Genetic analysis of forest species Eugenia uniflora L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA)(n) and (CA)(n) microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeir (a) over tildeo Preto, Tambau and S (a) over tildeo Jose do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guacu river basins, in state of S (a) over tildeo Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H(E) = 0.83) and a low genetic differentiation (mean F(ST) = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.

Genetic variability of Brazilian rice landraces determined by SSR markers

The objective of this study was to evaluate the genetic variability of rice (Oryza sativa) landraces collected in Brazilian small farms. Twelve simple sequence repeat (SSR) markers characterized 417 landraces collected in 1986, 1987 and 2003, in the state of Goiás, Brazil. The number of landraces with long and thin grain type increased in the evaluated period, probably due to market demand. Based on the molecular data, the genetic variability increased during this period and, as per to the factorial correspondence analysis, most of the accessions were grouped according to the year of collection. The incorporation of modern rice cultivars in landrace cultivation areas and the selection carried out by small farmers are the most probable factors responsible for increasing landrace genetic variability, during the evaluated period. Genotype exchange between farmers, selection practice and local environmental adaptation are able to generate novel adapted allele combinations, which can be used by breeding programs, to reinitiate the process.

Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers

The objectives of this work were to investigate the genetic variation in 79 soybean (Glycine max) accessions from different regions of the world, to cluster the accessions based on their similarity, and to test the correlation between the two types of markers used. Simple sequence repeat markers present in genomic (SSR) and in expressed regions (EST-SSR) were used. Thirty SSR primer-pairs were selected (20 genomic and 10 EST-SSR) based on their distribution on the 20 genetic linkage groups of soybean, on their trinucleotide repetition unit and on their polymorphism information content. All analyzed loci were polymorphic, and 259 alleles were found. The number of alleles per locus varied from 2-21, with an average of 8.63. The accessions exhibit a significant number of rare alleles, with genotypes 19, 35, 63 and 65 carrying the greater number of exclusive alleles. Accessions 75 and 79 were the most similar and accessions 31 and 35, and 40 and 78, were the most divergent ones. A low correlation between SSR and EST-SSR data was observed, thus genomic and expressed microsatellite markers are required for an appropriate analysis of genetic diversity in soybean. The genetic diversity observed was high and allowed the formation of five groups and several subgroups. A moderate relationship between genetic divergence and geographic origin of accessions was observed.

Genetic diversity of cultivated accessions and wild species of rubber tree using EST‑SSR markers

The objective of this work was to evaluate the efficiency of EST‑SSR markers in the assessment of the genetic diversity of rubber tree genotypes (Hevea brasiliensis) and to verify the transferability of these markers for wild species of Hevea. Forty‑five rubber tree accessions from the Instituto Agronômico (Campinas, SP, Brazil) and six wild species were used. Information provided by modified Roger's genetic distance were used to analyze EST‑SSR data. UPGMA clustering divided the samples into two major groups with high genetic differentiation, while the software Structure distributed the 51 clones into eight groups. A parallel could be established between both clustering analyses. The 30 polymorphic EST‑SSRs showed from two to ten alleles and were efficient in amplifying the six wild species. Functional EST‑SSR microsatellites are efficient in evaluating the genetic diversity among rubber tree clones and can be used to translate the genetic differences among cultivars and to fingerprint closely related materials. The accessions from the Instituto Agronômico show high genetic diversity. The EST‑SSR markers, developed from Hevea brasiliensis, show transferability and are able to amplify other species of Hevea.

Genetic diversity of sweet sorghum germplasm in Mexico using AFLP and SSR markers

The objective of this work was to evaluate the diversity and genetic relationships between lines and varieties of the sweet sorghum (Sorghum bicolor) germplasm bank of the National Institute for Forestry, Agriculture and Livestock Research, Mexico, using AFLP and SSR markers. The molecular markers revealed robust amplification profiles and were able to differentiate the 41 genotypes of sweet sorghum evaluated. Analysis of the frequency and distribution of polymorphic fragments allowed for the detection of unique (AFLP) and rare (SSR) alleles in several genotypes (RBSS‑8, RBSS‑9, RBSS‑25, RBSS‑32, and RBSS‑37), indicating that these markers may be associated with a feature that has not yet been determined or may be useful for the identification of these genotypes. The genetic relationships indicated the presence of at least two types of sweet sorghum: a group of modern genotypes used for sugar and biofuel production, and another group consisting of historic and modern genotypes used for the production of syrups. Sweet sorghum genotypes may be used to develop new varieties with higher sugar and juice contents.

