EST-derived Simple Sequence Repeat Research Articles

Gene flow between wild trees and cultivated varieties shapes the genetic structure of sweet chestnut (Castanea sativa Mill.) populations

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.

Functional characteristics of EST-SSR markers available for Scots pine

EST-derived simple sequence repeat markers (EST-SSRs) are important tools for studies on genetic diversity, phylogeny, evolution, comparative genomics, QTL analysis, and gene-based associations. We have searched the literature for known EST-SSRs used for Scots pine (Pinus sylvestris L.) – one of the world’s major forest species. Then, 91 of 102 EST-SSRs suggested for Scots pine studies were manually aligned against the reference genome of Pinus taeda L. as well as available genes of P. sylvestris. For 83 EST-SSRs, genome location and consensus putative functions of the associated genes were identified through conservative domain analysis (CDD), functional analysis of known homologs in terms of Gene Ontology annotations, and KEGG pathway analysis. Many of the markers were located in untranslated regions (mostly in 3’-UTR), as well as in coding sequences of Scots pine and loblolly pine genes. For eight markers whose EST sequences were known no genes could be identified in any of the species. Seven of these markers were located in P. taeda scaffold regions carrying no genes in the current genome assembly (v.1.0). The results can be used in the future to improve the choice of markers for population genetic research, studies of adaptive traits, and QTL mapping of P. sylvestris, as well as other pine species.

Quantitative Trait Loci Analysis Based on High-Density Mapping of Single-Nucleotide Polymorphisms by Genotyping-by-Sequencing Against Pine Wilt Disease in Japanese Black Pine (Pinus thunbergii).

Identifying genes/loci for resistance to pine wilt disease (PWD) caused by the pine wood nematode (PWN) is beneficial for improving resistance breeding in Pinus thunbergii, but to date, genetic information using molecular markers has been limited. Here, we constructed a high-density linkage map using genotyping-by-sequencing (GBS) and conducted quantitative trait loci (QTL) analysis for PWD resistance for the self-pollinated progeny of “Namikata 73,” which is the most resistant variety among resistant varieties of P. thunbergii, following inoculation tests with PWN. An S1 mapping population consisting of the 116 progenies derived from self-pollination of the resistant variety, “Namikata 73” (resistance rank 5 to PWN), was inoculated with PWN isolate Ka-4 and evaluated for disease symptoms. To construct a high-density linkage map, we used single-nucleotide polymorphisms (SNPs) identified by GBS based on next-generation sequencing technology and some anchor DNA markers, expressed sequence tag (EST)-derived SNP markers and EST-derived simple sequence repeat (SSR) markers, and genomic SSR markers. The linkage map had 13 linkage groups (LGs) consisting of 2,365 markers including 2,243 GBS-SNP markers over a total map distance of 1968.4 centimorgans (cM). Results from QTL analysis using phenotype data and the linkage map indicated that PWD resistance is controlled by a single locus located on LG-3, as identified in a previous study. This locus showed overdominant genetic action in the present study. With the confirmation of PWD1 in two different mapping populations (present study and a previous study), the locus associated with this region is thought to be a good target for marker-assisted selection in P. thunbergii breeding programs in order to obtain high levels of resistance to PWD caused by PWN.

Development of EST-Molecular Markers from RNA Sequencing for Genetic Management and Identification of Growth Traits in Potato Grouper (Epinephelus tukula)

Simple SummaryThe potato grouper is a novel aquaculture species in Taiwan. Due to the lack of genetic information concerning this species, we have developed molecular markers based on transcriptome sequencing and further characterized their association with gene diversity and growth traits of this species. Ultimately, these markers could be utilized as accurate and efficient tools for genetic management and marker-assisted selection of potato grouper with distinct growth traits.The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.

Characterization of new microsatellite markers from sugarcane (Saccharum officinarum) transcriptome

Microsatellites, or simple sequence repeats (SSR), developed by expressed sequence tag (EST) databases is an economical and efficient tools that can be used to perform genetic investigations at a functional level. Here, a new sugarcane database of transcriptome from our variety, GT35, was examined for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 51 new EST-SSRs were identified for possible use as potential genetic markers from no redundant SSR-positive ESTs, which were unmapped with the sequences available in the NCBI’EST database of sugarcane by BLASTN. Polymorphisms of the identified 51 EST-SSR markers were evaluated in 21 sugarcane genotypes, planted and collected in 2013 at Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences (SRI-GXAAS) in China. High polymorphisms were detected in terms of number of alleles ranging from 5-36 with an average of 16.8 per locus and polymorphism information content values ranging from 0.74 to 0.95 with a mean of 0.92. Average transferability to Erianthus arundinaceus and Narenga porphyrocoma was 23.9% and 34.4%, respectively. The ability to establish genetic relationship was analyzed by cluster analysis, the result of which revealed that the major grouping was in accordance with taxonomical classification. The development of new EST-SSR markers presented in this work will have important implications for genetic analysis and breeding.

Characterization and development of EST-derived SSR markers in Sinowilsonia henryi (Hamamelidaceae).

Premise of the study:Polymorphic microsatellite markers were developed to reveal the genetic diversity of extant populations and the mating system of Sinowilsonia henryi (Hamamelidaceae).Methods and Results:In this study, nuclear simple sequence repeat (SSR) markers were developed using the Illumina high-throughput sequencing technique (RNA-Seq). The de novo–assembled transcriptome generated a total of 64,694 unique sequences with an average length of 601 bp. A total of 2941 microsatellite loci were detected. Of the 121 tested loci, 13 loci were polymorphic and eight were monomorphic among 72 individuals representing three natural populations of the species. The number of alleles per locus ranged from one to four, and the observed and expected heterozygosity at population level were 0.00–1.00 and 0.10–0.66, respectively.Conclusions:The developed expressed sequence tag (EST)–SSRs will be useful for studying genetic diversity of S. henryi as well as assessing the mating system among Sinowilsonia species.

Development and characterization of EST-derived SSR markers in the cereal cyst nematode Heterodera avenae

The cereal cyst nematode Heterodera avenae is a pathogen of cereal crops and causes high yield losses worldwide. In this study, a collection of 37,348 expressed sequence tags (ESTs) of H. avenae was mined for EST-based simple sequence repeat (SSR) markers, which resulted in the identification of 5604 SSRs. A total of 210 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Eight SSR markers were finally identified and analyzed using 96 individual cysts of H. avenae sampled from three provinces in China. These loci were found to be moderately polymorphic with 3–8 alleles per locus. The observed and expected heterozygosity across the three populations ranged from 0.000 to 0.594 and from 0.000 to 0.731, respectively. The polymorphism information content (PIC) was medium and ranged from 0.080 to 0.562, with a mean of 0.409. The F ST ranged from 0.1034 to 0.1550, indicating moderate genetic differentiation among the three H. avenae populations. These EST-SSR markers can be used to study population genetic diversity and the dispersal route of H. avenae in China.

Genetic evaluation of the breeding population of a valuable reforestation conifer Platycladus orientalis (Cupressaceae).

Platycladus orientalis, a widespread conifer with long lifespan and significant adaptability. It is much used in reforestation in north China and commonly planted in central Asia. With the increasing demand for plantation forest in central to north China, breeding programs are progressively established for this species. Efficient use of breeding resources requires good understanding of the genetic value of the founder breeding materials. This study investigated the distribution of genetic variation in 192 elite trees collected for the breeding program for the central range of the species. We developed first set of 27 polymorphic EST-derived SSR loci for the species from transcriptome/genome data. After examination of amplification quality, 10 loci were used to evaluate the genetic variation in the breeding population. We found moderate genetic diversity (average He = 0.348) and low population differentiation (Fst = 0.011). Extensive admixture and no significant geographic population structure characterized this set of collections. Our analyses of the diversity and population structure are important steps toward a long-term sustainable deployment of the species and provide valuable genetic information for conservation and breeding applications.

Development and validation of EST-derived SSR markers and diversity analysis in cluster bean (Cyamopsis tetragonoloba)

Cluster bean/guar (Cyamopsis tetragonoloba), has an important place in industry because of its seeds, which contain galactomannan (guar gum) rich endosperm. Guar gum, an important ingredient of many products, is purely an export oriented commodity. Development of molecular markers for this crop is essential to accelerate breeding for guar gum content in seeds. A total of 100 novel primers pairs were developed from 16,476 expressed sequence tags (ESTs) sequence of cluster bean. A total of 50 primers pairs with function annotation of gum synthesis were selected and validated on a panel of 32 genotypes. Among the 50 primers 39 primers were amplified with a total of 45 loci. The polymorphic information content (PIC) ranged from 0.00 to 0.42 with an average of 0.13. With low polymorphic simple sequence repeats (SSRs) and narrow genetic base, most of the genotypes scattered into three clusters regardless of their geographical origin. Present study showed the existence of very low genetic diversity in cluster bean. The results indicated that there is need to explore SSR markers from whole genome or alternative marker systems like SNP (single nucleotide polymorphism) markers, for effective implication of markers in cluster bean breeding.

Analysis of inheritance mode in chrysanthemum using EST-derived SSR markers

To study the inheritance mode of hexaploid chrysanthemum (random or preferential chromosome pairing), a segregation analysis was carried out using SSR markers derived from chrysanthemum ESTs in the public domain. A total of 248 EST-SSR primer pairs were screened in chrysanthemum cultivars ‘Dancer’ and ‘Puma White’, of which 49 EST-SSRs were selected as polymorphic and informative markers. These polymorphic markers were used for genotyping a F1-pseudo test cross population derived from a cross between these two cultivars. The 49 EST-SSRs detected 210 marker alleles with an average number of 4.29 marker alleles per locus. For 180 of these polymorphic SSR marker alleles, segregation could be estimated using a χ2 goodness of fit test (α=0.05) with the expected segregation ratios for hexasomic or disomic inheritance. For 65 SSR marker alleles the segregation ratio was informative for the type of inheritance, 33 marker alleles gave a good fit to the expected segregation ratio for hexasomic inheritance and whereas 24 marker alleles gave a good fit for disomic inheritance showing a higher number of marker alleles supporting autopolyploid segregation in chrysanthemum. In addition, the observed ratio of non-simplex to simplex markers was 20:80 (25 vs. 99) supported hexasomic inheritance. Furthermore, random marker allele assortment was found within the six fully informative markers giving conclusive evidence for hexasomic inheritance in chrysanthemum at these chromosomal regions.

Development of EST-derived SSR markers using next-generation sequencing to reveal the genetic diversity of 50 chrysanthemum cultivars

Chrysanthemum plants are popular worldwide as cut flowers, potted, and in gardens. Several hundred cultivars have been commercialized, indicating that there is substantial genetic variations that can be manipulated under cultivations to produce a wide array of phenotypic variation. To study the genetic diversity of chrysanthemum cultivars in Korea, we first identified simple sequence repeats from chrysanthemum expressed sequence tags generated by FLX 454 sequencing. A total of 1109 ESTs out of 18,226 chrysanthemum ESTs were identified to carry SSRs. A total of 16 out of 46 primer pairs exhibited several polymorphisms among 50 chrysanthemum cultivars. The number of alleles per locus varied from 1 to 15, with an average of 6.25 alleles. The expected heterozygosity ranged from 0 to 0.8958, whereas polymorphism information content ranged from 0 to 0.8872. Based on polymorphisms using 16 SSR markers, a phylogenetic tree was generated revealing four groups within the 50 cultivars showing various levels of genetic diversity. The 16 polymorphic chrysanthemum SSR markers generated in this study would be useful for studies of the genetic conservation, diversity, and population structure of commercial chrysanthemum cultivars as well as closely related species.

