Inter-simple Sequence Repeat Fragments Research Articles

Genetic evaluation of Garcinia gummi-gutta L. (Roxb.) accessions based on inter simple sequence repeat markers

Garcinia gummi-gutta (Family: Clusiaceae) is a rich source of a natural anti-obesity agent, hydroxy citric acid (HCA). The present approach aimed to analyse the extent of genetic variability and structure of a population of 50 accessions of G. gummi-gutta from different geographical localities of Kerala state, India using inter simple sequence repeat (ISSR) markers. Among 30 primers tested, 21 showed clear, reproducible and scorable banding patterns. A total of 157 ISSR fragments were observed with an average of 7.48 bands for each primer. An average of 59.52% polymorphic bands were observed among the accessions, with the highest percentage was observed for UBC 809 (87.5%). Polymorphism information content (PIC) value of 0.355 indicating high degree of allele divergence within the individuals. The highest values for Simpson (0.99) and Shannon indices (1.02) substantiated the significant level of polymorphism within the population. Dendrogram with bootstrapping values (100) based on the neighbour joining method distributed the accessions into three main clusters. Relationships exhibited by the accessions discloses continuous geneflow happening between them irrespective of their geographical distances. STRUCTURE analysis revealed the probability of eight subpopulations based on Bayesian clustering. So, ISSR markers can be used as a powerful tool to differentiate closely related individuals.

Association of Genetic Structure and Diversity in Iranian Wild Germplasms of Mentha longifolia L. Based on Phenotypical, Biochemical, and Molecular Markers.

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.

Molecular characterization of endangered Iranian terrestrial orchids using ISSR markers and association with floral and tuber-related phenotypic traits.

The online version of this article (10.1007/s12298-020-00920-0) contains supplementary material, which is available to authorized users.

Genetic structure, molecular and phytochemical analysis in Iranian populations of Ruscus hyrcanus (Asparagaceae)

Ruscus hyrcanus Woronow a commercially important ornamental and medicinal plant belonging to the Asparagaceae family, has various applications in Iranian traditional medicine due to its diuretic, appetizing, cathartic, vasoconstrictor, anti-hemorrhage, ant-nephritis, anti-infection, aperient and anti-varicose properties. In the present study, morphological, phytochemical and molecular markers were investigated in twelve R. hyrcanus (RH) populations collected from the Hyrcanian ecoregion in the north of Iran. Phytochemical characteristics were remarkably variable among the studied populations, but little morphological variation was observed. The highest contents of ruscogenin and neoruscogenin in the underground parts of the plant were recorded in populations RH9 (Abbasabad 3.41 mg/g DW) and RH6 (Darab Kola 1.44 mg/g DW), respectively. A total of 66 bands were obtained from 11 inter-simple sequence repeat (ISSR) markers, 62 (93.94 %) of which were polymorphic. Although the ISSR evaluation indicated an inter-population genetic differentiation (GST = 0.27), most of the genetic diversity was intra-populational (PPB = 32.26–75.81 %, He = 0.11−0.24, I = 0.17−0.36). A UPGMA cluster analysis of 120 samples of R. hyrcanus was carried out to categorize the studied populations, based on a molecular data matrix calculated with Jaccard’s similarity coefficient. A multiple regression analysis of the relationships between the ISSR bands and morphological and phytochemical traits demonstrated that eight ISSR fragments were related to ruscogenin in the plant materials. Several ISSR markers showed association with some morphological traits. This study provided the comprehensive preliminary data on morphological, phytochemical, and genetic characteristics of R. hyrcanus populations in order to exploit genetic reserves conservation programs.

