Development and characterization of microsatellite markers for Vriesea simplex (Bromeliaceae) and cross-amplification in other species of Bromeliaceae

Ten polymorphic microsatellite loci were isolated and characterized for Vriesea simplex epiphytic and endemic species from the Brazilian Atlantic Rainforest. The number of alleles per locus ranged from 2 to 16. The observed and expected heterozygosity ranged from 0.000 to 0.414 and 0.068 to 0.795, respectively. All loci departed significantly from Hardy–Weinberg equilibrium. Linkage disequilibrium was not detected in any pair of loci. Transferability of 10 loci was positive across ten other Bromeliaceae species. The loci will be used for studying population genetic structure, mating system variation, and the origin and maintenance of reproductive barriers to gene exchange among sympatric Vriesea species.     

Development of 12 microsatellite markers for Aconitum brachypodum (Ranunculaceae), a critically endangered and endemic medicinal plant

In this study, 12 microsatellite markers were developed from two microsatellite-enriched libraries (AG, AC) of Aconitum brachypodum, which were constructed using a FIASCO method. Polymorphism of each locus was assessed in 24 individuals of A. brachypodum. Number of alleles per locus (N_A) ranged from 2 to 4. The average allele number of the microsatellites was 2.58 per locus. The observed (H_O) and expected (H_E) heterozygosities ranged from 0.125 to 0.875 and 0.117 to 0.612, respectively. Polymorphic information content (PIC) ranged from 0.110 to 0.531. Among the 12 microsatellite markers, three deviated from Hardy–Weinberg equilibrium significantly. These markers will facilitate further studies on the population genetics of A. brachypodum.     

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.     

Genetic diversity and variation in wild populations of dark sleeper (Odontobutis potamophila) in China inferred with microsatellite markers

Twenty-one highly variable microsatellite loci were used to investigate the genetic diversity and variation of Odontobutis potamophila in China. A total of 160 samples from five wild populations (Dangtu, Sheyang, Yuyao, Minjiang and Donxishan) were genotyped. All of the 21 microsatellite loci tested in this study showed polymorphism. The number of allele per locus ranged from 5.05 to 9.90. Locus 87a of Minjiang population had a 259-bp characteristic allele. The average observed heterozygosity and expected heterozygosity ranged from 0.33 to 0.62 and from 0.40 to 0.70, respectively. The pair-wise F_ST tests and NJ trees of the five O. potamophlia populations revealed that Dangtu, Sheyang, Yuyao and Dongxishan were genetically close to one another and distinct from Minjiang. Far genetic distances were observed among populations from distant geographical areas. This result provided guide for the use of O. potamophila breeds and the protection of the species.     

Genetic diversity and population structure of the endangered conifer Taxus wallichiana var. mairei (Taxaceae) revealed by Simple Sequence Repeat (SSR) markers

Taxus wallichiana var. mairei is an endangered conifer with important medicinal value in southern China. Nuclear SSR markers were employed to assess genetic diversity and structure of 13 geographically disjunct populations. The present study revealed a moderate genetic diversity (H_E = 0.538) and low genetic differentiation (F_ST = 0.159). And most populations encountered in severe inbreeding and bottleneck effect. No significant genetic structure was detected by IBD and Structure analysis, which was supported by AMOVA analysis. The present results could be ascribed to an earlier period of more pronounced gene flow when the species had a more continuous distribution. However, the 13 studied populations were divided into four clusters based on the UPGMA dendrogram; these clusters were almost congruent with their geographical distributions. Vital areas such as southern mountains of Sichuan basin, Nanling Mts. and the margin of this yew's distribution range had a high priority for conservation.     

