Species-specific Simple Sequence Repeats Research Articles

Brief restriction endonuclease digestion of genomic DNA improves PCR amplifiability and reproducibility of SSR loci: Augmenting exploration of hidden genetic variability in an endemic carp of South-East Asia

Majority of the microsatellite primers developed through painstaking, resource intensive research often fail to amplify due to secondary structures in the repeat rich target. In the present work, we describe the use of restriction endonuclease digestion of genomic DNA prior to PCR to counter this issue. The methodology, Restriction Endonuclease Digestion of genomic DNA to improve PCR Amplifiability and Repeatability of SSR loci (REDDAR-SSR), was tested on 40 species-specific Simple Sequence Repeat (SSR) loci in Osteobrama belangeri (F: Cyprinidae). Undigested and HpaII digested pooled genomic DNA samples were used as templates. Two primer concentrations (10 pM and 25 pM) and two annealing temperatures (56 °C uniformly and individual primer-specific annealing temperatures) were tested on both template types in sets of three, repeated thrice (360 reactions/condition). The digested templates exhibited significantly higher (P < 0.05) amplifiability (93.9 % against 24.16 % of undigested counterpart) as well as repeatability (94.47 % against 21.56 % of undigested counterpart). Primer concentration had no significant effect on outcomes (P > 0.05). The number of successfully amplified loci in our method was 38 compared to 18 by the conventional protocol. All 38 loci could also be reliably amplified in individual gDNA using the developed protocol out of which 9 loci were observed to be polymorphic. Notably, 4 of the polymorohic loci could be amplified only through REDDAR-SSR. Our protocol can potentially reduce expenses; resources & time vis-a-vis enhance the reach of SSR markers for genotyping species of commercial or conservational significance.

Contrasting effects of local environment and grazing pressure on the genetic diversity and structure of Artemisia frigida

Drylands count among the most globally extensive biomes, and while many desert and dry rangeland ecosystems are under threat, genetic structures of dryland species are still rarely studied. Artemisia frigida is one of the most widely distributed plant species in the temperate rangelands of Eurasia and North America, and it also dominates in many habitats of Mongolia due to its tolerance to low temperatures, drought and disturbance. Local environmental conditions and grazing pressure can influence species performance and affect spatial patterns of genetic diversity in contrasting ways, and our study set out to evaluate such effects on the genetic diversity and structure of A. frigida. We first developed new species-specific Simple Sequence Repeats (SSRs) markers using whole genome sequencing. We then analysed 11 populations of A. frigida that had been sampled along a large climatic gradient in Mongolia, which were sub-structured according to three levels of grazing intensity. Estimates of genetic diversity at the population level were high (HO = 0.56, HE = 0.73) and tended to increase with higher precipitation and soil nutrient availability. Grazing had no effect on genetic diversity, however, a high number of grazing-specific indicator alleles was found at grazed sites. Genetic differentiation among populations was extremely low (global GST = 0.034). Analysis of Molecular Variance revealed 5% variance between populations along the climatic gradient, with 3% of the variance being partitioned among different grazing intensity levels. We found no relationship between geographic and genetic distances, and thus no isolation by distance in this widely distributed species. The relatively low genetic structuring suggests that considerable gene flow exists among A. frigida populations across the rangelands of Mongolia, in spite of the pervasive grazing in the region.

Assessment of morphogenetic diversity in Garcinia gummi-gutta (L.) Roxb. using species-specific morphological and SSR markers

Garcinia gummi-gutta is an important tree crop valued for its dried fruit rinds as a souring agent in various dishes and source of hydroxycitric acid (HCA), an anti-obesity agent. In this study, the genetic variation among 35 accessions of G. gummi-gutta was analyzed by employing quantitative morphological traits and species-specific SSR markers. Recorded quantitative morphological data were statistically significant (p < 0.001) based on One way ANOVA. Considerable diversity observed among the accessions in growth and yield characters like the total number of fruits/tree, the mean number of fruits needed to weigh 1 kg, mean fresh and dry rind yield/tree; morphometric trait like single fruit weight. Molecular profiling was done by screening 24 simple sequence repeats (SSRs) primer from a set of 32 primers based on precise, intense and reproducible banding pattern. Total 39 alleles were obtained in the 24 SSRs with an average of 1.63 alleles/locus with an average of 86.11% polymorphism. An average Polymorphic Information Content (PIC) of 0.239 and an average Marker Index of 0.005, revealing the less allelic hetrozygosity and specificity of SSR primers for the selected accessions. The two dendrograms prepared based on Unweighted pair group method of arithmetic means (UPGMA), and Jaccard's similarity coefficient showed wide genetic variation among the accessions. Quality and productivity of G. gummi-gutta can be improved by selecting the better performing and diverse accession for future breeding programmes.