Genetic Diversity among Isolates of Phytophthora sojae in Anhui Province of China based on ISSR-PCR Markers

To explore genetic differentiation and the genetic relationships of Phytophthora sojae in Anhui Province, the inter-simple sequence repeat (ISSR) technique was used to analyze the genetic diversity of P. sojae. One hundred and sixty ISSR fragments were observed, including 129 (80.6%) polymorphic bands. This suggested that abundant genetic diversity existed among P. sojae in Anhui Province. The pairwise genetic similarity coefficients among the 62 strains ranged from 0.72 to 0.96, with a mean value of 0.85, indicating that there was a high level of genetic variation. Phytophthora sojae strains were divided into five clusters based on neighbor-joining (NJ) analysis, and the clustering was not related to geographic source, according to Mantel’s test (r = 0.3938). In addition, the clustering of strains from the same geographical source had little relationship to the year of collection. Analysis of molecular variance (AMOVA) showed that 16.65% of the genetic variation was derived from the collection area and 83.35% of the genetic variation was within-population variation. The genetic flow between different geographical sources ranged from 0.623 to 2.773, with a mean of 1.325, suggesting that gene exchange was frequent. Genetic distance and the genetic differentiation coefficient were not related to spatial distance.

Assessment of genetic diversity of <i>Prangos fedtschenkoi</i> (Apiaceae) and its conservation status based on ISSR markers

Prangos fedtschenkoi (Regel et Schmalh.) Korovin (Apiaceae) is an endemic species for moun-tainous Middle Asia, which is both a rare and useful plant. Organic extractions from this species are being used in pharmaceutics and cosmetology. In recent years, P. fedtschenkoi distribution area has considerably decreased, presumably, resulting from human activities such as agriculture, construction works, overgrazing and collection from wild for pharmaceutic purposes. Six populations were found in Uzbekistan and their genetic divergence and differentiation were studied with 10 inter-simple sequence repeat (ISSR) markers, selected out of 101. Totally 166 amplified ISSR fragments (loci) were revealed, of which 164 were polymorphic. Relatively mod-erate level of polymorphism was found at population level with polymorphic bands ranging from 27.71% to 47.59%. Mean P = 39.05%, Na=1.40,Ne=1.25, S.I. = 0.21, and He = 0.14 were revealed for all loci across six populations. AMOVA showed higher variation among populations (62%) than within them (38%). The Bayes-ian model determined 5 clusters, or genetic groups. The posteriori distribution of the Theta II estimator detected full model identifying high inbreeding, intensified by low gene flow (Nm = 0.3954). Mantel test confined pop-ulation 6 as distinct cluster corresponding to geographic remoteness (R = 0.5137, p ≤ 0.005). Results were used as the bases for developing conserve measures to restore populations.

Population Genetic Structure and Marker Trait Associations Using Morphological, Phytochemical and Molecular Parameters in Habenaria edgeworthii-a Threatened Medicinal Orchid of West Himalaya, India.

Habenaria edgeworthii Hook. f. ex Collett is an important terrestrial orchid used in different Ayurvedic formulations. In the present study, variations among morphological, phytochemical and molecular markers were assessed. A significant difference was observed among populations using morphological traits. Inter-simple sequence repeat (ISSR) data revealed lower genetic diversity at population level (He=0.207) as compared to species level (He=0.334). Analysis of molecular variance (AMOVA) indicates 74% variation among populations and 26% within population. Tuber extracts showed significantly (p<0.05) higher total phenolics and flavonoids among the populations. Antioxidant activity determined by 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays exhibited considerable antioxidant potential. Furthermore, the associations between molecular and morphological and phytochemical attributes were studied using multiple regression analysis (MRA). Several ISSR fragments were associated with some morphological and phytochemical traits. These ISSR fragments can be useful for breeding programme of the species when no other genetic information, such as linkage maps and quantitative trait loci, is available.

Genetic Relationship Among 36 Genotypes of &amp;lt;i&amp;gt;Ceiba pentandra&amp;lt;/i&amp;gt; (L.) as Revealed by RAPD and ISSR Markers

