Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

Nine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

Isolation of 10 microsatellite markers for mongoose lemurs (Eulemur mongoz)

We present here 10 new microsatellite markers for the mongoose lemur (Eulemur mongoz), nine of which were isolated from E. mongoz and one from Lemur catta. At least 60 individuals were genotyped for each of the 10 loci. Mendelian inheritance at each locus was tested by genotyping five captive families with known pedigrees. All loci were polymorphic and demonstrated simple Mendelian inheritance. The microsatellite markers presented here will be useful for genetic surveys of wild E. mongoz, to gain insights into the genetic background of the captive population, and to aid in the conservation of the species’ genetic diversity.     

The Pleistocene demography of an alpine juniper of the Qinghai‐Tibetan Plateau: tabula rasa,cryptic refugia or something else?

Aim Numerous palaeoecological and genetic studies have shown that different tree species responded in very different ways to Pleistocene climatic oscillations. Some were forced into small refugia far from their current range, while others were able to survive in small refugia close to, or even within, their current natural range. In this study we examine the Pleistocene demography of a juniper species (Juniperus przewalskii, Cupressaceae) from the Qinghai‐Tibetan Plateau. Location The Qinghai‐Tibetan Plateau (QTP). Methods Eight nuclear loci were sequenced in 141 individuals from 20 natural populations distributed across the entire natural range of J. przewalskii, and coalescent analysis was used to test demographic hypotheses. Results The overall nucleotide diversity in the sample was low (π_sil = 0.0029), with few rare alleles and pronounced population genetic structure (F_ST = 0.181). We detected a division previously found using chloroplast DNA markers: all segregating sites in populations from the central part of the QTP appear to be a subset of those found around the edge of the plateau, confirming the relatively young age of the former. In contrast to the middle Pleistocene bottlenecks detected in boreal tree species, the coalescent‐based analyses failed to reject the standard neutral model for the juniper species considered here. Main conclusions Juniperus przewalskii did not undergo marked changes in population sizes during the Pleistocene, although this species seems to have experienced recent, post‐glacial expansion. This finding is largely consistent with the limited number of previous studies on conifer species of the QTP, but contradicts findings of studies on boreal species. These findings have wide implications for understanding plant species’ responses to past climatic oscillations on the high‐elevation QTP.     

Genetic diversity and structure of Cordyceps sinensis populations from extensive geographical regions in China as revealed by inter-simple sequence repeat markers

Cordyceps sinensis is one of the most valuable medicinal caterpillar fungi native to China. However, its productivity is extremely limited and the species is becoming endangered. The genetic diversity of eighteen C. sinensis populations across its major distributing regions in China was evaluated by inter-simple sequence repeat (ISSR) markers. A total of 141 markers were produced in 180 individuals from the 18 populations, of which 99.3% were polymorphic. The low average of Shannon (0.104) and Nei index (0.07) of the 18 populations indicates that there are little genetic variations within populations. For all 18 populations, estimates of total gene diversity (H_T), gene diversity within populations (H_S), coefficient of genetic differentiation (G_ST), and gene flow (Nm) were 0.170, 0.071, 0.583, and 0.357, respectively. This pattern suggests that the genetic diversity of C. sinensis is low and most of the ISSR variations are found among populations with little gene exchange. The 18 populations are divided into five groups based on the genetic distance and the grouping pattern matches with the geographic distribution along the latitudinal gradient. The five groups show obvious difference in the G_ST and Nm values. Therefore, the genetic diversification of C. sinensis populations may be determined by geographic isolation and the combined effects of life history characters and the interaction with host insect species. The information illustrated by this study is useful for selecting in situ conservation sites of C. sinensis.     

Development and polymorphism of simple sequence repeat DNA markers for Bruguiera gymnorrhiza (L.) Lamk

Microsatellite markers of Bruguiera gymnorrhiza were developed. Fifty‐four of 161 clones were found to contain microsatellite repeats. Primer pairs were designed for 20 of these clones according to their sequence data. Of these, seven primers showed polymorphism for 32 individuals from Iriomote Island, Japan. Two to five alleles per locus were detected, and the observed heterozygosities ranged from 0.031 to 0.500. Cross‐species amplification using five of the seven primers also worked well for B. cylindrica and B. parviflora. Because our previous study reported very low levels of genetic diversity for allozymes in the same Iriomote population, these microsatellite markers should be a powerful tool for various kinds of genetic analysis.     

Using minimum DNA marker loci for accurate population classification in rice (Oryza sativa L.)

