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Calotropis procera (Aiton) Dryand is a medicinally and economically important woody shrub used to cure various diseases. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from C. procera and studied the cross-species transferability of the selected markers. We have identified 34,355 potential EST-SSRs from C. procera and classified them based on the type of repeat motifs and Gene Ontology (GO) annotation. Virtual primers for 17,036 EST-SSRs were designed, and 55 primer pairs representing di-, tri-, tetra-, penta-, and hexa-nucleotide repeats were used for the validation of the EST-SSRs. A total of 48 out of 55 EST-SSR primers showed clear amplification in Calotropis procera. To validate EST-SSRs at the nucleotide level, four randomly selected EST-SSR primer sets were amplified and sequenced. Sequence alignment confirmed the presence of SSRs in the amplified primers. The polymorphisms of the 16 primer pairs were studied, and it was observed that 15 primer sets produced polymorphic bands. Out of these 15 pairs of primers, we identified 141 alleles with an average of 2.56 alleles per locus. The allelic diversity was estimated for 15 EST-SSR markers using three accessions of Calotropis. The study has succeeded in the development of novel EST-SSR markers from Calotropis procera and establishment of the molecular marker resources that can serve as a useful genomic tool for further development of germplasm resources and effective marker-assisted breeding and domestication programs for Calotropis procera. [ABSTRACT FROM AUTHOR]

Lentil is nutritionally important crop for human diet and enriched with quality protein, complex carbohydrates, fibers, essential minerals, and vitamins. However, genetic improvement of lentil is hampered largely due to unattributed and unexploited genetic and genomic resources. To administer genomic resources in lentil, we have identified 9949 EST-SSR loci from lentil RNA Seq data and validated 50 of them using 234 genotypes representing various Lens species and 34 accessions of 12 different legumes. Out of 50 EST-SSRs, 46 were polymorphic with polymorphic information content (PIC) ranging from 0.16–0.74. The transferability of these markers exhibited varied levels from 45.1 to 71.3% across the cultivated/wild species of lentil and from 10.8 to 54.3% across the twelve legume genera. On the basis of total identified EST-SSRs, mononucleotide (51%) repeat proportion was high followed by trinucleotide (30%) and dinucleotide (14%) repeat. Population structure and cluster analysis classified all the studied genotypes into 4 groups. However, principal coordinate analysis (PCA) was able to group genotypes based on their area of collection. Annotation of all the 46 polymorphic marker sequences revealed that most of the markers linked to genes involved in metabolism of plants. Further, polymorphic markers were also used for linkage mapping in F3 population where 4 markers were found to be linked with a map distance of 72.5 cM. The newly developed markers represent an impressive tool for characterization of germplasm, genetic linkage mapping, phylogenetic studies, as well as to determine disparity in taxonomic status of subspecies of the genus Lens. [ABSTRACT FROM AUTHOR]

The genetic improvement of trees for freezing tolerance is one of the most important goals to extend the plantations to colder areas. RNA-Seq technology has become a key tool in transcriptome studies. It can quantify overall expression levels for each gene simultaneously with high efficiency and speed through in silico gene expression, where differentially expressed genes can be identified by measuring the reads mapped for each transcript. In this study, the results of ESTs libraries from two Eucalyptus globulus genotypes showing contrasting differences in frost tolerance after cold acclimation using mRNA-Seq and in silico gene expression are discussed. A total of 14,265 non-redundant transcripts were predicted, where 163 corresponded to upregulated and 537 to downregulated genes. Pathway analyses of upregulated transcripts indicated that differences in frost tolerance might be regulated by the tree response to chemical and osmotic stimulus and organic substances, principally by overexpressing proteins that respond to hormone stress. These results suggest that genes coding for dehydrins, outer envelope, and voltage-dependent anion channel proteins are likely to participate in the regulation of the cold acclimation process and may have an important role in frost tolerance. The transcription factor analysis allowed identifying that those most differentially expressed in a resistant genotype were participating in the regulation of transcription, hormone regulation, photosynthesis, and response to stress. Additionally, the screening of polymorphic EST-SSR in silico and the validation of these markers in a reference population lead to identify a polymorphic EST-SSR with potential use for plant breeding and genotype discrimination. [ABSTRACT FROM AUTHOR]