Comparison of gSSR and EST-SSR markers for analyzing genetic variability among tomato cultivars (Solanum lycopersicum L.).

In order to study genetic variability and develop better strategies for the utilization of 48 tomato cultivars from America, China, the Netherlands, and Portugal, genomic simple sequence repeat (gSSR) and EST-derived SSR (EST-SSR) markers were applied. In all, 15 of 82 gSSR and 18 of 115 EST-SSR markers showed polymorphic loci. There were 995 and 2072 clear fragments amplified by polymorphic gSSR and EST-SSR markers, respectively. The total and average number of alleles detected by EST-SSRs (75, 4.2) was more than gSSRs (54, 3.6) as a result of some multi-locus EST-SSRs. A lower polymorphism information content value was found in gSSRs (0.529) compared to EST-SSRs (0.620). Similarity coefficient matrixes of the 48 tomato cultivars were established based on the gSSRs and EST-SSRs, and UPGMA dendrograms were constructed from the gSSRs and EST-SSRs similarity coefficient matrixes. A high similarity was observed between the gSSRs and EST-SSRs dendrograms. Genetic variability of four tomato populations from different countries showed that the observed number of alleles and Nei's genetic diversity were highest in the American population, and the effective number of alleles was highest in the Dutch population. The estimated genetic structure showed some tomato cultivars from different countries shared a common genetic background, which might be related to gene flow. It was inferred that both gSSR and EST-SSR markers were effective to assess genetic variability of tomato cultivars, and the combination of both markers could be more effective for genetic diversity analysis in tomato.

Breeding malting barley for consistently low grain protein to sustain production against predicted changes from global warming

Climate change, as summarised by global warming, poses a substantial challenge to malting barley (Hordeum vulgare L.) production and the malting industries. This is because, in sensitive varieties, heat stress causes grain protein content (GPC) to exceed the maximum acceptable value for malting purposes. One way to avoid the problem is to use cultivars with inherently low-GPC and hence reduce the risk of approaching a GPC above 12 %. The present study aimed to characterise the genetic locus on 5HS linked to GPC in two-rowed barley and to combine it with the locus on 6H identified in six-rowed barley. Saturation mapping with EST-derived SSR markers identified four new markers (UMB704, K03390, UMB707, and HvSMEh297) within the target QTL region, which provides functional markers for selection. Over a 3-year period, doubled haploid lines differing in the presence/absence of the marker alleles at the 5HS locus were characterised for physiological differences related to GPC accumulation. Both genotype groups flowered at about the same time, but during grain filling, the low-GPC group accumulated less nitrogen in the spikes, and more dry matter, than the high-GPC group. The 5HS and 6H QTLs were combined into two-row backgrounds, and preliminary field evaluation showed that almost all of the recombinants had GPC levels that were lower than the commercial check (Schooner) by up to 4 % units. Of the 155 F2-derived F5 lines, 20 had grain protein levels that were lower than that of the low-GPC check (VB0229) by 1–2 %. This represents a new germplasm pool that will be useful for variety development, not just to lower GPC, but also to stabilise GPC variability across different environments.

Construction of full-length cDNA library and development of EST-derived simple sequence repeat (EST-SSR) markers in Senecio scandens.

Senecio scandens Buch.-Ham. ex D. Don (Compositae) is a crucial source of Chinese traditional medicine with antibacterial properties. We constructed a cDNA library and obtained expressed sequence tags (ESTs) to show the distribution of gene ontology annotations for mRNAs, using an individual plant with superior antibacterial characteristics. Analysis of comparative genomics indicates that the putative uncharacterized proteins (21.07%) might be derived from "molecular function unknown" clones or rare transcripts. Furthermore, the Compositae had high cross-species transferability of EST-derived simple sequence repeats (EST-SSR), based on valid amplifications of 206 primer pairs developed from the newly assembled expressed sequence tag sequences in Artemisia annua L. Among those EST-SSR markers, 52 primers showed polymorphic amplifications between individuals with contrasting diverse antibacterial traits. Our sequence data and molecular markers will be cost-effective tools for further studies such as genome annotation, molecular breeding, and novel transcript profiles within Compositae species.

Characterization of EST-derived and non-EST simple sequence repeats in an F₁ hybrid population of Vitis vinifera L.

Among different classes of molecular markers, expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping in F1 hybrid populations of Vitis vinifera L. Recently, because of the availability of an enormous amount of data for ESTs in the public domain, the emphasis has shifted from genomic SSRs to EST-SSRs, which belong to transcribed regions of the genome and may have a role in gene expression or function. The objective of this study was to assess the polymorphisms among 94 F1 hybrids from "Early Rose" and "Red Globe" using 25 EST-derived and 25 non-EST SSR markers. A total collection of 362,375 grape ESTs that were retrieved from the National Center for Biotechnology Information (NCBI) and 2522 EST-SSR sequences were identified. From them, 205 primer pairs were randomly selected, including 176 pairs that were EST-derived and 29 non-EST SSR primer pairs, for polymerase chain reaction amplification. A total of 131 alleles were amplified using 50 pairs of primers; 78 alleles were amplified using EST-derived SSR primers and 53 were from non-EST SSR primers. At most, 6 and 5 alleles were amplified by EST-derived and non-EST SSR primers, respectively. The EST-derived SSR markers showed a maximum polymorphic information content (PIC) value of 1 and a minimum of 0.33 while non-EST SSR markers had maximum and minimum PIC values of 1 and 0.25, respectively. The average PIC value was 0.56 for EST-derived SSR markers and 0.45 for non-EST SSR markers.

SNP discovery and high-density genetic mapping in faba bean (Vicia faba L.) permits identification of QTLs for ascochyta blight resistance

Ascochyta blight, caused by the fungus Ascochyta fabae Speg., is a common and destructive disease of faba bean (Vicia faba L.) on a global basis. Yield losses vary from typical values of 35–40% to 90% under specific environmental conditions. Several sources of resistance have been identified and used in breeding programs. However, introgression of the resistance gene determinants into commercial cultivars as a gene pyramiding approach is reliant on selection of closely linked genetic markers. A total of 14,552 base variants were identified from a faba bean expressed sequence tag (EST) database, and were further quality assessed to obtain a set of 822 high-quality single nucleotide polymorphisms (SNPs). Sub-sets of 336 EST-derived simple sequence repeats (SSRs) and 768 SNPs were further used for high-density genetic mapping of a biparental faba bean mapping population (Icarus×Ascot) that segregates for resistance to ascochyta blight. The linkage map spanned a total length of 1216.8cM with 12 linkage groups (LGs) and an average marker interval distance of 2.3cM. Comparison of map structure to the genomes of closely related legume species revealed a high degree of conserved macrosynteny, as well as some rearrangements. Based on glasshouse evaluation of ascochyta blight resistance performed over two years, four genomic regions controlling resistance were identified on Chr-II, Chr-VI and two regions on Chr-I.A. Of these, one (QTL-3) may be identical with quantitative trait loci (QTLs) identified in prior studies, while the others (QTL-1, QTL-2 and QTL-4) may be novel. Markers in close linkage to ascochyta blight resistance genes identified in this study can be further validated and effectively implemented in faba bean breeding programs.

