Lathyrus quinquenervius EST-SSR primer group developed based on transcriptome sequencing, method and application

A technology of transcriptome sequencing and lathyrus, applied in the field of molecular markers and bioinformatics, can solve the problem of lack of EST-SSR primers and achieve low-cost results

Inactive Publication Date: 2017-11-14
山西省农业科学院农作物品种资源研究所 +1
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  • Abstract
  • Description
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Problems solved by technology

[0008] Due to the large genome (8.2Gb) of lathyrus, the whole genome

Method used

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  • Lathyrus quinquenervius EST-SSR primer group developed based on transcriptome sequencing, method and application
  • Lathyrus quinquenervius EST-SSR primer group developed based on transcriptome sequencing, method and application
  • Lathyrus quinquenervius EST-SSR primer group developed based on transcriptome sequencing, method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: RNA-Seq analysis and SSR primer design

[0027] 1. Determination of transcriptome data

[0028] Using the Tianenze column plant RNAout kit, the total RNA of the roots, stems, and leaves of the two varieties of Lathyrus RQ23 and RQ36 at the seedling stage was used to purify the mRNA by capturing the mRNA with oligo dT magnetic beads; using fragmentation buffer containing divalent cations The captured mRNA was broken into 200-300 nt fragments, the first cDNA strand was synthesized using random primers containing 6 bases and fragmented mRNA, and then the second cDNA strand was synthesized using DNA polymerase I and RNase H. The product was purified by QIAquick PCR Extraction Kit, eluted with EB buffer, and the overhanging end was repaired by exonuclease and polymerase. Add "A" to the 3' end of the cDNA fragment and connect it with the sequencing adapter, use AMPure XPbeads to purify the adapter-added system, then perform PCR amplification, and finally use 2% ag...

Embodiment 2

[0047] Example 2 Verification of SSR primers

[0048]43 Lathyrus germplasm resources from different sources recorded in Table 2 were used for planting, and 2 g of leaves at the seedling stage were collected respectively, and the leaves were pulverized by a high-throughput pulverizer, and Shanghai Sangong DNA extraction kit (Ezup column type) was used for planting. Plant Genomic DNA Extraction Kit, B518261-0050) for leaf DNA extraction, DNA quality detection and concentration determination on a microplate reader, and DNA was diluted to a final concentration of 30ng / μL, and stored at -20°C. Using 43 materials DNA and 284 randomly selected pairs of SSR primers to carry out PCR amplification to verify the polymorphism of the primers. The PCR reaction system was 10 μL in total, including 1.5 μL (30ng / μL) of genomic DNA, 5 μL of 2× Taq PCR MasterMix (Tiangen, KT:121221), and 2 μL (0.002nmol / μL) of forward and reverse primers in total. The PCR amplification reaction program was: pre...

Embodiment 3

[0052] Example 3: Construction of 43 Fingerprints of Lathyrus Germplasm Resources

[0053] Using 284 pairs of primers to PCR amplification results of DNA from 43 Lathyrus germplasm resources, 87 pairs of polymorphic SSR markers were selected (see Table 3 for 87 pairs of polymorphic SSR markers), and 87 pairs of primers for 43 The polymorphism scanning data of these materials was used to screen the best markers to distinguish 43 Lathyrus germplasms by IDAnalysis 4.0 software, and finally 8 effective markers were obtained to distinguish 43 Lathyrus germplasms.

[0054] The PCR amplification and polyacrylamide gel electrophoresis results of 43 materials were read by 8 pairs of primers. The 8 pairs of primers can amplify different band patterns respectively, among which SSR No.013 is 6 band patterns, which are labeled separately 1-6; SSR No.253 is 5-band type, respectively marked as 1-5; SSR No.58 is 5-band type, respectively marked as 1-5; SSR No.203 is 4-band type, respectively ...

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Abstract

The invention provides a lathyrus quinquenervius EST-SSR primer group developed based on transcriptome sequencing, a method and application. The method comprises the steps of obtaining a set of all root, stem and leaf transcripts of two lathyrus quinquenervius varieties of different flower colors at a seedling stage to form an original sequence database; removing low-quality sequencing data with a joint and forming a high-quality filtered sequence database; splicing high-quality filtered sequences, reducing the transcripts and carrying out BLAST comparison on the transcripts and a reference protein database NR, reserving the optimal comparison result and determining the maximal sequence as a Unigene representative sequence; carrying out SSR locus scanning in Unigene by using MISA 1.0; and carrying out SSR primer design by adopting primer 3.0, screening SSR primers and carrying out fingerprint map construction on 43 parts of lathyrus quinquenervius materials. The lathyrus quinquenervius EST-SSR primer group is convenient, fast, accurate, low in cost, and a new thought is provided for the development of lathyrus quinquenervius EST-SSR primers and germplasm resources identification.

Description

technical field [0001] The invention belongs to the technical field of molecular markers and bioinformatics, and in particular relates to a primer set, method and application for developing the EST-SSR of lathyrus based on transcriptome sequencing. Background technique [0002] Lathyrus ( Lathyrus sativus L.), also known as grass pea, horse tooth bean, grass pea and triangular bean, English name Grass pea, chromosome 2n=14, is the only cultivated species of the genus Lathyrus L. An ancient legume crop that originated in regions such as West Asia and the Middle East. Lathyrus is mainly planted in Gansu, Ningxia, Inner Mongolia, northern Shaanxi and northern Shanxi in China. Lathyrus not only can eat seeds, but also the plants can be used as pasture. At the same time, Lathyrus has the characteristics of drought resistance, barren resistance, waterlogging resistance, salt and alkali resistance, and resistance to diseases and insect pests. Crop of choice. There are many wil...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2600/156C12Q2525/151
Inventor 郝晓鹏王燕杨涛刘荣畅建武宗续晓赵建栋关现民
Owner 山西省农业科学院农作物品种资源研究所
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