Although tea leaves are rich in secondary metabolites, not enough transcriptomic information is available to aid understanding of the molecular mechanisms underlying plant growth, development, and secondary metabolite production. In this study, a total of 437,908 reads were generated from the tea leaf transcriptome using 454 sequencing. De novo assembly yielded 25,637 unigenes, 22,872 of which were annotated by BLAST searches against public databases. Most of these unigenes mapped to carbohydrate metabolism, energy metabolism and secondary metabolite biosynthetic pathways. Some abundant transcripts related to photomorphogenesis and development in plants, including ubiquitin/26S proteasome, lipid transfer protein, PPR-containing protein, small GTPase, expansin, transport inhibitor response 1 and thioredoxin, were identified in the transcriptome. Most of the genes encoding the main enzymes involved in flavonoid, caffeine and theanine biosynthesis were also found, and six MYB and two bHLH genes known to regulate flavonoid synthesis were identified. ABC transporter and glutathione S-transferase, generally responsible for secondary metabolite transport, and CYP450, broadly involved in oxidation steps in secondary metabolism, were also present in a large number of unigenes. Additionally, 3,767 EST-SSRs were identified as potential molecular markers in our unigenes. A total of 100 PCR primer pairs used in initial screening tests among 20 tea genotypes successfully identified 36 polymorphic loci. Overall, the tea leaf transcriptome sequences generated in this study reveal novel gene expression profiles and offer important clues for further study of the molecular mechanism of tea leaf growth, development and secondary metabolite synthesis. The thousands of EST-SSR markers identified will facilitate marker-assisted selection in tea breeding. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cassava ( Manihot esculenta Crantz) is a very important staple and industrial crop in tropical and subtropical regions of the world. The paucity of markers is a serious limitation in marker-assisted breeding. A total of 35,992 expressed sequence tags (ESTs) from cassava, which were clustered in 13,173 unigenes, were used in this study. A total of 1,889 microsatellites were identified, with an average density of one simple sequence repeats (SSRs) every 4.40 kb. Of the 1,058 designed EST-SSRs from cultivars SC06, TMS60444, and W14, 431 were polymorphic. Then, 31 randomly selected EST-SSRs from the 431 polymorphic EST-SSRs were used to evaluate the genetic diversity of 76 cassava accessions. A total of 93 alleles were identified, and the number detected for each EST-SSR ranged from one to four. Based on the 93 alleles, the 76 cassava accessions could be classified into six groups, and the genetic similarity coefficient ranged from 0.55 to 0.94. This study demonstrated the potential of EST-derived SSRs in cassava. The resources developed in this study enriched the available molecular markers for cassava. [ABSTRACT FROM AUTHOR]

A genetic linkage map of cassava has been generated with total of 355 molecular markers, 231 amplified fragment length polymorphisms (AFLPs), 41 simple sequence repeats (SSRs), 48 sequence-related amplified polymorphisms (SRAPs), and 35 expressed sequence tag (EST)-SSRs segregating from an F1 population of an intraspecific cross between SC6, as female parent, and Mianbao, as male parent. The genetic map consisted of 18 linkage groups and spanned a 1,707.9 cM genetic distance, with the average marker interval being 4.81 cM. Thirty-five EST-SSR markers that were developed by our laboratory were mapped onto this map. The genetic map generated in this study will enrich the information of structural genomics in cassava, facilitating to identify quantitative trait loci (QTL) of interest and to serve as a useful complement for construction of an integrated map in the future. [ABSTRACT FROM AUTHOR]