Assessment of genetic diversity among androdioecious ancient Osmanthus fragrans trees by SSR markers

The well known ornamental plant, Osmanthus fragrans, is one of the rare androdioecious species in nature. Determining the genetic diversity of this species may assist in its conservation and genetic improvement. Data of 10 ancient tree populations of O. fragrans were used to assess its genetic diversity and population structure with microsatellite markers. A high level of genetic diversity was observed with POPGENE. As anticipated the heterozygosity of ancient hermaphrodite trees was higher than male ones, although the number of hermaphrodites was far fewer than males. Abundant gene exchange between males and hermaphrodites was detected, and trees from the same population often clustered together. Androdioecy is an important factor that determines the genetic structure of O. fragrans and there is an urgent need to conserve ancient O. fragrans germplasms especially the hermaphrodites.     

Distribution, stand characteristics and habitat of a critically endangered plant Euryodendron excelsum H. T. Chang (Theaceae): implications for conservation

In the present study, the distribution, population size and structure, habitat and stand disturbance of a critically endangered plant Euryodendron excelsum H. T. Chang were examined using field investigations. A total of 179 individual plants, including 23 adult trees, were found. The population was distributed in 10 highly isolated and fragmented patches. Half of the 10 patches had less than 10 individual plants and two patches had only one remnant adult; there were no adult plants in two of the patches. Although the overall population structure trends to a reversed 'J' shape, two fragile stages of E. excelsum limit its natural recruitment and regeneration: seed germination and seedling growth toward adulthood. The major threats for the survival of this species are its specific small population size and the high frequency of anthropogenic destruction. Based on the results, some strategies were put forward for the conservation of E. excelsum populations.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Development of microsatellite markers in Qualea grandiflora (Vochysiaceae) and transferability to congeneric species,typical trees of the Brazilian savanna

This study aimed to isolate and characterize microsatellite markers for Qualea grandiflora and to test their transferability to congeneric species Qualea multiflora and Qualea parviflora. These three species are widespread in the Cerrado, the largest, richest and probably the most threatened tropical savanna in the world. We characterized ten markers in 40 individuals belonging to two populations of Q. grandiflora and eight markers in 20 individuals belonging to one population of Q. multiflora and Q. parviflora. In Q. grandiflora, considering all 40 analyzed individuals, the number of alleles per locus ranged from eight to 21, and the average was 11.60. The mean number of alleles per locus was 8.8 and 7.3 in each population. The observed and expected heterozygosities (H_o and H_e) within populations varied from 0.235 to 0.944 and from 0.225 to 0.932, respectively. In Q. multiflora the number of alleles varied from two to 11 with an average of 5.75; the H_o ranged from 0.150 to 0.950, while H_e ranged from 0.191 to 0.817. In Q. parviflora, considering the seven polymorphic loci, the number of alleles ranged from two to 13, with an average of 7.5, while H_o ranged from 0.211 to 0.944, and H_e ranged from 0.193 to 0.906. The polymorphism level of the microsatellite markers here described enable them as powerful tools for future population genetic studies in these species, helping to answer ecological and evolutionary questions.     

Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders  genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (N_a), observed heterozygosity (H_o), expected heterozygosity (H_e), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01035-w.     

Isolation and characterization of microsatellite markers for Achyranthes bidentata (Amaranthaceae) using next-generation sequencing platform

Achyranthes bidentata Blume (Amaranthaceae) is a perennial herb that is widely distributed in India, Java, China, and Japan. The natural resources of A. bidentata in its geo-authentic product area have rapidly declined in recent years because of the over-collection of its roots. To devise adequate conservation and management strategies for this species, its genetic diversity and population structure should be characterized. Roche 454 pyrosequencing combined with magnetic bead enrichment was used to develop microsatellite markers for A. bidentata. A total of 903 microsatellite loci were identified from 42,004 individual sequence reads. One hundred microsatellite loci were selected to test the primer amplification efficiency across 16 individuals from two A. bidentata populations. Of these tested markers, 8 yielded polymorphic amplification products, 29 yielded single alleles. For polymorphic primer pairs, the number of alleles per locus ranged from 2 to 4, with an average of 2.75. The observed and expected heterozygosities ranged from 0.353 to 0.671 and 0.250 to 0.938, respectively. The inbreeding coefficient varied from −0.692 to 0.627. This set of markers will provide useful tools for examining genetic diversity and population structure, and aid in better understanding of the conservation of A. bidentata.     