Because of the rich diversity among rice accessions grown around the world in distinct environments, traditional methods using morphology, cross compatibility and geography for classifying rice accessions according to different sub-populations have given way to use of molecular markers. Having a few robust markers that can quickly assign population structure to germplasm will facilitate making more informed choices about genetic diversity within seedbanks and breeding genepools. WHICHLOCI is a computer program that selects the best combination of loci for population assignment through empirical analysis of molecular marker data. This program has been used in surveys of plant species, for fish population assignment, and in human ancestry analysis. Using WHICHLOCI, we ranked the discriminatory power of 72 DNA markers used to genotype 1,604 accessions of the USDA rice core collection, and developed panels with a minimum number of markers for population assignment with 99% or higher accuracy. A total of 14 markers with high discriminatory power, genetic diversity, allelic frequency, and polymorphic information content were identified. A panel of just four markers, RM551, RM11, RM224 and RM44, was effective in assigning germplasm accessions to any of five sub-populations with 99.4% accuracy. Panels using only three markers were effective for assignment of rice germplasm to specific sub-populations, tropical japonica, temperate japonica, indica, aus, and aromatic. Assignment to tropical japonica, temperate japonica, or indica sub-populations was highly reliable using 3–4 markers, demonstrated by the high correlation with assignment using 72 markers. However, population assignment to aus and aromatic groups was less reliable, possibly due to the smaller representation of this material in the USDA core collection. More reference cultivars may be needed to improve population assignment to these two groups. This study demonstrated that a small number of DNA markers is effective for classification of germplasm into five sub-populations in rice. This will facilitate rapid screening of large rice germplasm banks for population assignment at a modest cost. The resulting information will be valuable to researchers to verify population classification of germplasm prior to initiating genetic studies, maximizing genetic diversity between sub-populations, or minimizing cross incompatibility while maximizing allelic diversity within specific sub-populations.     

Isolation and characterization of microsatellite loci in the sugar beet cyst nematode Heterodera schachtii

We report the isolation of five microsatellites loci from the sugar beet cyst nematode (Heterodera schachtii). Multilocus genotypes were obtained on individual larvae freshly emerged from cysts. Allelic diversity ranged from four to 27 among the five loci. The primers were tested for cross‐species amplification in six other species of phytoparasitic nematodes of the Heterodera genus. Those molecular markers will be used to study the genetic structure of this obligatory parasite and how it is affected by the use of resistant plants.     

Microsatellite markers for <Emphasis Type="Italic">Juglans cinerea</Emphasis> L. and their utility in other Juglandaceae species

Ten polymorphic microsatellite markers were found to amplify in butternut (Juglans cinerea; Juglandaceae). These microsatellite loci were found to amplify across most of nine other species and five hybrids examined. Loci were highly polymorphic, with 18 to 32 alleles per locus across species. These nuclear microsatellite markers will be useful in examining genetic diversity within and among populations of butternut, and in distinguishing butternut from interspecific hybrids.     

Isolation and characterization of 12 microsatellite loci for cross-species amplification in the cyprinid gudgeons Squalidus chankaensis and S. japonicus

Twelve dinucleotide markers were successfully isolated and characterized from a microsatellite‐enriched genomic library obtained for the gudgeon Squalidus chankaensis biwae. These markers were also available for the congeners S. c. tsuchigae and S. japonicus from Japan, which had five to 46 alleles and an expected heterozygosity ranging from zero to 0.946. Linkage equilibrium was observed at all loci, and most loci did not show significant deviation from Hardy–Weinberg equilibrium. The isolated microsatellite markers will be useful for genetic diversity studies of Squalidus populations.     

Population genetic diversity in the polyploid complex of wheatgrasses using isoenzyme and RAPD data

Thirty five bands (alleles) from six enzyme systems and fifty seven random amplified polymorphic DNA (RAPD) fragments were selected to analyse the genetic diversity of 33 polyploid wheatgrasses (Triticeae) populations of species Thinopyrum junceiforme and Elytrigia pycnantha, and two hybrids, one pentaploid and one novel 9-ploid. Dice’s similarity coefficient, the UPGMA-derived phenograms from RAPD, and allozymes markers showed that the clustering of wheatgrass populations was based on ploidy level. These markers had similar levels of diversity between populations, with high genetic similarity within the same ploidy-level and within population’s individuals. The tetraploid Th. junceiforme populations are closely related, with a large similarity distances varied from 0.8 to 1. Based on the isozyme and RAPD analyses, diploid taxa are related to polyploids with similarity coefficients 0.4.     

Development of microsatellite markers for Diaphanosoma dubium (Crustacea,Cladocera) and application to seasonal population dynamics

Diaphanosoma, the “tropical Daphnia”, is common and ubiquitous in South China. Like other ctenopods, Diaphanosoma has a reproductive mode similar to Daphnia’s, but its resting eggs are rarely observed and lack an ephippium. With limited dispersal and reduced buffer effect from resting egg banks, Diaphanosoma is expected to have a population genetic structure different from that of temperate Daphnia. To facilitate genetic comparison, we developed microsatellite markers using next-generation sequencing for the most common species in tropical and subtropical East Asia, Diaphanosoma dubium. Thirty-one polymorphic microsatellite markers were obtained, and 29 of them were efficient for the congeneric species D. excisum, D. orghidani, D. mongolianum and D. chankensis. The markers allowed intra- and interspecific genetic analysis, including population structure, hybridization and introgression. We used 11 selected microsatellite markers to analyze spatial and temporal heterogeneity of genetic diversity in four (sub)tropical D. dubium populations from two large reservoirs and two temporary ponds. In contrast to temperate Cladocera, higher genetic diversity in summer rather than in spring suggested weak contribution from resting eggs in spring. Clustering of DAPC and STRUCTURE analyses indicated a clear-cut genetic structure in the four populations. Variation partitioning revealed that water storage and depth were key factors in genetic differentiation. Within large reservoirs, we detected backward (reversing time) gene flow from resting egg banks. We conclude that resting eggs have an effective contribution to the genetic diversity in large water bodies during growing seasons and that large water bodies can host higher genetic diversity in summer due to environmental heterogeneity and high carrying capacity. Spatial and temporal heterogeneity of genetic diversity detected by our microsatellite markers showed the newly developed markers can be applied for further study of populations of D. dubium and other species of Diaphanosoma at a contemporary scale.

     

Analysis of the genetic diversity of Bemisia tabaci populations in Shanxi province, China

Five different primer combinations were used for the analysis of 152 B biotype Bemisia tabaci (Gennadius) individuals and five Trialeurodes vaporairiorum individuals collected from 19 counties and seven host plants in Shanxi province in China, respectively. The main objective of the present study was to use AFLP markers to determine the genetic diversity of B. tabaci populations collected from Shanxi Province. The use of these primer combinations allowed the identification of 127 polymorphic bands (52.26%) from 60 to 500 bp. The average number of polymorphic bands per primer was 25.4 while the range for the five primers was 20–32. The average degree of heterozygosity was 0.251, while the range for the five primers was 0.204–0.289. The results suggested definite genetic diversity among different B. tabaci populations. Cluster analysis showed that B. tabaci populations were firstly scattered to three genetic groups according to the regions, then every genetic group was scattered to several subgroups according to the host plants, which revealed the genetic variability of B biotype B. tabaci populations has been not only among different regions, but also among different host plants in Shanxi Province.     

Genetic diversity analysis of Rhododendron aureum Georgi (Ericaceae) located on Changbai Mountain using ISSR and RAPD markers

Rhododendron aureum Georgi (Ericaceae) is a perennial alpine shrub endemic to Changbai Mountain in China. We used ISSR and RAPD markers to describe the diversity and genetic structure within and among four natural populations located at different altitudes. DNA from 66 individuals was amplified with ten ISSR markers and seven RAPD markers. High genetic diversity was observed by these two techniques at the species level. The genetic diversity of populations increased with altitudinal gradients from low to high. The coefficient of gene differentiation (G_ST 0.3652 in ISSR and 0.2511 in RAPD) and AMOVA analysis revealed that most genetic diversity was distributed within populations (61.96% in ISSR and 70.23% in RAPD). The estimate of gene flow based on G_ST was 0.8690 in ISSR and 1.4910 in RAPD. The UPGMA clustering results using ISSR and RAPD showed that all individuals from the same altitude were gathered together, and the two populations (TYD2a and YHLa) from middle altitudes always clustered together. Compared with populations from different altitudes, similar genetic diversity and low genetic differentiation were obtained from populations at the same altitudes, as revealed by ISSR markers. In addition to the reproductive strategy of R. aureum, these data highlight that local environmental conditions may play an important role in shaping the diversity and genetic structure of this species.     

Microsatellite markers in Fijian crested iguanas (Brachylophus vitiensis) also amplify Fijian banded iguanas (B. fasciatus)

Five microsatellite markers were isolated from the Fijian crested iguana (Brachylophus vitiensis) using an enrichment technique. These loci also amplified the Fijian banded iguana (B. fasciatus). All five loci were polymorphic with between 2–7 and 4–7 alleles per locus in B. vitiensis and B. fasciatus, respectively. Heterozygosity (H_O) values ranged from 0.069 to 0.875. These markers were useful in resolving kinship relationships and will aid future endeavours by the captive management program to conserve genetic diversity.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

Congruence between morphological and molecular markers inferred from the analysis of the intra-morphotype genetic diversity and the spatial structure of Oxalis tuberosa Mol.

Oxalis tuberosa is an important crop cultivated in the highest Andean zones. A germplasm collection is maintained ex situ by CIP, which has developed a morphological markers system to classify the accessions into morphotypes, i.e. groups of morphologically identical accessions. However, their genetic uniformity is currently unknown. The ISSR technique was used in two experiments to determine the relationships between both morphological and molecular markers systems. The intra-morphotype genetic diversity, the spatial structures of the diversity and the congruence between both markers systems were determined. In the first experience, 44 accessions representing five morphotypes, clearly distinct from each other, were analyzed. At the molecular level, the accessions exactly clustered according to their morphotypes. However, a genetic variability was observed inside each morphotype. In the second experiment, 34 accessions gradually differing from each other on morphological base were analyzed. The morphological clustering showed no geographical structure. On the opposite, the molecular analysis showed that the genetic structure was slightly related to the collection site. The correlation between both markers systems was weak but significant. The lack of perfect congruence between morphological and molecular data suggests that the morphological system may be useful for the morphotypes management but is not appropriate to study the genetic structure of the oca. The spatial structure of the genetic diversity can be related to the evolution of the species and the discordance between the morphological and molecular structures may result from similar selection pressures at different places leading to similar forms with a different genetic background.     

Genetic diversity of Ligula intestinalis (Cestoda: Diphyllobothriidea) based on analysis of inter‐simple sequence repeat markers

In order to investigate the genetic diversity of Ligula intestinalis populations, nine inter‐simple sequence repeat (ISSR) markers were applied to populations from nine geographical areas around the world and 10 host species. The 110 loci selected from the ISSR patterns produced revealed high variability among the analysed samples, with a polymorphism of 100% and a global coefficient of gene differentiation estimated by Nei’s index (G_ST) of 0.776. Major genetic differentiation was found to be correlated to five broad geographical regions (Europe, China, Canada, Australia and Algeria). Nevertheless, no significant genetic variation was found among European isolates, although they originated from disparate geographical localities and/or unrelated hosts. Classical classification methods: maximum parsimony and factorial correspondence analysis were compared with an advanced statistical method: the self‐organizing map (SOM). The results demonstrated that the ISSR approach is rapid and inexpensive and provides reliable markers to assess genetic diversity of L. intestinalis. Furthermore, SOM artificial neuronal networks are considered to provide an efficient alternative tool for mapping the genetic structures of parasite populations.     

Microsatellite-based assessment of genetic diversity in stripe rust resistant NUWYT candidate lines

Stripe rust is one of the most devastating diseases, caused by Puccinia striiformis f. sp. tritici, affecting a huge amount of wheat crops worldwide. In this study, the genetic diversity of 16 National Uniform Wheat Yield Trial (NUWYT) candidate lines was evaluated by using 22 screened microsatellite markers. These lines were found resistant for stripe rust at adult plant stage. These wheat microsatellite markers identified a total of 38 alleles, with an average of 2.3 alleles per microsatellite locus. The number of alleles ranged from one to five alleles and the highest number of alleles were associated with B genome (25), as compared to D (11) and A (2) genomes. The allelic polymorphism index content (PIC) reflecting the gene diversity of these microsatellite markers ranged from 0.00 to 0.66, with an average of 0.27. The maximum PIC value of 0.66 was observed for xgwm 159-5B and 0.64 for xgwm 413-1B. The gene diversity ranged from 0.00 to 0.71, with an average of 0.30. The genetic similarity matrix was used to construct a dendrogram and the cluster analysis was performed by the use of unweighted pair-group method with arithmetic average algorithm. This divided the entire 16 candidate lines into three main clusters on the basis of their similarity. Our results indicate that the genetic diversity among the 16 candidate NUWYT lines was very narrow.