Characterizing the genetic diversity of the Andean blueberry (Vaccinium floribundum Kunth.) across the Ecuadorian Highlands.

The Ecuadorian páramo, a high altitude tundra-like ecosystem, is a unique source of various ecosystem services and distinct biodiversity. Anthropogenic activities are associated with its fragmentation, which alters ecological factors and directly threatens resident species. Vaccinium floribundum Kunth., commonly known as Andean blueberry or mortiño, is a wild shrub endemic to the Andean region and highly valued in Ecuador for its berries, which are widely used in food preparations and hold an important cultural value. Since it is a wild species, mortiño could be vulnerable to environmental changes, resulting in a reduction of the size and distribution of its populations. To evaluate the extent of these effects on the mortiño populations, we assessed the genetic diversity and population structure of the species along the Ecuadorian highlands. We designed and developed a set of 30 species-specific SSR (simple sequence repeats) markers and used 16 of these to characterize 100 mortiño individuals from 27 collection sites. Our results revealed a high degree of genetic diversity (HE = 0.73) for the Ecuadorian mortiño, and a population structure analyses suggested the existence of distinct genetic clusters present in the northern, central and southern highlands. A fourth, clearly differentiated cluster was also found and included individuals from locations at higher elevations. We suggest that the population structure of the species could be explained by an isolation-by-distance model and can be associated with the geological history of the Andean region. Our results suggest that elevation could also be a key factor in the differentiation of mortiño populations. This study provides an extensive overview of the species across its distribution range in Ecuador, contributing to a better understanding of its conservation status. These results can assist in the development of conservation programs for this valuable biological and cultural resource and for the páramo ecosystem as a whole.

The Landscape of Repetitive Elements in the Refined Genome of Chilli Anthracnose Fungus Colletotrichum truncatum.

The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and “two-speed” genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

The Complete Chloroplast Genome Sequences of Aconitum pseudolaeve and Aconitum longecassidatum, and Development of Molecular Markers for Distinguishing Species in the Aconitum Subgenus Lycoctonum.

Aconitum pseudolaeve Nakai and Aconitum longecassidatum Nakai, which belong to the Aconitum subgenus Lycoctonum, are distributed in East Asia and Korea. Aconitum species are used in herbal medicine and contain highly toxic components, including aconitine. A. pseudolaeve, an endemic species of Korea, is a commercially valuable material that has been used in the manufacture of cosmetics and perfumes. Although Aconitum species are important plant resources, they have not been extensively studied, and genomic information is limited. Within the subgenus Lycoctonum, which includes A. pseudolaeve and A. longecassidatum, a complete chloroplast (CP) genome is available for only one species, Aconitum barbatum Patrin ex Pers. Therefore, we sequenced the complete CP genomes of two Aconitum species, A. pseudolaeve and A. longecassidatum, which are 155,628 and 155,524 bp in length, respectively. Both genomes have a quadripartite structure consisting of a pair of inverted repeated regions (51,854 and 52,108 bp, respectively) separated by large single-copy (86,683 and 86,466 bp) and small single-copy (17,091 and 16,950 bp) regions similar to those in other Aconitum CP genomes. Both CP genomes consist of 112 unique genes, 78 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes. We identified 268 and 277 simple sequence repeats (SSRs) in A. pseudolaeve and A. longecassidatum, respectively. We also identified potential 36 species-specific SSRs, 53 indels, and 62 single-nucleotide polymorphisms (SNPs) between the two CP genomes. Furthermore, a comparison of the three Aconitum CP genomes from the subgenus Lycoctonum revealed highly divergent regions, including trnK-trnQ, ycf1-ndhF, and ycf4-cemA. Based on this finding, we developed indel markers using indel sequences in trnK-trnQ and ycf1-ndhF. A. pseudolaeve, A. longecassidatum, and A. barbatum could be clearly distinguished using the novel indel markers AcoTT (Aconitum trnK-trnQ) and AcoYN (Aconitum ycf1-ndhF). These two new complete CP genomes provide useful genomic information for species identification and evolutionary studies of the Aconitum subgenus Lycoctonum.

