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Development and characterization of polymorphic genic-SSR markers in Larix kaempferi.

ABSTRACT: New simple sequence repeat (SSR) markers were developed in the Japanese larch (Larix kaempferi) using unigene sequences for further genetic diversity studies and the genetic improvement of breeding programs. One thousand two handred and thirty five (1235) primer pairs were tested and 165 successfully identified in L. kaempferi. Out of the amplified candidate markers, 145 (90.6%) exhibited polymorphism among 24 individuals of L. kaempferi, with the number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) averaging at 4.510, 0.487, 0.518 and 0.459, respectively. Cross-species amplification of randomly selection of 30 genic-SSRs among the 145 polymorphic ones showed that 80.0% of the SSRs could be amplified in Larix olgensis, 86.7% could be amplified in Larix principi-rupprechtii and 83.0% could be amplified in Larix gmelinii. High rates of cross-species amplification were observed. The genic-SSRs developed herein would be a valuable resource for genetic analysis of Larix kaempferi and related species, and also have the potential to facilitate the genetic improvement and breeding of larch.

SUBMITTER: Chen XB

PROVIDER: S-EPMC6272221 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Development and characterization of polymorphic genic-SSR markers in Larix kaempferi.

Chen Xing-Bin XB Xie Yun-Hui YH Sun Xiao-Mei XM

Molecules (Basel, Switzerland) 20150408 4

New simple sequence repeat (SSR) markers were developed in the Japanese larch (Larix kaempferi) using unigene sequences for further genetic diversity studies and the genetic improvement of breeding programs. One thousand two handred and thirty five (1235) primer pairs were tested and 165 successfully identified in L. kaempferi. Out of the amplified candidate markers, 145 (90.6%) exhibited polymorphism among 24 individuals of L. kaempferi, with the number of alleles per locus (Na), observed heter ...[more]

PMID: 25856058

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Project description:Hybrids of larch (Larix kaempferi × Larix olgensis) are important afforestation species in northeastern China. They are routinely propagated via rooted stem cuttings. Despite the importance of rooting, little is known about the regulation of adventitious root development in larch hybrids. 454 GS FLX Titanium technology represents a new method for characterizing the transcriptomes of non-model species. This method can be used to identify differentially expressed genes, and then two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses can be used to analyze their corresponding proteins. In this study, we analyzed semi-lignified cuttings of two clones of L. kaempferi × L. olgensis with different rooting capacities to study the molecular basis of adventitious root development.We analyzed two clones; clone 25-5, with strong rooting capacity, and clone 23-12, with weak rooting capacity. We constructed four cDNA libraries from 25-5 and 23-12 at two development stages. Sequencing was conducted using the 454 pyrosequencing platform. A total of 957832 raw reads was produced; 95.07% were high-quality reads, and were assembled into 45137 contigs and 61647 singletons. The functions of the unigenes, as indicated by their Gene Ontology annotation, included diverse roles in the molecular functions, biological processes, and cellular component categories. We analyzed 75 protein spots (-fold change ? 2, P ? 0.05) by 2D-DIGE, and identified the differentially expressed proteins using MALDI-TOF/TOF MS. A joint analysis of transcriptome and proteome showed genes related to two pathways, polyamine synthesis and stress response, might play an important role on adventitious root development.These results provide fundamental and important information for research on the molecular mechanism of adventitious root development. We also demonstrated for the first time the combined use of two important technologies as a powerful approach to advance research on non-model, but otherwise important, larch species.

Project description:BACKGROUND: Sesame (Sesamum indicum L.) is one of the most important oil crops; however, a lack of useful molecular markers hinders current genetic research. We performed transcriptome sequencing of samples from different sesame growth and developmental stages, and mining of genic-SSR markers to identify valuable markers for sesame molecular genetics research. RESULTS: In this study, 75 bp and 100 bp paired-end RNA-seq was used to sequence 24 cDNA libraries, and 42,566 uni-transcripts were assembled from more than 260 million filtered reads. The total length of uni-transcript sequences was 47.99 Mb, and 7,324 SSRs (SSRs ?15 bp) and 4,440 SSRs (SSRs ?18 bp) were identified. On average, there was one genic-SSR per 6.55 kb (SSRs ?15 bp) or 10.81 kb (SSRs ?18 bp). Among perfect SSRs (?18 bp), di-nucleotide motifs (48.01%) were the most abundant, followed by tri- (20.96%), hexa- (25.37%), penta- (2.97%), tetra- (2.12%), and mono-nucleotides (0.57%). The top four motif repeats were (AG/CT)n [1,268 (34.51%)], (CA/TG)n [281 (7.65%)], (AT/AT)n [215 (5.85%)], and (GAA/TTC)n [131 (3.57%)]. A total of 2,164 SSR primer pairs were identified in the 4,440 SSR-containing sequences (?18 bp), and 300 SSR primer pairs were randomly chosen for validation. These SSR markers were amplified and validated in 25 sesame accessions (24 cultivated accessions, one wild species). 276 (92.0%) primer pairs yielded PCR amplification products in 24 cultivars. Thirty two primer pairs (11.59%) exhibited polymorphisms. Moreover, 203 primer pairs (67.67%) yielded PCR amplicons in the wild accession and 167 (60.51%) were polymorphic between species. A UPGMA dendrogram based on genetic similarity coefficients showed that the correlation between genotype and geographical source was low and that the genetic basis of sesame in China is narrow, as previously reported. The 32 polymorphic primer pairs were validated using an F2 mapping population; 18 primer pairs exhibited polymorphisms between the parents, and 14 genic-SSRs could be integrated into 9 main linkage groups. CONCLUSIONS: 2,164 genic-SSR markers have been developed in sesame using transcriptome sequencing. 276 of 300 validated primer pairs successfully yielded PCR amplicons in 24 cultivated sesame accessions. These markers increase current SSR marker resources and will greatly benefit genetic diversity, qualitative and quantitative trait mapping and marker-assisted selection studies in sesame.

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Development and characterization of polymorphic genic-SSR markers in Larix kaempferi.

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Development and characterization of polymorphic genic-SSR markers in Larix kaempferi.

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