Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversity assessment of the rice blast pathogen (Magnaporthe grisea).

This present study is the first report of the application of the retrotransposon-microsatellite amplified polymorphism (REMAP) technique in fungi. Genome fingerprinting has a major role in the characterization of population structure and in the analysis of the variability in fungi. Retrotransposon-microsatellite amplified polymorphism assay was used in virulent isolates of a rice blast pathogen (Magnaporthe grisea) as a new assay system for genetic variability studies that overcomes the limitations of previous techniques. The high polymorphism observed in REMAP could be due to past or recent actions of retrotransposon in M. grisea. Retrotransposon-microsatellite amplified polymorphism, with its superior marker utility, was concluded to be the marker of choice for characterizing M. grisea isolates.     

Assessment of genetic diversity among and within Carthamus species using sequence-related amplified polymorphism (SRAP) markers

Due to precise evaluation of genetic diversity of Carthamus species, sixty-two genotypes consisting fifty-two from five wild (C. oxyacanthus M. Bieb, C. lanatus L., C. dentatus Vahl, C. boissieri Halácsy, C. glaucus M.B.) and ten from cultivated species (C. tinctorius L.) were selected for evaluation of the genetic diversity in Carthamus species. A total of 238 (81.2 %) polymorphic bands were detected by 12 SRAP primer combinations with an average of 22 bands per combination. Me4-Em1 and Me5-Em2 primer combinations were known as the most informative SRAP markers based on the PIC values (0.34) where they distinguished all studied Carthamus species. Cluster analysis classified all accessions into five main groups among which clusters containing cultivated individuals were distinctly separated from those containing wilds. The most and the least genetic variation based on analysis of molecular variance, were detected within (76.90 %) and among (22.84 %) groups, respectively. The obtained results suggested that C. dentatus, C. glaucus and C. boissieri species may be classified in one section including C. dentatus in one and C. glaucus and C. boissieri in another subsection. The results also revealed high genetic similarity between C. oxyacanthus and C. tinctorius despite their different morphological characteristics.     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.Communicated by B. Friebe     

Assessment of genetic diversity and relatedness among Tunisian almond germplasm using SSR markers

Genetic diversity of 50 Tunisian almond (Prunus dulcis Mill.) genotypes and their relationships to European and American cultivars were studied. In total 82 genotypes were analyzed using ten genomic SSRs. A total of 159 alleles were scored and their sizes ranged from 116 to 227 bp. The number of alleles per locus varied from 12 to 23 with an average of 15.9 alleles per locus. Mean expected and observed heterozygosities were 0.86 and 0.68, respectively. The total value for the probability of identity was 4 × 10(-13) . All SSRs were polymorphic and they were able all together to distinguish unambiguously the 82 genotypes. The Dice similarity coefficient was calculated for all pair wise and was used to construct an UPGMA dendrogram. The results demonstrated that the genetic diversity within local almond cultivars was important, with clear geographic divergence between the northern and the southern Tunisian cultivars. The usefulness of SSR markers for almond fingerprinting, detection of synonyms and homonyms and evaluation of the genetic diversity in the Tunisian almond germplasm was also discussed. The results confirm the potential value of genetic diversity preservation for future breeding programs.     

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03–0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers

The silkworm, Bombyx mori is a beneficial insect of great economic importance in China for its silk production. In this study, we obtained 11 cleaved amplified polymorphic sequence (CAPS) markers and one PCR polymorphism marker from the genes of the silkworm, B. mori. A backcross progeny analysis showed that all these molecular markers were segregated in a Mendelian fashion and that polymorphisms were co-dominant. These markers were used to investigate the genetic diversity among 29 strains of B. mori from China, Japan and Europe. Cluster analysis, based on the genetic similarities calculated from CAPS data, grouped these strains roughly according to their geographical origin. One group contained silkworm strains from Europe and some of the Japanese strains were interspersed into the Chinese groups, whereas other Japanese strains clustered together.     