A Genome-Wide Survey of the Microsatellite Content of the Globe Artichoke Genome and the Development of a Web-Based Database.

The recently acquired genome sequence of globe artichoke (Cynara cardunculus var. scolymus) has been used to catalog the genome’s content of simple sequence repeat (SSR) markers. More than 177,000 perfect SSRs were revealed, equivalent to an overall density across the genome of 244.5 SSRs/Mbp, but some 224,000 imperfect SSRs were also identified. About 21% of these SSRs were complex (two stretches of repeats separated by <100 nt). Some 73% of the SSRs were composed of dinucleotide motifs. The SSRs were categorized for the numbers of repeats present, their overall length and were allocated to their linkage group. A total of 4,761 perfect and 6,583 imperfect SSRs were present in 3,781 genes (14.11% of the total), corresponding to an overall density across the gene space of 32,5 and 44,9 SSRs/Mbp for perfect and imperfect motifs, respectively. A putative function has been assigned, using the gene ontology approach, to the set of genes harboring at least one SSR. The same search parameters were applied to reveal the SSR content of 14 other plant species for which genome sequence is available. Certain species-specific SSR motifs were identified, along with a hexa-nucleotide motif shared only with the other two Compositae species (sunflower (Helianthus annuus) and horseweed (Conyza canadensis)) included in the study. Finally, a database, called “Cynara cardunculus MicroSatellite DataBase” (CyMSatDB) was developed to provide a searchable interface to the SSR data. CyMSatDB facilitates the retrieval of SSR markers, as well as suggested forward and reverse primers, on the basis of genomic location, genomic vs genic context, perfect vs imperfect repeat, motif type, motif sequence and repeat number. The SSR markers were validated via an in silico based PCR analysis adopting two available assembled transcriptomes, derived from contrasting globe artichoke accessions, as templates.

Analysis of genetic diversity and population structure of oil palm (Elaeis guineensis) from China and Malaysia based on species-specific simple sequence repeat markers.

Genetic diversity and patterns of population structure of the 94 oil palm lines were investigated using species-specific simple sequence repeat (SSR) markers. We designed primers for 63 SSR loci based on their flanking sequences and conducted amplification in 94 oil palm DNA samples. The amplification result showed that a relatively high level of genetic diversity was observed between oil palm individuals according a set of 21 polymorphic microsatellite loci. The observed heterozygosity (Ho) was 0.3683 and 0.4035, with an average of 0.3859. The Ho value was a reliable determinant of the discriminatory power of the SSR primer combinations. The principal component analysis and unweighted pair-group method with arithmetic averaging cluster analysis showed the 94 oil palm lines were grouped into one cluster. These results demonstrated that the oil palm in Hainan Province of China and the germplasm introduced from Malaysia may be from the same source. The SSR protocol was effective and reliable for assessing the genetic diversity of oil palm. Knowledge of the genetic diversity and population structure will be crucial for establishing appropriate management stocks for this species.

Development of SSR Markers for Genetic Analysis of Silverleaf Nightshade (Solanum elaeagnifolium) and Related Species

Silverleaf nightshade (2n = 2x = 24) is a serious weed in the Solanaceae, for which no specific molecular markers are currently available. In order to investigate the extent and distribution of genetic diversity among accessions of silverleaf nightshade, we developed 23 simple sequence repeat (SSR) markers from publicly available nucleotide and EST databases for silverleaf nightshade. Eleven of them were single-locus polymorphic markers. The number of alleles among these loci ranged from 2 to 4. The observed and expected heterozygosity ranged from 0 to 0.97 and 0.07 to 0.64, respectively. Fourteen SSR markers enabled to amplify alleles in morphologically similar species quena. These results proved that the SSR markers that we developed could be useful for (1) determining genetic diversity and structure among natural populations of silverleaf nightshade and (2) identifying silverleaf nightshade and quena ecotypes. This is the first set of species-specific SSR markers identified in silverleaf nightshade, which could contribute to the better understanding of genetic diversity of silverleaf nightshade and related species.

