populations of Gycyrrhiza uralensis

Fourteen efficient inter-simple sequence repeat (ISSR) primers were screened and optimized for detecting the genetic diversity in wild populations of Glycyrrhiza uralensis Fisch. By using these primers, 249 polymorphic bands out of a total of 270 (92.2 %) were generated from 70 individuals of 4 wild G. uralensis populations sampled from Inner Mongolia Province of China. Nei’s gene diversity (h) and Shannon index (I) calculated from the data matrix of the ISSR phenotypes revealed a high level of genetic diversity with h = 0.268 and I = 0.415 within this plant. Analysis of molecular variation (AMOVA) showed that most of the genetic variation (81 %) occurred within the populations, whereas the variance among populations was only 19 %. The UPGMA tree based on Nei’s unbiased genetic diversity illustrated that populations from Bulage and Bayanwusu were genetically close related, while the population from Shanghaimiao was found to be the most diverse from the other three. The high genetic diversity implies that the wild resources of this species could be restored soon if an appropriate and efficient protection strategy was employed. Our results also provided an optimized method for evaluating genetic diversity of G. uralensis using ISSR markers which was useful for further investigation.     

小苍兰种质遗传多样性的ISSR分析

利用ISSR(Inter Simple Sequence Repeat)分子标记对12份小苍兰(Freesia refracta)种质进行了遗传多样性分析研究。从34条ISSR引物中筛选出了12条适宜的引物。这12条引物中每条引物可扩增出5~11条DNA片段,共扩增了96个条带,其中多态性片段62条,平均每条引物可产生5.2条多态性片段,多态性条带比率(PPB)为64.6%。经NTSYS-pc分析,12份小苍兰种质间的遗传距离(GD)的变化范围为0.123~0.907,平均为0.442。根据Nei’s相似系数建立了UPGMA聚类图,在相似系数为0.56时,可将紫色花系的小苍兰种质与其它种质分开,形成两个组。结果表明,ISSR分子标记可有效地分析小苍兰种质资源的遗传多样性和亲缘关系,为小苍兰的杂交育种和新品种保护提供理论基础。     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

ISSR and DAMD markers revealed high genetic variability within Flavoparmelia caperata in Western Himalaya (India)

Flavoparmelia caperata (L.) Hale is medicinally very important and possesses antifungal and antibacterial activities. F. caperata is the only species found in India. Inter simple sequence repeat (ISSR) and Directed amplification of minisatellite DNA (DAMD) methods were used to analyze the genetic variability within F. caperata from the Western Himalayan region of India. Eleven ISSR and 10 DAMD primers produced 139 and 117 polymorphic bands, and detected 91.44 and 82.34 % polymorphisms, respectively. Cumulative band data generated for ISSR and DAMD markers resulted in 86.86 % polymorphism across all the accessions of F. caperata. The average Polymorphic information content (PIC) value obtained with ISSR, DAMD, and cumulative band data were 0.28, 0.27, and 0.27, respectively. The clustering of the F. caperata accessions in the UPGMA dendrogram showed that these accessions are intermingled with each other in different subclusters irrespective of their geographical affiliations. The pattern of genetic variations within F. caperata accessions could be due to free exchange of spores that might have taken place among these accessions in the wild. ISSR and DAMD markers efficiently and reliably resulted in discrete banding patterns and polymorphic profiles. These markers despite targeting different regions of genome, revealed almost similar levels of polymorphism across all the accessions. The wide range of genetic distance and high level of polymorphism detected by ISSR and DAMD reflected a high genetic variability among the different accessions of F. caperata.     