Development of new EST-derived SSRs in Salvia miltiorrhiza (Labiatae) in China and preliminary analysis of genetic diversity and population structure

Salvia miltiorrhiza, known as Dan-shen in China, is a well-known and important medicinal herb in Asia. Its root and rhizome has been used to treat cardiovascular diseases in China for centuries. Recently, it had been widely cultivated in China due to a rapid decline in wild resources. However, the germplasm resources of different cultivated regions are extremely variable. Here, new simple sequence repeats (SSRs) from expressed sequence tags (ESTs) were mined to analyze the frequency and distribution of SSRs, assess the validity of developed EST-SSR marker and identify new EST-SSR marker for the preparatory investigation of the population structure in cultivated S. miltiorrhiza. This study showed that 5160 unique ESTs were accumulated from database, 760 SSRs combined with 41 types of SSR motifs were mined. High polymorphism and transferability were presented in newly developed EST-SSR marker. It also showed that cultivated S. miltiorrhiza were mainly divided into three large groups. The results suggested that S. miltiorrhiza may has high abundance of SSRs in transcribed regions of genome and the developed EST-SSR marker may be useful for analysis of genetic diversity and population structure.

SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.).

BackgroundField pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs.ResultsIn this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance.ConclusionThe SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

EST-SSR and SSR analyses of genetic diversity in diploid cotton genotypes from Iran

There are 21 diploid species/genotypes of the cotton genus Gossypium L. that are cultivated in many regions of Iran. Due to continuous artificial selection and cultivation practice there is a decrease in its genetic diversity causing genetic erosion. Therefore, cotton breeders continue to make various cross combinations among available cultivars. Genetic diversity analysis was considered in 21 cotton genotypes by using SSR and EST-derived SSR markers. Amplification of the EST-SSR and SSR markers produced 19 reproducible alleles in the cotton plants studied. The mean number of effective alleles was 1.12, and the mean value of Shannon’s information index was 0.10. The mean value for genetic polymorphism was 20 % in cotton plants studied. AMOVA test showed that only 2 % of total genetic variation is accounted in 3 populations groups, of which 98 % is due to genetic variations among accessions in each population group. Neighbor Joining tree and PCoA plot showed genetic affinity between plants from different populations. STRUCTURE result confirmed AMOVA test and showed presence of genetic diversity within each presumed population groups. Reticulogram showed gene exchange among some of the cotton plants. Gene exchange/allelic similarity were observed between VTDL (G. arboreum) and Semnan (G. herbaceum) genotypes and between some interspecific hybrids.

A Novel Set of EST-Derived SSR Markers for Pear and Cross-Species Transferability in Rosaceae

Pear is a fruit species with worldwide distribution belonging to the Rosaceae family. In this study, transcriptomes of 12 different tissues of pear were newly assembled, with a total of 17,353 SSR loci identified. Within these, repetitive sequences with dinucleotide and trinucleotide motifs were more abundant than other types. We developed 194 EST-SSR makers from the assembled sequences to test on seven pear varieties from different genetic backgrounds, on which 132 EST-SSR primers had amplicons. There were 108 primers with dinucleotide and trinucleotide motifs detecting a total of 311 alleles, with an average per primer of 2.88 and 2.86, respectively. These had better quality than primers with tetranucleotides, pentanucleotides, and hexanucleotides. Fifty polymorphic markers divided 36 pear genotypes into two groups with a similarity coefficient of 0.62. Results showed that Japanese pears had a comparatively close relationship with Chinese pears, indicating the great potential of the novel EST-SSR makers in future evaluations of pear germplasm. Furthermore, 54 EST-SSR markers were selected to test transferability to 96 other Rosaceae cultivars from eight species, including Japanese apricot, plum, apricot, peach, cherry, apple, strawberry, and loquat. Seventeen EST-SSR primers had PCR amplification products in at least one of the 96 accessions, with the highest transferability was from pear to apple at 29.63 %. Transferability from pear to Japanese apricot, plum, apricot, peach, cherry, strawberry, and loquat was 9.26, 14.81, 11.11, 12.96, 5.56, 3.7, and 12.96 %, respectively. Therefore, the newly developed EST-SSR markers can contribute to exploring the genetic relationship, evolution and comparative genomics, and gene introgressions in the Rosaceae family.