<i>Ceiba pentandra</i> (L.) Gaertn (trade name Ceiba) of the family Bombacaceae is an important multi-purpose tree species in Ghana and demand for it is rising daily. As a result, it has been included as one of the species for the National Forest Plantation Development Programme of Ghana as part of the efforts towards its restoration. Seedlings for the programme are being raised from seed collected from the wild without regard for their genetic quality and its future adverse effects. The absence of adequate information on the genetic diversity within the species therefore serves as a potential threat to its long term sustainable management and efficient genetic conversation. The phylogenetic relationships of 36 genotypes of <i>C. pentandra</i> from natural range of the species in five ecological zones (populations) of Ghana were therefore analyzed using 10 polymerase chain reaction (PCR) markers (5 random amplified polymorphic DNAs (RAPDs) and 5 inter simple sequence repeats (ISSRs)). The principal component analysis (PcoA) defined by axis 1 and 2 accounted for 67.15% of the variation observed. Cluster analysis using GeneStat Discovery showed that, 14 (38.89%), 21 (58.33%) and 4 (11.11%) of the accessions had a coefficient of similarity of 1 from the RAPD, ISSR and the combined RAPD and ISSR polymorphism respectively. The most distantly related accessions from the RAPD polymorphism analysis were BAW 1 and KON 8 with a similarity coefficient of 0.06 whereas those from the ISSR polymorphism were BAW 10 and ASE 1 with 21% similarity between them. Similarly, BUF 1 and KUE 1 were the most dissimilar accessions from the combined RAPD and ISSR fragments analysis with a similarity coefficient of 0.23. Nine (25%) accessions with high degree of dissimilarity between them were identified in the study. These accessions could serve as good candidates for conservation as seed trees and in breeding programs of <i>C. pentandra</i> in Ghana. Implications of the findings for effective management of the genetic resources of the species were also discussed.

Genetic divergence through joint analysis of morphoagronomic and molecular characters in accessions of Jatropha curcas.

The aim of this study was to investigate the genetic divergence between accessions of Jatropha curcas through joint analysis of morphoagronomic and molecular characters. To this end, we investigated 11 morphoagronomic characters and performed molecular genotyping, using 23 inter-simple sequence repeat (ISSR) primers in 46 accessions of J. curcas. We calculated the contribution of each character on divergence using analysis of variance. The grouping among accessions was performed using the Ward-MLM (modified location model) method, using morphoagronomic and molecular data, whereas the cophenetic correlation was obtained based on Gower's algorithm. There were significant differences in all growth-related characteristics: number of primary and secondary branches per plant, plant height, and stem diameter. For characters related to grain production, differences were found for number of fruit clusters per plant and number of inflorescence clusters per plant and average number of seeds per fruit. The greatest phenotypic variation was found in plant height (59.67- 222.33 cm), whereas the smallest variation was found in average number of seeds per fruit (0-2.90), followed by the number of fruit clusters per plant (0-8.67). In total, 94 polymorphic ISSR fragments were obtained. The genotypic grouping identified six groups, indicating that there is genetic divergence among the accessions. The most promising crossings for future hybridization were identified among accessions UFRB60 and UFVJC45, and UFRB61 and UFVJC18. In conclusion, the joint analysis of morphoagronomic characters and ISSR markers is an efficient method to assess the genetic divergence in J. curcas.

Chromosome Number, Genomic Variation, and Molecular Markers to Assess Genetic Diversity of Brachiaria Species

ABSTRACTThe genus Brachiaria contains approximately 100 identified species, including economically important tropical grasses; however, there is not much accessible information about the genetics or genomes of the genus. In this study, we determined the chromosome number and genome size by cytological analysis and flow cytometry, respectively, and estimated the molecular variability using intersimple sequence repeat (ISSR) markers for 28 accessions representing 11 Brachiaria species. The chromosome number of 10 species agreed with previous reports, and the chromosome number for B. xantholeuca was evaluated (2n = 4x = 36) for the first time. Genome size expressed as C values differed among these species (0.89 to 4.02 pg/2C) and increased with increasing ploidy levels. In contrast, the genome size given as a Cx value tended to decrease with increasing ploidy levels in polyploid plants. A total of 441 ISSR fragments were generated with 10 primers to assess the molecular variability of Brachiaria species. All 11 Brachiaria species clustered into three groups with the neighbor‐joining (NJ) method. We found no correlation between the clusters and the region from where the plants were collected. Nevertheless, the clustering of accessions was clearly correlated with ploidy level and reproductive mode (sexual and apomictic) except for a few accessions. Furthermore, evolution of the Brachiaria genome and genetic diversity within the genus were discussed.