Population structure and genetic diversity in bottle gourd [Lagenaria siceraria (Mol.) Standl.] germplasm from India assessed by ISSR markers

Genetic diversity analysis was undertaken in 42 geographically distant genotypes accessions of bottle gourd (Lagenaria siceraria) from India northeastern (14) and northern region (28) using inter-simple sequence repeat (ISSR) markers. A total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00 % band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Nei’s gene diversity/heterozygosity, resolving power, Shannon’s information index and gene flow were estimated under experiment. Jaccard’s similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA cluster analysis based on this matrix showed clustering into six groups. Jaccard’s coefficient of similarity values ranged from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity. The Bayesian model-based approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among the 42 genotypes. This is the first report on the assessment of genetic variation using ISSR markers in this medicinal vegetable plant, and this study of diversity analysis will be helpful in analyzing future hybrid breeding strategy and devising effective germplasm exploration and conservation strategy.     

烟草种质资源遗传多样性的IRAP和ISSR标记比较分析

采用IRAP和ISSR两种分子标记方法,对国内外28份烟草种质资源进行聚类分析,并对两种方法的结果予以比较。结果如下：（1）用IRAP方法对供试材料进行PCR扩增,共得到214条条带,其中187条具有多态性,多态性比率达87．4％。当L1取值0．495时,可将28份材料划分为3个类群;L2取值0．34时,又可将第3类群划分为3个亚类群和3个单一品种组成的独立个类;（2）用ISSR方法进行PCR扩增时,共得到334条条带,其中250条有多态性,多态性比率为75％。当L1取值0．41时,可将28份烟草种质划分为2个类群和2个独立个类Coker176、BaSmaUovina,第一类群（Ⅰ）为来自美国的两个品种RG11和K730,其它24个品种为第二类群（Ⅱ）;（3）IRAP和ISSR两种方法的相关系数为0．823（r0．01=0．478）,说明这两种方法的结果高度相关,均可用来进行烟草种质资源遗传多样性的分析。用IRAP方法得到的品种间遗传相异系数（GD）平均为0．423,高于ISSR方法,而且条带更稳定、多态性比率更高,更易揭示不同品种材料的遗传差异。     

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.     

Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR (inter-simple sequence repeat) markers.The results showed that genetic variation was relatively higher among the accessions.A total of 593 bands were amplified by 12 ISSR primers,of which 535 bands (90.2%) were polymorphic.Eleven to 80 polymorphie bands were amplified from each prime,with an average of 44.6 bands.The interspecies GS (genetic similarity)value ranged from 0.430 to 0.866,and the average was 0.620.Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers.The different accessions in a species were clustered together,but they had genetic variation in molecular levels.There was obvious interspecies genetic variation.Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships.ISSR markers are useful in analyzing interspecies variation in Kengyilia.     

Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR (inter-simple sequence repeat) markers. The results showed that genetic variation was relatively higher among the accessions. A total of 593 bands were amplified by 12 ISSR primers, of which 535 bands (90.2%) were polymorphic. Eleven to 80 polymorphic bands were amplified from each prime, with an average of 44.6 bands. The interspecies GS (genetic similarity) value ranged from 0.430 to 0.866, and the average was 0.620. Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers. The different accessions in a species were clustered together, but they had genetic variation in molecular levels. There was obvious interspecies genetic variation. Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships. ISSR markers are useful in analyzing interspecies variation in Kengyilia. __________ Translated from Guihaia, 2006, 26 (4): 375–380 [译自: 广西植物]     

A comparative analysis of genetic diversity in medicinal Chrysanthemum morifolium based on morphology, ISSR and SRAP markers

The diversity and genetic relationship among 29 populations of Chrysanthemum morifolium, one of Chrysanthemum indicum and one of Chrysanthemum nankingense from China were analyzed using morphological traits and molecular markers. Twenty morphological traits were scored as well as 182 ISSR marker-fragments, as amplified by 22 primers [the percentage of polymorphic bands (PPB): 81.87%], and 243 SRAP marker-fragments as generated by 26 primer pairs (PPB: 75.72%). Mantel’s test indicated significant correlation (r = 0.624) of morphological trait and SRAP. By contrast, the morphological trait showed low correlation with ISSR (r = 0.246). Cluster analysis showed groupings of the accessions according to all four methods correlated well with their geographic region of origin, and most populations from the south of China were classified into one cluster and most populations from the north of China were classified into another cluster. Finally, an appropriate strategy for conserving the C. morifolium germplasm was proposed.     