Genetic diversity and structure of Manila clam (Ruditapes philippinarum) populations from Liaodong peninsula revealed by SSR markers

Manila clam (Ruditapes philippinarum) is an important commercially marine bivalve, and its wild populations have been severely declining in the coast of China during the last decade. In this study, a set of 7 genomic simple sequence repeat (SSR), and 5 expressed sequences tag (EST)-derived SSR markers were analyzed on eight wild R. philippinarum populations to assess the genetic diversity and population differentiation. A total of 114 alleles were detected on 12 loci, and the number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 2.00 to 10.88 for each sample. The average of observed and expected heterozygosity ranged from 0.386 to 0.550, and from 0.533 to 0.707, respectively. Pairwise F_ST values indicated that all population pairs had significant genetic differentiation (overall F_ST = 0.242, P < 0.01). Cluster analysis using unweighted pair group method with the arithmetic mean (UPGMA) separated the eight populations into two groups. This study will shed light on the domestication and cultivation on population genetic diversity of R. philippinarum, and also provide the foundation for conservation of R. philippinarum germplasm resources in clam breeding practices.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Genetic diversity analysis by microsatellite markers in four captive populations of the sika deer (Cervus nippon)

The Chinese sika deer (Cervus nippon) is a rare and vulnerable animal in China for medical use. In this study, the genetic diversity and population genetic structure of 113 Chinese sika deer from 4 populations (Linyi Farm, LF; Linyi Park, LP, Yangzhou Farm, YF; Yangzhou Zoo, YZ) were investigated with 14 microsatellite loci. Eighty-three alleles were detected at the 14 loci in all populations. The expected heterozygosity ranged from 0.257 to 0.863 and the observed heterozygosity from 0.226 to 0.821. The polymorphism information content at different loci ranged from 0.217 to 0.825. The results of the HWE (Hardy–Weinberg equilibrium) tests indicated that only four loci (CEH-5, BL42, Mber70, and CEH-2) were in HWE (P > 0.01). The mean number of alleles per population ranged from 3.21 to 5.64, observed and expected heterozygosities ranged from 0.568 to 0.685, respectively. Positive inbreeding coefficient (F_IS) values were found in every population. F_ST values ranged from 0.101 in the LF to 0.155 in the YZ. The genetic identity ranged from 0.1236 to 0.1645. The genetic distance ranged from 0.4746 to 0.6025. The results of this study indicate moderate genetic variation and polymorphism across the loci. Appropriate breeding strategies should be designed for deer in captivity.     

Analysis of genetic structure in a sample of coffee (Coffea arabica L.) using fluorescent SSR markers

The knowledge of population structure is important to determine the degree of linkage disequilibrium, which allows the selection of genotypes for association mapping. Using 47 SSR markers, the genetic variability and population structure of 68 accessions of C. arabica (wild and cultivated) and of three diploid species used as reference were evaluated. The analysis was done with the distance method and the structure model. The structure analysis inferred nine subpopulations (k = 9), for which the greatest values of probability were obtained. Three of the groups corresponded to the three diploid species as expected. There were six groups identified within C. arabica. The genetic subdivisions within C. arabica were based on geographical origin, degree of domestication, and dispersal history of coffee. One group consisted entirely of cultivated genotypes, where intense population bottleneck were associated with a founder effect. This was the most homogeneous group, as demonstrated by the reduced distance between cultivars in the dendrogram. Three of the cultivated genotypes, originating from Sudan, were separated into an independent group, presumably due to selective adaptation to a different set of environmental conditions. Another group consisted of genotypes of the type “ennarea” that were grown and cultivated in isolation on the shores of the Tana lake. The semi-wild genotypes clustered into three different groups. This type of analysis provides a strong evidence of population structure in C. arabica. Based on these findings, it is possible to better identify a balanced sample of diverse plants in germplasm.     

