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湿地松转录组SSR分析及EST-SSR标记开发(PDF)

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

Issue:
2020年02期
Page:
75-83
Column:
研究论文
publishdate:
2020-03-31

Article Info:/Info

Title:
Analysis of SSR information in transcriptome and development of EST-SSR molecular markers in Pinus elliottii Engelm.
Article ID:
1000-2006(2020)02-0075-09
Author(s):
YI Min1 ZHANG Lu1 LEI Lei1 CHENG Zishan1 SUN Shiwu2 LAI Meng1*
(1. 2011 Collaboration Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, Jiangxi Agricultural University, Nanchang 330045, China; 2. Baiyun Mountain Forest Farm in Qingyuan District, Ji'an 343062, China)
Keywords:
Pinus elliottii Engelm.(slash pine) transcriptome SSR primer EST-SSR moleculcy marlcer mono-nucleotide di-nucleotides tri-nucleotides
Classification number :
S722.8; Q319
DOI:
10.3969/j.issn.1000-2006.201907017.
Document Code:
A
Abstract:
【Objective】Slash pine(Pinus elliotii Engelm.)is a high-quality resin-producing species widely distributed in southern China. Despite it being an important economically important species, genomic and transcriptomic data on this species is scarce, which has hampered its genomic studies. To date, SSR markers used in molecular studies on P. elliottii were mainly those from other related species or developed by using limited gene sequence resources from public databases, which have low polymorphism rate and high generality. In order to solve these problems, we used transcriptome data to develop EST-SSR markers for slash pine. Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed in order to lay the foundation for molecular marker-assisted selection of P. elliottii.【Method】The SSR loci from the transcriptome sequences were analyzed by MicroSAtellite(MISA), and statistical analyses were conducted for the distribution and characteristics of SSR loci. The parameters were set as follows: the SSRs were considered to contain mono-, di-, tri-, tetra-, penta- and hexa-nucleotides with minimum repeat numbers of 10, 6, 5, 5, 5 and 5, respectively. The 120 pairs of EST-SSR primers were designed using Primer 3. Agarose electrophoresis was used for initial check, and capillary electrophoresis was used for separation and detection of the polymorphisms in the primers. In order to study their genetic diversity, 113 samples of families were collected from three seed orchards in South America and from a seed stand in Ji'an,Jiangxi Province.【Result】A total of 79 574 unigenes with 3 818 SSR loci were detected through transcription of slash pine genes. SSR sites occurred with a frequency of 4.80%(number of SSR/number of searching sequences), with an average of one SSR per 18.27 kb. A total of 3 373 EST sequences were screened for SSRs with a frequency of 4.24%(number of sequences with SSRs/number of searching sequences). A total of 2 980 sequences contained single SSRs of different motif types, and 393 sequences contained more than two SSRs. Among the 3 818 potential EST-SSRs, six types of motifs were identified: mononucleotide(63.54%)which had the highest frequency, followed by dinucleotide(19.15%),trinucleotide(16.27%), tetranucleotide(0.52%), pentanucleotide(0.13%)and hexanucleotide repeats(0.31%). The number of repeats of the different SSR motifs varied from 5 to 22, with the exception of mononucleotides. The frequency of five repeats was the highest among all repeats(35.80%), followed by that of six repeats(29.98%)and seven repeats(14.23%). Only 2.73% of ten repeats were found. Among the dinucleotide repeats, AT/AT was the most common motif(12.86%), followed by AG/CT(4.09%)and AC/GT(2.12%). Among the trinucleotide repeats, AAT/ATT was the most common motif, accounting for 3.64% of the total trinucleotide repeats, followed by the AAG/CTT(3.20%). Among all mapped SSRs, the EST-SSR that belonged to the unknown region accounted for 24.59%. SSRs in different genomic regions(5'UTR, 3'-UTR and CDS)showed distinct patterns of distribution. At the genomic level, 3'UTRs had the highest density of SSRs, followed by 5'UTR and CDS. Among the 120 primer pairs, twenty-four pairs(containing 13 di-, 7 tri and four tetranucleotides)showed polymorphism, which accounted for 4.8% of the total number of primer pairs. Eighty-one alleles were tested from twenty-four pairs of fluorescence primers, and the number of alleles ranged from 2 to 9 with a mean value of 3.38. Polymorphic information content ranged from 0.103 to 0.726, with an average of 0.349.【Conclusion】A total of 3 818 SSRs were identified from transcriptome sequencing of P. elliottii,with AT/AT and AAT/ATT the most common repeats.The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats.We concluded that it is feasible to develop the SSR markers based on the P. elliottii transcriptomic sequence, and our results provide new information on genetic diversity analysis and molecular marker-assisted selection of P. elliottii, as well as a basis for SSR marker development in other species

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