Genetic diversity within a newly identified population of Adenophora liliifolia (L.) A.DC. in Romania: implications for conservation

Adenophora liliifolia is a relict plant species, endangered at European level. Its occurrence in Romania is limited to a very few sites. The amount of genetic diversity of plant species is a valuable indicator of population, being the baseline in developing proper strategies for their conservation. Inter-simple sequence repeats (ISSRs) markers were used to analyze polymorphism in A. liliifolia genome and to evaluate the genetic diversity and accordingly, the state of a recently identified population. Five ISSR primers, specially designed for plants, and containing different simple sequence repeat motifs were tested. A total of 52 ISSR fragments were generated of which 41 were polymorphic (78.84%) and 32 (61.53%) specific to Adenophora genus. The value of Shannon’s index of genotypic diversity was 0.812. Jaccard similarity coefficient was calculated for pair wise comparisons among all individuals and ranged from 0.17 to 0.83. The genetic variability between individuals was 78.84% which suggests a relative high genetic differentiation. Although the level of genetic variability is moderate to high, the population is declining and exposed to demographic stochasticity. A possible cause is species germination requirements hampered by modification in vegetation structure and abundance. The population survival and reinforcement is conditioned by urgent measures for forest management in order to reduce herbaceous and shrubby vegetation and to limit mowing and grazing. Ex situ conservation measures are also proposed.

Comparative analysis of ISSR marker polymorphism in populations of yak (Bos mutus) and in F1 hybrids between yak and cattle in the Sayan-Altai region

The genetic variability in seven yak populations from the Sayan-Altai region and in F1 hybrids between yak and cattle (khainags) was investigated with the help of a technique that involves the use of inter simple sequence repeat (ISSR) markers generated with PCR primers (AG)9C and (GA)9C. Samples for the analysis were collected in Mongolia, Tuva, and Altai from 2008 through 2012. The examined yak populations differed in in the presence/absence of ISSR fragments, as well as in their frequency. In total, 46 ISSR fragments were identified using two marker systems; the proportion of polymorphic loci constituted 76% and 90% for the AG-ISSR and GA-ISSR markers, respectively. For the total sample of yaks, total genetic diversity (Ht), within-population diversity (Hs), and interpopulation diversity (Gst) constituted 0.081, 0.044, and 0.459 for the AG-ISSR and 0.137, 0.057, and 0.582 for the GA-ISSR markers, respectively. Based on ISSR finger printing, species- and breed-specific DNA patterns were described for the three groups of animals (yaks, cattle, khainags). For the domestic yak, the species-specific profile was represented by eight ISSR fragments. Genetic relationships between the yak populations, cattle breeds, and khainags were examined with the help of four different approaches used in the analysis of population structure: estimation of phylogenetic similarity, multidimensional scaling, principal component analysis, and cluster analysis. Clear evidence on the differentiation of the populations examined at the interspecific, as well as at intraspecific, level were obtained. Similar (relative); as well as remote (isolated), yak populations were identified. Khainags occupy an intermediate position between yak and cattle. However, the data on the ISSR-PCR marker polymorphism (genome polymorphism, population structure).indicate that part of the analyzed khainag genome was more similar to the yak genome than to the cattle genome.

Mutagenesis in olive (Olea europaea L.) calli caused by sodium azide and detection of mutants using ISSR and RAPD markers

SummaryIn vitro callus formation was established in the olive (Olea europaea L.) cultivar ‘Kronaki’ and in vitro mutagenesis of calli was carried out using five different concentrations (1.0 – 5.0 mM) of sodium azide (NaN3). Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to detect genetic polymorphism among the olive mutants produced. Six RAPD and seven ISSR primers were used to amplify DNA segments from the genomes of untreated and NaN3-treated calli. The optimum medium for callus formation was 0.5× Murashige and Skoog (MS) medium, supplemented with 2 mg l–1 αnaphthaleneacetic acid and 0.5 mg l–1 2-isopentenyl-adenine. A total 60 RAPD amplicons and 60 ISSR fragments were detected. The sizes of the PCR amplicons varied from 200 – 3,000 bp in length. Fifteen of the 60 RAPD (40%) and ten of the 60 ISSR (16.6%) fragments were polymorphic. The results showed that 4.0 mM and 5.0 mM NaN3 were the most effective doses for mutagenesis. Based on RAPD, ISSR, and combined RAPD and ISSR marker analysis, it was shown that all NaN3 treatments caused detectable levels of mutation. The use of NaN3 for in vitro mutagenesis in olive calli resulted in genetic dissimilarity values of 0.14 – 0.21, and represents a useful way to induce mutants in olive which could be exploited in the future for trait improvement and in olive breeding programmes.

Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium 'Earsakul' mutants.

In vitro mutagenesis of Dendrobium 'Earsakul' was carried out by incubating the protocorm-like bodies in 0-5 mM sodium azide for 1 h. Twenty-eight putative mutants were evaluated for genetic variability compared to untreated control plants using inter-simple sequence repeat (ISSR) analysis. Polymorphic fragments were produced by 9 of 12 ISSR primers. A total of 173 amplified ISSR fragments varying in size from 140 to 5000 bp were obtained, 39 of which were polymorphic (22.5%). Of the 28 putative mutants, 15 (53.6%) showed altered genetic profiles compared to control and were identified as mutants. These results suggest that sodium azide can be effectively utilized to generate mutants in Dendrobium 'Earsakul', and ISSR provides a powerful tool that allows efficient early detection of these mutants. The identified mutants are currently being multiplied for further evaluation of their horticultural characteristics.

Development of an ISSR based STS marker for sex identification in pointed gourd (Trichosanthes dioica Roxb.)

Pointed gourd (Trichosanthes dioica Roxb.), a perennial dioecious plant of the family Cucurbitaceae has recently emerged as an important cash crop due to its great medicinal and nutritive value. Breeding programmes in pointed gourd have several constraints such as poor germination, vegetative means of propagation and dioecy. Identification of male and female at the seedling stage is a pre-requisite for the genetic improvement of this species. Screening of genomic DNA with inter simple sequence repeat (ISSR) primers was used for early detection of male and female plants in this study. Using pooled DNA from male and female genotypes and 40 ISSR primers, a putative male specific marker TdMSM was produced. It was converted into a sequence tagged marker TdSTSM which could successfully amplify 720bp product in male but not in the female. Southern blotting confirmed it as a single copy locus found only in male plant. Further, the TdSTSM marker was used to accurately detect 9 males and 6 females from 15 individuals of pointed gourd with unknown sex. Thus, the STS primers designed from ISSR fragment TdMSM could be used as an effective and reliable molecular marker for early sex diagnosis in T. dioeca.

Exploring the genetic diversity of Cynodon radiatus (Poaceae) accessions using ISSR markers

Abstract Inter-simple sequence repeat (ISSR) markers were applied to determine the genetic relationship among 29 Cynodon radiatus accessions from different regions of China. Fifty ISSR primers were screened, and 14 primers were selected for their capability to produce clear and reproducible patterns of multiple bands to amplify specific C. radiatus genomic sequences. A total of 189 ISSR fragments from 300 bp to 3000 bp were amplified, of which 98.94% were polymorphic. Genetic similarity coefficients among the 29 accessions ranged from 0.45 to 0.90, with an average of 0.68. Cluster analysis by 2 methods, unweighted pair-group method with arithmetic averages and principle coordinate analysis, showed that the C. radiatus accessions clustered into 4 distinct groups. A high level of variation exists in C. radiatus accessions. The present study shows that the ISSR technique is a powerful method for the identification and molecular classification of C. radiatus accessions, and for the determination of their genetic relationships.

Genetic variation, population structure and identification of yellow catfish, Mystus nemurus (C&amp;V) in Thailand using RAPD, ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei's gene diversity (H) and Shannon's information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.

