Supplementary Materials Supplementary Data supp_18_6_471__index. The map consisted of 97 loci, comprising 37 novel EST-SSRs and 60 released SSRs, distributed on 23 linkage groupings and covered 842.9 cM with a mean interval of 11.9 cM and 4 loci per linkage group. Although the amounts of linkage groupings go beyond the haploid amount (18), but with a TH-302 kinase inhibitor few common markers between homologous linkage groupings with the prior map indicated that the F1 map in this study is appropriate for further study in marker-assisted selection. is usually a perennial cross-pollinating and monoecious plant that belongs to the Euphorbiaceae family. The observation of tetravalents during meiosis has lead to the conclusion that is a stabilized amphidiploid (2= 4= 36).1 However, the pattern of marker ratios segregating in a population of over 100 trees suggests that behaves as a diploid (2= 36).2 Several research groups have developed molecular markers to study the genetic diversity of in GenBank, restricting the quality of research that can be performed on this important plant species. Previous transcriptome studies of have been limited in range, focusing mainly on latex in order to gain insight into the rubber biosynthesis pathways.20C22 Of the available ESTs, 11 256 ESTs are from latex, 1091 ESTs are from bark and 18 ESTs are from leaves. In addition to gene discovery, EST resources enable the identification of markers such as EST-SSRs and single-nucleotide polymorphism. Since TH-302 kinase inhibitor these markers are directly linked to functional genes, they are useful for assessing genetic diversity and mapping phenotypic traits. Feng using 87 EST-SSR markers. The result provided evidence for cross-taxa transferability and indicated moderate polymorphisms of EST-SSR markers in species.3 However, additional markers are desirable to enable quality research into the genetic basis of commercially relevant traits that can be used in marker-assisted breeding programs. Genomic and transcriptomic resources for can greatly benefit from the software of the recent high-throughput sequencing technology, such as the 454 pyrosequencer,23 which has been instrumental in the development of genetic databases for several economical crops.24C27 The purpose of the present study is therefore to sequence the transcriptome of the shoot apical tissue, which is a highly dynamic structure, to discover genes, expand the EST database and develop EST-SSR markers that can be used for assessing TH-302 kinase inhibitor genetic diversity, constructing linkage maps and identifying traits of commercial interest. 2.?Materials and methods 2.1. Plant materials The shoot apical meristem (SAM; 1-cm long from the vegetative shoot apex) of (clone RRIM600) was collected for RNA extraction from an experimental field at the Rubber Research Institute of Thailand, Ministry of Agriculture and Cooperatives, Thailand. The sample was immediately frozen in liquid nitrogen and stored at ?80C until RNA extraction. For the analysis of SSR markers, leaf samples from 20 clones of and 1 accession of (Supplementary data, File S1) were collected and DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). For genetic linkage map construction, 81 samples of an F1 mapping populace were developed from a cross between RRIM600 as a female parent and RRII105 as a male parent. The plants were grown at Chachoengsao Rubber Research Center Office of Agriculture Research and Development, Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand. DNA samples were extracted using a DNeasy Plant Mini Kit (Qiagen). The concentration of each sample was calculated from the OD measurement using a nanodrop ND1000 (NanoDrop Technologies). 2.2. cDNA library preparation and sequencing Total RNA was extracted using Concert? Plant RNA Reagent (Invitrogen). 2 hundred TH-302 kinase inhibitor nanograms of the poly-A mRNA sample was isolated using a truly mRNA Purification Package (Stratagene) and fragmented in 10 fragmentation buffer (0.1 M ZnCl2, 0.1 M TrisCHCl, pH 7.0) in 70C for 30 s. The response was stopped with the addition of 2 l of 0.5 M EDTA and 28 l of 10 mM TrisCHCl, pH 7.5. The mRNA sample was cleaned using Agencourt RNAClean reagent (Beckman Coulter), washed with 200 l of 70% EtOH, surroundings dried and eluted in 20 l of TLR4 10 mM TrisCHCl, pH 7.5. Fragmented mRNA samples had been changed into double-stranded cDNA with the cDNA Synthesis Program Kit (Roche SYSTEMS) using random primers and AMV Reverse Transcriptase. A cDNA library for 454 pyrosequencing was ready based on the October 2008 edition of the cDNA Fast Library Preparation.