Optimal use of SSR markers for varietal identification of upland cotton

Abstract: The objective of this work was to identify polymorphic simple sequence repeat (SSR) markers for varietal identification of cotton and evaluation of the genetic distance among the varieties. Initially, 92 SSR markers were genotyped in 20 Brazilian cotton cultivars. Of this total, 38 loci were polymorphic, two of which were amplified by one primer pair; the mean number of alleles per locus was 2.2. The values of polymorphic information content (PIC) and discrimination power (DP) were, on average, 0.374 and 0.433, respectively. The mean genetic distance was 0.397 (minimum of 0.092 and maximum of 0.641). A panel of 96 varieties originating from different regions of the world was assessed by 21 polymorphic loci derived from 17 selected primer pairs. Among these varieties, the mean genetic distance was 0.387 (minimum of 0 and maximum of 0.786). The dendrograms generated by the unweighted pair group method with arithmetic average (UPGMA) did not reflect the regions of Brazil (20 genotypes) or around the world (96 genotypes), where the varieties or lines were selected. Bootstrap resampling shows that genotype identification is viable with 19 loci. The polymorphic markers evaluated are useful to perform varietal identification in a large panel of cotton varieties and may be applied in studies of the species diversity.

Genetic diversity among alfalfa genotypes (Medicago sativa L.) of non-dormant cultivars using SSR markers and agronomic traits

The aim of this study was to assess genetic diversity among 40 alfalfa (Medicago sativa L.) genotypes of different non-dormant (FD=8) cultivars. Biomass yield, regrowth speed and reaction to spring black stem, lepto leaf spot, and rust were evaluated. Analyses of variances were performed using a mixed model to examine the agronomic variation among individuals. A principal component analysis on standardized agronomic data was performed. Agronomic data were also used to calculate Gower's distance and UPGMA algorithm. For the molecular analysis, six SSR markers were evaluated and 84 alleles were identified. The genetic distance was estimated using standard Nei's distance. Average standard genetic diversity was 0.843, indicating a high degree of variability among genotypes. Finally, a generalized procrustes analysis was performed to calculate the correlation between molecular and agronomic distance, indicating a 65.4% of consensus. This value is likely related to the low number of individuals included in the study, which might have underestimated the real phenotypic variability among genotypes. Despite the low number of individuals and SSR markers analyzed, this study provides a baseline for future diversity studies to identify genetically distant alfalfa individuals or cultivars.

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ∼25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.

Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

Rationalization of taro germplam collections in the Pacific Island region using simple sequence repeat (SSR) markers

A regional (Oceania) core collection for taro germplasm has been developed based on phenotypic and molecular characterization. In total, 2199 accessions of taro germplasm have been collected by TaroGen (Taro Genetic Resources: Conservation and Utilisation) from 10 countries in Oceania: Papua New Guinea, Solomon Islands, Vanuatu, New Caledonia, Fiji, Palau, Niue, Tonga, Cook Islands and Samoa. Our objective was to select 10% from each country to contribute to a regional core. The larger collections from Papua New Guinea, Vanuatu and New Caledonia were analysed based on phenotypic characters, and a diverse subset representing 20% of these collections was fingerprinted. A diverse 20% subsample was also taken from the Solomon Islands. All accessions from the other six countries were fingerprinted. In total, 515 accessions were genotyped (23.4% overall) using taro specific simple sequence repeat (SSR) markers. DNA fingerprint data showed that great allelic diversity existed in Papua New Guinea and the Solomon Islands. Interestingly, rare alleles were identified in taros from the Solomon Islands province of Choiseul which were not observed in any of the other collections. Overall, 211 accessions were recommended for inclusion in the final regional core collection based on the phenotypic and molecular characterization.