Estimation of genetic variation in Cynodon dactylon accessions using the ISSR technique

Genetic variation in 55 accessions of Cynodon dactylon was estimated using inter-simple sequence repeat (ISSR) markers. The plant materials used in this study originated from 17 countries. A total of 236 ISSR fragments were generated with 14 primers. Fragment sizes ranged from 200 to 3000 bp. All scorable bands were polymorphic in nature and none of the primers used produced monomorphic bands, indicating a high level of genetic variation in this grass. The accessions were found to be clustered into eight major groups through the unweighted pair-group method with arithmetic averages. Genetic similarity coefficients (GSC) among the 55 accessions ranged from 0.52 to 0.95. The results clearly indicate that a high level of variation exists in Cynodon accessions. This study shows that the ISSR technique is a reliable tool for differentiating Cynodon accessions and for determining the genetic relationships among them.

Variety identification and genetic relationships of mungbean and blackgram in Thailand based on morphological characters and ISSR analysis

Genetic diversity and relatedness were measured in 17 mungbean (Vigna radiata (L.) Wilczek) and 5 blackgram (Vigna mungo (L.) Hepper) genotypes by means of inter-simple sequence repeat (ISSR) analysis and morphological characters. Unweighted pair-group method arithmetic average (UPGMA) analysis of 19 morpho-agronomic characters showed clear separation of the genotypes into 3 major groups; cluster I containing 15 Thai cultivated mungbean varieties and breeding lines, cluster III containing 4 Thai cultivated blackgram varieties, and cluster II containing a mungbean wild relative (subspecies sublobata), a blackgram wild relative (subspecies silvestris) together with an Indian mungbean landrace. Pair wise coefficients of genetic similarity between all genotypes ranged from 0.17 to 0.84 with an average of 0.52. In total, 341 ISSR fragments were amplified for the two Vigna species by ISSR analysis using 18 ISSR primers. The polymorphism revealed with the ISSR primers was 90.6%. The number of amplified fragments varied from 9 to 32, with a size range of 200 - 1500 bp. The average numbers of fragments per primer and polymorphic fragments per primer were 18.94 and 17.17, respectively. Polymorphism information content (PIC) values ranged from 0.23 to 0.37 with an average of 0.31 across all the genotypes. Pair wise coefficients of ISSR-based genetic similarity between all genotypes ranged from 0.70 to 0.99 with an average of 0.86, suggesting quite narrow genetic base of mungbean and blackgram in Thailand that might limit continued breeding success. UPGMA analysis based on ISSR exhibited 2 major clusters; cluster I containing all mungbean genotypes and cluster II containing all blackgram genotypes. It appeared that ISSR was more effective for classification at the species level although no clear separation at the subspecies level was found. All 22 mungbean and blackgram genotypes can be effectively distinguished by only 6 ISSR primers with the highest PIC values, suggesting the applicability of ISSR analysis for variety identification.

Differentiation of homokaryons and heterokaryons of Agaricus bisporus with inter-simple sequence repeat markers

Morphologically different fifteen slow growing single spore isolates (SSIs) were screened from germinated basidiospores of Agaricus bisporus; assumed to be homokaryons, and subjected to growth rate, spawn run, fruiting and inter-simple sequence repeat (ISSR) analysis for confirmation of true homokaryons. The present studies are the first report of fingerprinting on differentiating homo- and hetero-karyotic SSIs using ISSR markers. The patterns were highly polymorphic and very reproducible. Among 40 primers tested, 7 ISSR primers were selected for the analysis of genomic DNA and generated a total of 54 ISSR fragments, sufficient to differentiate the 15 isolates from each other. ISSR fingerprinting detected 46.30% polymorphic loci. All appressed homokaryons carried a subset of ISSR markers found in the heterokaryons, and these were not able to produce a fruiting body. A test of cross-fertility and the following fruiting trial proved that 3 of the 15 SSIs with different ISSR fingerprints were homokaryons. None of the discarded SSIs have any missing bands present in the parental heterokaryotic control; these are heterokaryons derived from non-sister nuclear pairs. It is revealed that homokaryons are probably restricted to the assumed morphological classes of SSIs. These results demonstrate that ISSR markers provide an efficient alternate for identification of homokaryons and suggest these markers be considered as new tools for the survey of Agaricus species.