Elaeis oleifera Genomic-SSR Markers: Exploitation in Oil Palm Germplasm Diversity and Cross-Amplification in Arecaceae

Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (Ho) (0.164) and highly positive fixation indices (Fis) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.

ASSESSMENT OF GUAVA-DERIVED SSR MARKERS FOR THE MOLECULAR CHARACTERIZATION OF MYRTACEAE FROM DIFFERENT ECOSYSTEMS IN VENEZUELA

Simple sequence repeat (SSR) markers have proven to be a very powerful tool to analyze genetic variability and relatedness at different hierarchical levels, for genetic map generation and validation as well as for many other applications. However, despite the fact that the development of species-specific SSR markers involves a laborious and costly task, there is also a lot of information concerning the successful use of SSR markers derived from one species and employed with other closely related taxa. In this research we report on the use of SSR developed for Psidium guajava, for the characterization of individuals belonging to the same genus and to the same family (Myrtaceae). The plant samples used in this work were collected in two Venezuelan sharply different and geographically isolated ecosystems. One of these ecosystems is a mountainous region in the Venezuelan Andes, in the state of Merida, and the other, is located south to the Orinoco River, situated in the state of Amazonas and characterized by typical rain forest vegetation. Both regions are ecosystems rich in Myrtaceae species, some of which are shared by both regions, whilst others are exclusive of one of these. Besides the morphological observations, an analysis with more than 16 pairs of guava-derived SSR primers was carried out. All primer combinations were assessed in terms of efficiency of amplification, number of loci, size of the amplification products, and degree of polymorphism. Due to the geographical distance between the sampled regions and our research lab, particularly those proceeding from Amazonas, and the low germination efficiency of most of the collected seeds, dry leaf sampled were used as a source of DNA. Successful amplifications of informative loci validate the transfer of most of the guava-derived SSR primers to other Myrtaceae species.

Genetic reticulation and interrelationships amongcitrullusspecies as revealed by joint analysis of shared AFLPs and species-specific SSR alleles

Thirty-one accessions ofCitrullusspp. belonging toCitrullus lanatusvar.lanatus,C. lanatusvar.citroidesandCitrullus colocynthiswere subjected to phylogenetic analysis using combined datasets of amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs). Tree topologies inferred by neighbour-joining analysis have resolved the phylogenic relationships among the species with special reference to established taxonomic classification. In this study, we have clearly resolved species boundaries of various taxa ofcitroides,lanatusandcolocynthisinto three well-supported clusters. Clustering pattern of principal component analysis with the shared polymorphisms using the subsets of data between any two taxon combinations helped to elucidate the introgression and interrelationships among the species. We report two major groups ofC. lanatustaxa, one of which has undergone wide introgressions with the taxa ofC. lanatusvar.citroidesandC. colocynthis. In this paper, we identified 583 AFLP bands that are polymorphic within the var.lanatusofC. lanatus, which is the largest set ever reported. The species-specific diagnostic SSRs and polymorphic AFLPs that are informative within and between the taxa reported in this paper would be immensely useful for future studies of these economically important genera.

Molecular Identification Keys for Cultivars and Lines of Cornus florida and C. kousa Based on Simple Sequence Repeat Loci

Flowering dogwood (Cornus florida) and kousa dogwood (C. kousa) are popular ornamental species commonly used in the horticultural industry. Both trees are valued for their beautiful floral display and four-season appeal. Species-specific simple sequence repeat (SSR) loci were used to genotype and assess genetic diversity of 24 flowering dogwood cultivars and breeding lines and 22 kousa dogwood cultivars. Genetic diversity was determined by allele sharing distances and principal coordinate analysis and was high in both species. Molecular identification keys were developed for cultivars and breeding lines of each species using a few polymorphic SSRs loci (four in C. florida and five in C. kousa). Most (18 of 24) of the flowering dogwood and all (22 of 22) kousa dogwood accessions could be distinguished from each other using these SSRs; those that could not were resolved using DNA amplification fingerprinting. The reliability of both keys was assessed using five anonymous cultivars for each dogwood species, which were correctly identified using the molecular keys. The genetic information presented here will be useful for identification and verification of cultivars for nurseries and as molecular markers for breeders and researchers.