Current range characteristics of Swiss stone pine (Pinus cembra L.) along the Carpathians revealed by chloroplast SSR markers.

We investigated the diversity pattern of nine Swiss stone pine (Pinus cembra L.) populations along the Carpathian range including the High Tatras, by using six chloroplast DNA microsatellites (cpSSR). Our aim was to detect genetically distinct regions by clustering of populations, and to tackle possible historical colonization routes. Our analysis referred to an investigated geographical range with the two most distant populations situated at about 500 air km. We found that the most diverse populations are situated at the two edges of the investigated part, in the Retezat Mts. (South Carpathians) and the High Tatras, and diversity decreases towards the populations of the Eastern Carpathians. Hierarchical clustering and NMDS revealed that the populations of the South Carpathians with the Tatras form a distinct cluster, significantly separated from those of the Eastern Carpathians. Moreover, based on the most variable chloroplast microsatellites, the four populations of the two range edges are not significantly different. Our results, supported also by palynological and late glacial macrofossil evidences, indicate refugial territories within the Retezat Mts. that conserved rich haplotype composition. From this refugial territory Pinus cembra might have colonized the Eastern Carpathians, and this was accompanied by a gradual decrease in population diversity. Populations of the High Tatras might have had the same role in the colonizing events of the Carpathians, as positive correlation was detected among populations lying from each other at a distance of 280 km, the maximum distance between neighbouring populations.

Development, characterization and use of genomic SSR markers for assessment of genetic diversity in some Saudi date palm ( Phoenix dactylifera L.) cultivars

Background: The present study was undertaken towards the development of SSR markers and assessing genetic relationships among 32 date palm ( Phoenix dactylifera L.) representing common cultivars grown in different geographical regions in Saudi Arabia. Results: Ninety-three novel simple sequence repeat markers were developed and screened for their ability to detect polymorphism in date palm. Around 71% of genomic SSRs were dinucleotide, 25% tri, 3% tetra and 1% penta nucleotide motives. Twenty-two primers generated a total of 91 alleles with a mean of 4.14 alleles per locus and 100% polymorphism percentage. A 0.595 average polymorphic information content and 0.662 primer discrimination power values were recorded. The expected and observed heterozygosities were 0.676 and 0.763 respectively. Pair-wise similarity values ranged from 0.06 to 0.89 and the overall cultivars averaged 0.41. The UPGMA cluster analysis recovered by principal coordinate analysis illustrated that cultivars tend to group according to their class of maturity, region of cultivation, and fruit color. Analysis of molecular variations (AMOVA) revealed that genetic variation among and within cultivars were 27% and 73%, respectively according to geographical distribution of cultivars. Conclusions: The developed microsatellite markers are additional values to date palm characterization tools that can be used by researchers in population genetics, cultivar identification as well as genetic resource exploration and management. The tested cultivars exhibited a significant amount of genetic diversity and could be suitable for successful breeding program. Genomic sequences generated from this study are available at the National Center for Biotechnology Information (NCBI), Sequence Read Archive (Accession numbers. LIBGSS_039019).

Novel and highly informative Capsicum SSR markers and their cross-species transferability.

2016

Mapping highly informative SSR markers in the genome of Magnaportheoryzae from wheat.

2016

Extension of the core map of common bean with EST-SSR, RGA, AFLP, and putative functional markers

Microsatellites and gene-derived markers are still underrepresented in the core molecular linkage map of common bean compared to other types of markers. In order to increase the density of the core map, a set of new markers were developed and mapped onto the RIL population derived from the `BAT93` x `Jalo EEP558` cross. The EST-SSR markers were first characterized using a set of 24 bean inbred lines. On average, the polymorphism information content was 0.40 and the mean number of alleles per locus was 2.7. In addition, AFLP and RGA markers based on the NBS-profiling method were developed and a subset of the mapped RGA was sequenced. With the integration of 282 new markers into the common bean core map, we were able to place markers with putative known function in some existing gaps including regions with QTL for resistance to anthracnose and rust. The distribution of the markers over 11 linkage groups is discussed and a newer version of the common bean core linkage map is proposed.