Evaluation of genetic diversity within asparagus germplasm based on morphological traits and ISSR markers

Asparagus officinalis L. is a dioecious perennial plant globally known for its fine flavor and high nutritional value. An evaluation of genetic diversity in 46 asparagus accessions was carried out based on morphological and inter-simple sequence repeat (ISSR) markers. The result show that the coefficient of variation for 20 morphological characteristics is between 12.45 and 62.22%. Factor analysis revealed that nine factors explained 83.37% of the total variance. At Euclidean distance of 135.7, 46 accessions were divided into two clusters. Genetic similarity coefficient (GSC) based on ISSR data ranged from 0.60 to 0.97, suggesting a relatively abundant genetic base. Furthermore, the 46 asparagus accessions could also be grouped into three major clusters at a GSC of 0.74. And there is no significant relation between the two marker systems using the Mantel test. Clustering based on morphological traits compared with that based on ISSR data was not consistent, however, some common groupings were observed between two dendrograms. Therefore the results elucidated asparagus germplasm genetic background and determined hybrid parents, which will facilitate optimal application of asparagus germplasm resources and provide additional data for genetic improvement.

     

Construction of a rat genetic map by using randomly amplified microsatellite polymorphism (RAMP) markers

Many rat strains have been employed in the genetic study of quantitative traits such as blood pressure. In such genetic studies, it is essential to prepare rat genetic maps fine enough to identify the genes regulating quantitative traits. However, it is not an easy task to isolate a sufficient number of genetic markers polymorphic between a particular pair of rat strains. In this study, we applied the randomly amplified microsatellite polymorphism (RAMP) method, a simple method to identify co-dominant markers (Wu et al. Nucleic Acids Res 22, 3257, 1994), to isolate markers polymorphic between the stroke-prone spontaneously hypertensive rat and the Wistar-Kyoto rat, a genetically hypertensive strain and its normotensive control strain, which share a common genetic background. We successfully identified 111 RAMP markers distributed throughout the rat genome after screening 3046 sets of primers. We also showed that we could isolate ordinary simple-sequence-length-polymorphism markers by cloning RAMP markers. The RAMP method is a simple and efficient way to identify co-dominant genetic markers on mammalian genomes. Received: 10 October 1997 / Accepted: 16 March 1998     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Genetic relationships among wild and cultivated blackberries (Rubus caucasicus L.) based on amplified fragment length polymorphism markers

Abstract

Rubus is a large genus of flowering plants in the rose family, Rosaceae, subfamily Rosoideae. The blackberries, as well as various other Rubus species with mounding or rambling growth habits, are often called brambles. Little information is available on the genetic diversity of wild-grown blackberries. The objective of this study was to determine the genetic relationships among nine promising (high-yield capacity, free of pest and diseases, better fruit traits) wild blackberry (Rubus caucasicus L.) selections and the well-known cultivar, “Chester” by using amplified fragment length polymorphism (AFLP) markers. Genotypes were evaluated with three selective primer-enzyme combinations, producing a total of 223 AFLP fragments with 53% polymorphism ratio. Clustering of genotypes using unweighted pair-group method of arithmetic average cluster analysis clearly separated groups of wild blackberry genotypes while the variety “Chester” was clustered independently. Wild selections represented a distinct germplasm source on the basis of the estimated genetic distance among them. Genetic diversity data from this study will be helpful in using and exploiting the wild genetic material for breeding purposes as well as for further research.     

Genetic characterization of Iranian native Bombyx mori strains using amplified fragment length polymorphism markers

Genetic relationships within and among seven Iranian native silkworm strains was determined by DNA fingerprinting by using amplified fragment length polymorphism (AFLP) markers. In total, 189 informative AFLP markers were generated and analyzed. Estimates of Nei's gene diversity for all loci in individual strains showed a higher degree of genetic similarity within each studied strain. The highest and the least degrees of gene diversity were related to Khorasan Pink (h = 0.1804) and Baghdadi (h = 0.1412) strains, respectively. The unweighted pair-group method with arithmetic average dendrogram revealed seven strains of silkworm, Bombyx mori (L.), resolving into two major clusters. The highest degree of genetic similarity was related to Baghdadi and Harati White, and the least degree was related to Guilan Orange and Harati Yellow. The genetic similarity estimated within and among silkworms could be explained by the pedigrees, historical and geographical distribution of the strains, effective population size, inbreeding rate, selection intensity, and gene flow. This study revealed that the variability of DNA fingerprints within and among silkworm strains could provide an essential basis for breeders in planning crossbreeding strategies to produce potentially hetrotic hybrids in addition to contributing in conservation programs.     