Convergence of Goals: Phylogenetical,Morphological, and Physiological Characterization of Tolerance to Drought Stress in Tall Fescue (Festuca arundinacea Schreb.)

The aim of this study is to find Iranian tall fescue accessions that tolerate drought stress and investigation on phylogenetical, morphological, and physiological characterization of them. For this propose, inter-simple sequence repeats (ISSR) markers were used to examine the genetic variability of accessions from different provinces of Iran. Of 21 primers, 20 primers generated highly reproducible fragments. Using these primers, 390 discernible DNA fragments were produced with 367 (93.95 %) being polymorphic. The polymorphic information content (PIC) values ranged from 0.948 to 0.976, with a mean PIC value of 0.969. Probability identity (PI) and discriminating power (D = 1 ? PI) among the primers ranged from 0.001 to 0.004 and 0.998 to 0.995, respectively. A binary qualitative data matrix was constructed. Data analyses were performed using the NTSYS software and the similarity values were used to generate a dendrogram via UPGMA. To study the drought stress, plants were irrigated at 25 % FC condition for three times. Fresh leaves were collected to measure physiological characters including: superoxide dismutase, catalase, and peroxidase activities and proline and total chlorophyll content at two times, before and after stress application. Relative water content, fresh and dry weight ratio, survival percentage, and visual quality were evaluated after stress. Morphological and physiological characters were assessed in order to classify accessions as either tolerant or sensitive using Ward’s method of Hierarchical cluster analysis in SPSS software. The results of present study demonstrated that the ISSR markers are useful for studying tall fescue genetic diversity. Convergence of morphological and physiological characterizations during drought stress and phylogenetic relationship results showed that accessions can be grouped into four clusters; drought-tolerant accessions that collected from west of Iran, drought-tolerant accessions collected from northwest of Iran, drought semi-tolerant accessions collected from center of Iran, and drought-sensitive accessions collected from north of Iran. Data presented could be used to classify the tall fescue accessions based on suitability of cultivation in the regions studied or the regions with the similar environmental condition.     

黄独遗传多样性研究

采用ISSR标记技术研究了我国14个黄独样品的遗传多样性.从55条简单重复序列引物中筛选出9条多态性引物,共扩增出70条带,其中67条多态性带,多态性比率为95.71%,平均每条引物扩增出7.8条带.黄独原变种内Nei s基因多样性(h)为0.294 9,有效等位基因数(Ne)为1.491 1,Shannon多样性指数(I)为0.444 8.种水平h为0.326 3,Ne为1.552 9,I为0.488 3.基因分化系数(Gst)为0.782 1,基因流(Nm)为0.139 3.聚类分析表明来自海南省和台湾省的样品与我国内陆的样品较早分离.据此可将来自我国内陆的样品分为5组.ISSR聚类分析基本上支持依据形态特征对黄独变种的划分.同时实验结果也表明,云南可能是黄独在我国的分化中心.     

Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers

Iran has a long history of acid lime cultivation and propagation. In this study, genetic variation in 28 acid lime accessions from five regions of south of Iran, and their relatedness with other 19 citrus cultivars were analyzed using Simple Sequence Repeat (SSR) and Inter-Simple Sequence Repeat (ISSR) molecular markers. Nine primers for SSR and nine ISSR primers were used for allele scoring. In total, 49 SSR and 131 ISSR polymorphic alleles were detected. Cluster analysis of SSR and ISSR data showed that most of the acid lime accessions (19 genotypes) have hybrid origin and genetically distance with nucellar of Mexican lime (9 genotypes). As nucellar of Mexican lime are susceptible to phytoplasma, these acid lime genotypes can be used to evaluate their tolerance against biotic constricts like lime “witches’ broom disease”.     