Characterization of microsatellite DNA markers in a critically endangered species,Mekong giant catfish,Pangasianodon gigas

Microsatellite DNA markers for a critically endangered Mekong giant catfish (Pangasianodon gigas Roberts and Vidthayanon, 1991) were developed from fin clips collected from captive fish using (GT)₁₅ probe. The number of alleles per locus ranged from two to four. The expected heterozygosities ranged from 0.13 to 0.68. Also, these primers were successfully amplified in four closely related species, Pangasius bocourti, Pangasius conchophilus, Pangasius larnaudii and Pangasius sanitwongsei with the number of alleles per locus ranged from 1 to 13, 1 to 16, 1 to 12 and 1 to 4, respectively. These markers should prove to be very useful for the evaluation of genetic diversity for this species and other related Pangasius species.     

Population genetic structure and interspecific differentiation between Acer davidii Franchi. and A. morrisonense Hayata (Aceraceae) based on SSR markers

Acer davidii Franch. and A. morrisonense Hayata are two important forest tree species (Aceraceae) endemic to mainland China and the Taiwan area, respectively. To investigate population structure and interspecific differentiation between them, we characterized a set of novel microsatellite markers using Illumina sequencing technology. The cross-species amplification analysis showed that 11 out of 21 polymorphic SSR primers of A. davidii also exhibited polymorphisms in A. morrisonense. At the species level, A. davidii has a slightly higher genetic diversity (mean observed heterozygosity, H_O = 0.180) than A. morrisonense (H_O = 0.119). AMOVA showed that most of the variations in A. davidii occurred among individuals within populations and within individuals. Bayesian clustering analyses demonstrated that the two maple species formed two clear genetic lineages. PCoA showed that A. davidii and A. morrisonense were significantly divided into two genetic groups. In addition, asymmetrical weaker gene flow was detected among the two forest tree species. These results suggest that the long term geographic isolation between the mainland and Taiwan may have resulted in a high level of genetic differentiation between these two important maple species.     

Identification and cross-species amplification of EST derived SSR markers in different bamboo species

The availability of expressed sequence data derived from gene discovery programs became an alternative source of mining simple sequence repeat (SSR) and developing inexpensive genetic markers for the crop improvements. In present study, 10 express sequence tags (EST)-SSR markers were identified from Bambusa oldhamii public sequence data base. Transferability to 25 species of Bambusoideae ranged from 30% to 100%. The number of alleles detected per locus ranged from 2 to 10. All the newly identified SSR markers were found to be moderately to highly polymorphic with an average Polymorphic Information Content (PIC) value of 0.54. As these loci represents transcribed region and recorded high level of cross transferability and reliable amplification across the species, demonstrating the utility of these markers for functional and genetic analyses of bamboo species.     

SSR markers development and their application in genetic diversity evaluation of garlic (Allium sativum) germplasm

Garlic (Allium sativum), an asexually propagated vegetable and medicinal crop, has abundant genetic variation. Genetic diversity evaluation based on molecular markers has apparent advantages since their genomic abundance, environment insensitivity, and non-tissue specific features. However, the limited number of available DNA markers, especially SSR markers, are insufficient to conduct related genetic diversity assessment studies in garlic. In this study, 4372 EST-SSR markers were newly developed, and 12 polymorphic markers together with other 17 garlic SSR markers were used to assess the genetic diversity and population structure of 127 garlic accessions. The averaged polymorphism information content (PIC) of these 29 SSR markers was 0.36, ranging from 0.22 to 0.49. Seventy-nine polymorphic loci were detected among these accessions, with an average of 3.48 polymorphic loci per SSR. Both the clustering analyses based on either the genotype data of SSR markers or the phenotypic data of morphological traits obtained genetic distance divided the 127 garlic accessions into three clusters. Moreover, the Mantel test showed that genetic distance had no significant correlations with geographic distance, and weak correlations were found between genetic distance and the phenotypic traits. AMOVA analysis showed that the main genetic variation of this garlic germplasm collection existed in the within-population or cluster. Results of this study will be of great value for the genetic/breeding studies in garlic and enhance the utilization of these garlic germplasms.