Assessment of genetic diversity patterns in Chilean quinoa (Chenopodium quinoa Willd.) germplasm using multiplex fluorescent microsatellite markers

Quinoa (Chenopodium quinoa Willd.) is a staple seed crop in the Andean region of South America. Improving quinoa productivity is a primary food-security issue for this region, and has been part of the impetus for the establishment of several new quinoa breeding programs throughout the Andean region. Chilean quinoa has been characterized as morphologically diverse and bifurcated into coastal and highland ecotypes. The success of emerging breeding programs will rely heavily on the development of core germplasm collections and germplasm evaluation—especially of the coastal quinoa ecotypes that are often neglected in traditional breeding programs. Thus, the objective of this study was to characterize and quantify the genetic diversity within 28 Altiplano and 31 coastal Chilean accessions of quinoa using microsatellite markers. To facilitate the analysis, we also report the development of seven sets of fluorescent multiplexed microsatellite PCR reactions that result in genetic information for 20 highly polymorphic microsatellite loci. A total of 150 alleles were detected among the quinoa accession, ranging from 2 to 20 alleles per locus and an average 7.5 allele/locus. Both cluster (UPGMA) and principal component analyses separated the accessions into two discrete groups. The first group contained quinoa accessions from the north (Andean highlands) and the second group consisted of accessions from the south (lowland or coastal). Three accessions from Europe were classified into the southern quinoa group. The data obtained in the diversity analyses highlights the relationships within and among northern and southern Chilean quinoa accessions and provides the quinoa scientific community with a new set of easy to use and highly informative genetic markers.     

Assessment of genetic diversity of landraces of Dendrocalamus hamiltonii using AFLP markers and association with biochemical traits

Fermented bamboo shoots are popular traditional food items of various ethnic groups of the northeastern India, especially in Manipur State. Dendrocalamus hamiltonii is an economically important bamboo species used to produce fermented bamboo shoots. We studied genetic variability of this bamboo species in Chandel and Imphal-East (commercial production districts), using AFLP molecular markers. Each of the selected primers detected polymorphisms and 1614 (95.8%) were found to be polymorphic. Cluster analysis based on Dice similarity coefficients using UPGMA differentiated the populations into two major groups. Principal coordinate analysis based on the AFLP data clearly separated the populations according to their genetic diversity and antioxidant activity. Four primers were tested through multiple regression analysis to identify marker-trait association between AFLP data and biochemical attributes, i.e., antioxidant activity and total cyanide content. The 273 bp generated by EcoRI-AAG(Joe)/MseI-CTC showed high positive correlation with antioxidant activity (r = 0.729, P < 0.01). The 396 bp generated by EcoRI-AAC(Ned)/MseI-CTG were negatively correlated with cyanide content (r = -0.694, P < 0.01). Thus, we found association of DNA markers with antioxidant activities and total cyanide content. These results could be of use for the identification of superior genotypes with desirable traits.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

A comparative analysis of genetic polymorphism in wild and cultivated barley from Tibet using isozyme and ribosomal DNA markers.

This study was conducted to address some of the issues concerning the possible significance of Tibet in the origin and evolution of cultivated barley. A total of 1757 barley accessions from Tibet, including 1496 entries of Hordeum vulgare ssp. vulgare (HV), 229 entries of the six-rowed wild barley H. vulgare ssp. agriocrithon (HA), and 32 entries of the two-rowed wild barley H. vulgare ssp. spontaneum (HS), were assayed for allozymes at four esterase loci. A subsample of 491 accessions was surveyed for spacer-length polymorphism at two ribosomal DNA loci. Genetic variation is extensive in these barley groups, and the amount of genetic diversity in cultivated barley of this region is comparable with that of cultivated barley worldwide. The level of genetic variation of HA is significantly lower than the other two barley groups, and there is also substantial heterogeneity in the level of polymorphism among different agrigeographical subregions. However, little genetic differentiation was detected among the three barley groups (HV, HA, and HS), as well as among different agrigeographical subregions. Comparison of the results from this and previous studies indicated a strong differentiation between Oriental and Occidental barley, thus favoring the hypothesis of a diphyletic origin of cultivated barley.