38份瓠瓜种质资源遗传多样性的ISSR分析

采用ISSR分子标记技术对源自中国7个省份的38份瓠瓜种质进行遗传多样性分析。12个ISSR引物共扩增出96条多态性带,平均每个引物扩增的多态性带数为8条,多态性比率平均为83.5%。聚类分析将供试的38份种质分为4个类群8组,主坐标分析将其分为4个类群10组。ISSR分子标记的分类结果与瓠瓜的农艺性状分类和地理分布有一定的相关性。     

RAPD和ISSR标记对水稻化感种质资源遗传多态性的分析

运用RAPD和ISSR技术分析水稻化感种质资源的遗传多态性。从供试材料中筛选到具有多态性的RAPD引物12条,ISSR引物7条。RAPD引物共扩增到85条清晰的多态性条带,多态性条带比率为69．4％。ISSR引物共扩增到34条清晰的多态性条带,多态性条带比率为53．0％。对两种标记结果进行UPGMA聚类分析,结果极其类似,呈极显著的正相关(r=0．74)。聚类结果表明,地理位置相近的品种聚为一类。部分具有较强化感作用潜力的水稻品种亲缘关系很近,表明控制其化感作用性状的基因可能是等位的相同基因。而部分化感作用潜力差异显著的水稻品种聚为一类,这是由于人类在长期高产品种的定向选择过程中,水稻化感作用性状不被注意而丢失,遗传基础日益狭窄的原因。     

基于ISSR数据探讨卷叶凤尾藓(Fissidens dubius P.Beauv.)遗传多样性

卷叶凤尾藓(Fissidens dubius P.Beauv.)是凤尾藓科凤尾藓属植物,该种分布广泛,形态变异强烈。为了解其遗传多样性及种群遗传结构特点,本研究利用ISSR分子标记对采集于浙江、福建、广西、四川、辽宁5个省区的卷叶凤尾藓14个自然种群的遗传多样性进行了评价。结果显示:筛选出的12对引物共扩增出259条清晰、重复性高的条带,其中多态性位点有248个,多态位点百分率为95.75%;种群总的Nei's基因多样性指数为0.2327,Shannon's信息指数为0.3701,说明卷叶凤尾藓遗传多样性水平较高;14个种群的遗传分化系数(Gst)为0.7078,种群间的基因流(Nm)为0.1864,表明大部分遗传变异(72.01%)存在于种群间,27.99%的遗传变异存在于种群内,即卷叶凤尾藓种群间遗传分化明显。基于ISSR数据的聚类分析表明,在遗传距离50为分组阈值处,14个种群可被分为6组(G1~G6):G1为来自于浙江省不同采集地点的8个种群(JHBS除外);G2由浙江金华北山种群(JHBS)组成;G3包括福建武夷山(WYS)和天宝岩种群(TBY);G4、G5、G6分别由广西龙胜县花坪种群(GX)、四川龙池种群(LC)、辽宁白石砬子保护区种群(BSLZ)组成,表明卷叶凤尾藓种群间的遗传分化主要由地理距离造成,种群内的遗传分化可能与其生境的异质性有关。     

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.     

Analysis of molecular genetic diversity in a representative collection of foxtail millet [Setaria italica (L.) P. Beauv.] from different agro-ecological regions of India

Foxtail millet [Setaria italica (L.) P. Beauv.], an important crop of East Asia is known for its drought tolerance and was once an indispensible crop of vast rainfed areas in semi-arid regions in India. In India it is cultivated in Andhra Pradesh, Karnataka, Maharashtra, Tamil Nadu, Rajasthan, Madhya Pradesh, Uttar Pradesh and north eastern states. The grain finds use in several local recipes such as roti (bread), jaula, singal, sirol. Foxtail millet grain contains 12.3 % protein, 4.7 % fat, 60.6 % carbohydrates, and 3.2 % ash. The present study was conducted to analyse the genetic diversity among foxtail accessions from different states of India and a few exotic accessions using RAPD and ISSR techniques and identify diverse accessions for use in variety improvement programmes. A set of 125 foxtail millet accessions selected from 11 different agro-ecological regions of India were analyzed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker techniques. A total of 146 (115 RAPD and 31 ISSR) scoreable markers were generated with 16 RAPD and four ISSR primers. The dendrogram generated using Nei’s genetic distances and principal component analyses revealed presence of two clusters and two subclusters in group I. The accessions from Andhra Pradesh, Karnataka, Maharashtra and Uttarakhand were more diverse since they were distributed in both the clusters. There was no clear geographical differentiation observable. The bootstrap support for the major groups identified was strong (above 80 %) indicating good statistical support. The average value of Nei and Li’s genetic distance was lowest (0.081) for accessions from West Bengal while the collections from Karnataka showed highest dissimilarity (average genetic distance = 0.239). The average genetic distance for all 125 accessions together was 0.177 indicating presence of only moderate genetic diversity in the collections. The analysis of molecular variance indicated that only 2.76 % variation was explained by variations among the groups and 11.55 % among populations within groups. However the percentage of variation observed within populations was high (85.68). The value of Fst was observed to be very low (0.028) indicating low differentiation of the accessions analysed. The population genetic analysis carried out indicates that highest number of alleles per locus (1.745 ± 0.438) was observed for Andhra Pradesh with 35 accessions. When four eco-geographic regions were considered, the southern region comprising AP, Karnataka and TN showed the highest number of alleles per locus (1.787 ± 0.411). The value of Gst was lowest for south (0.123) and highest for central west (0.455). This indicated that all the landraces from south share common alleles. The gene flow between the accessions from different regions was also observed to be high with the highest migration (3.557) recorded for south.     

Evaluation of Diversity Based on Morphological Variabilities and ISSR Molecular Markers in Iranian <Emphasis Type="Italic">Cynodon dactylon</Emphasis> (L.) Pers. Accessions to Select and Introduce Cold-Tolerant Genotypes

The main goals of the present study were to screen Iranian common bermudagrasses to find cold-tolerant accessions and evaluate their genetic and morphological variabilities. In this study, 49 accessions were collected from 18 provinces of Iran. One foreign cultivar of common bermudagrass was used as control. Morphological variation was evaluated based on 14 morphological traits to give information about taxonomic position of Iranian common bermudagrass. Data from morphological traits were evaluated to categorize all accessions as either cold sensitive or tolerant using hierarchical clustering with Ward’s method in SPSS software. Inter-Simple Sequence Repeat (ISSR) primers were employed to evaluate genetic variability of accessions. The results of our taxonomic investigation support the existence of two varieties of Cynodon dactylon in Iran: var. dactylon (hairless plant) and var. villosous (plant with hairs at leaf underside and/or upper side surfaces or exterior surfaces of sheath). All 15 primers amplified and gave clear and highly reproducible DNA fragments. In total, 152 fragments were produced, of which 144 (94.73%) being polymorphic. The polymorphic information content (PIC) values ranged from 0.700 to 0.928. The average PIC value obtained with 15 ISSR primers was 0.800, which shows that all primers were informative. Probability identity (PI) and discriminating power between all primers ranged from 0.029 to 0.185 and 0.815 to 0.971, respectively. Genetic data were converted into a binary data matrix. NTSYS software was used for data analysis. Clustering was done by the unweighted pair-group method with arithmetic averages and principle coordinate analysis, separated the accessions into six main clusters. According to both morphological and genetic diversity investigations of accessions, they can be clustered into three groups: cold sensitive, cold semi-tolerant, and cold tolerant. The most cold-tolerant accessions were: Taft, Malayear, Gorgan, Safashahr, Naein, Aligoudarz, and the foreign cultivar. This study may provide useful information for further breeding programs on common bermudagrass. Selected genotypes can be evaluated for other abiotic stresses such as drought and salinity.