Large-scale advances in SSR markers with high-throughput sequencing in Euphorbia fischeriana Steud

BACKGROUND: Euphorbia fischeriana Steud is a very important medicinal herb and has significant medical value for healing cancer, edema and tuberculosis in China. The lack of molecular markers for Euphorbia fischeriana Steud is a dominant barrier to genetic research. For the purpose of developing many simple sequence repeat (SSR) molecular markers, we completed transcriptome analysis with the Illumina HiSeq 2000 platform. RESULTS: Approximately 9.1 million clean reads were acquired and then assembled into approximately 186.3 thousand nonredundant unigenes, 53,146 of which were SSR-containing unigenes. A total of 76,193 SSR loci were identified. Of these SSR loci, 28,491 were detected at the terminal position of ESTs, which made it difficult to design SSR primers for these SSR-containing sequences, and the residual SSRs were thus used to design primer pairs. Analyzing the results of these markers revealed that the mononucleotide motif A/T (44,067, 57.83% of all SSRs) was the most abundant, followed by the dinucleotide type AG/CT (9430, 12.38%). Using 100 randomly selected primer pairs, 77 primers were successfully amplified in Euphorbia fischeriana Steud, and 79 were successfully amplified in three other related species. The markers developed displayed relatively high quality and cross-species transferability. CONCLUSIONS: The large number of EST-SSRs exploited successfully in Euphorbia fischeriana Steud for the first time could provide genetic information for research on linkage maps, variety identification, genetic diversity analysis, and molecular marker-assisted breeding.

Use of EST database markers from M. truncatula in the transferability to other forage legumes.

In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60oC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. A total of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNA from 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55oC and 32.3% at 60oC annealing temperature. 24 alleles of the total 50 (48%) at 55oC and 27 of 51 (53%) at 60oC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where Sequential Agglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.

Characterization of novel genic SSR markers in Linum usitatissimum (L. ) and their transferability across eleven Linum species

Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3 percent) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1 percent to 92.7 percent in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies.