CN108531609B - Set of golden monkshood EST-SSR primers and kit developed based on transcriptome sequencing - Google Patents

Abstract

The invention discloses a set of golden monkshood EST-SSR primers and a kit developed based on transcriptome sequencing. The invention provides 17 pairs of golden monkey EST-SSR labeled primers which are shown as SEQ ID NO: 1-SEQ ID NO:34, wherein the EST-SSR labels have polymorphism in 9 golden monkey individuals, can identify different golden monkey individuals by combined use, are stable SSR labels, and can be used for genetic diversity analysis, genetic relationship identification and the like of golden monkey germplasm resources.

Description

Set of golden monkshood EST-SSR primers and kit developed based on transcriptome sequencing

Technical Field

The invention relates to a set of golden monkey EST-SSR primers and a kit developed based on transcriptome sequencing, belonging to the technical field of molecular biological molecular markers.

Background

Microsatellite markers (STRs), also known as Short Tandem Repeats (STRs) or simple sequence repeats (simple sequence repeats), are simple repeats that are uniformly distributed in eukaryotic genomes, are composed of tandem repeats of 2-6 nucleotides, and are widely used because the number of repeats of a repeat unit is highly variable and abundant among individuals. The co-dominant feature of microsatellite markers enables the marker to be used for researching alleles and distinguishing homozygotes or heterozygotes of diploids (or polyploids), which cannot be realized by dominant markers such as AFLP, RAPD and the like. In addition, the specific primer amplification of the microsatellite has good repeatability and fidelity, and is convenient for communication among laboratories. At present, microsatellite markers are widely applied to gene linkage and genetic map construction, genetic diversity research, pedigree and development research, disease detection and variety identification, parent analysis and individual and pure line detection.

SSR markers are divided into genome SSR markers and expressed sequence tag SSR markers (EST-SSR). Compared with genome SSR markers, the EST-SSR markers have better conservation and higher efficiency in closely related species. Moreover, the SSR marker based on expression data is based on a gene expression sequence of a certain period and is directly related to gene functions and trait phenotypes, so that the SSR marker has important research value.

The golden monkey (Rhizophicus roxellana) belongs to Primates (Primates), Macacae (Cercopathicidae), Macacae subfamily (Colobinae) and Rhizophicus (Rhizophicus), is a special species in China, is a grade I important protection wild animal in China, and is recorded as an easy-to-risk (IUCN, 2011) by the name of International Union for Conservation of Nature (IUCN). At present, the golden silk monkeys are only distributed in a plurality of mutually isolated regions (3 regions of northern Sichuan, southern and Gansu, Qinling mountain of Shaanxi and Shennongjia of Hubei), and the research on the genetic diversity and the population structure of the golden silk monkeys has very important significance for protecting rare or endangered germplasm resources in China. The SSR markers currently used for genetic diversity detection of the golden monkey are mostly from other closely related primates, but the method relying on the transfer characteristics of the SSR markers in the same genus or the same family is easy to fail in transfer due to mutation of flanking sequences. Although the whole genome sequencing of golden monkey has been completed, the SSR marker specific to golden monkey is an important simple and feasible method for identifying genetic diversity and genetic relationship of golden monkey, and has not been developed yet. The development of a group of SSR marker primers and reagents special for golden monkeys is imminent.

Disclosure of Invention

Aiming at the current situation that the current demand is urgent but the exclusive SSR marker of golden monkey is lacked, the invention mainly aims to provide a group of primers for developing the EST-SSR marker of golden monkey based on transcriptome sequencing. The invention searches EST-SSR sites from exon sequences of a genome, designs corresponding PCR amplification primers, firstly carries out ePCR verification through the exon, finally carries out ePCR on a transcript obtained by carrying out non-reference assembly by utilizing sequencing data of 4 golden monkeys, and screens to obtain 17 pairs of EST-SSR markers with polymorphism and the primers thereof. The EST-SSR markers are used for successfully verifying the polymorphism in 9 golden monkey individuals. The invention provides an accurate, efficient and rapid way for researching genetic diversity and genetic relationship of the golden monkey.

A set of golden monkey EST-SSR primer pairs developed based on transcriptome sequencing, wherein the primers have 17 pairs in total and are respectively as follows:

the 1 st pair of primers has the sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

the 2 nd pair of primers has the sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4;

the 3 rd pair of primers has the sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6;

the 4 th pair of primers has the sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the 5 th pair of primers has the sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the 6 th pair of primers has the sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12;

the 7 th pair of primers has the sequences shown as SEQ ID NO. 13 and SEQ ID NO. 14;

the 8 th pair of primers has the sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16;

the 9 th pair of primers has the sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18;

the 10 th pair of primers has the sequences shown as SEQ ID NO. 19 and SEQ ID NO. 20;

the 11 th pair of primers has the sequences shown as SEQ ID NO. 21 and SEQ ID NO. 22;

the 12 th pair of primers has the sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24;

the 13 th pair of primers has the sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26;

14 th pair of primers, the sequences of which are shown as SEQ ID NO. 27 and SEQ ID NO. 28;

15 th pair of primers, the sequences of which are shown as SEQ ID NO. 29 and SEQ ID NO. 30;

the 16 th pair of primers has the sequences shown as SEQ ID NO. 31 and SEQ ID NO. 32;

the 17 th pair of primers has the sequences shown in SEQ ID NO. 33 and SEQ ID NO. 34.

The second purpose of the invention is to provide a kit for researching genetic diversity and genetic relationship of golden monkey, which comprises one or more pairs of primers.

The primers or kits of the invention employ one or more pairs of primers in use.

The invention has the following beneficial technical effects: the invention provides a group of monkey-free special EST-SSR labeled primers and a kit developed based on transcriptome sequencing for the first time, and provides an accurate and efficient mode for researching genetic diversity and genetic relationship identification of golden monkeys.

Drawings

FIG. 1 shows the results of electrophoresis of two pairs of primers obtained by screening according to the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention as claimed. All kit operations are carried out according to instructions, and the non-instruction part of the transcriptome library building process is carried out according to a standard process.

Example 1

First, extraction of total RNA and construction of transcriptome library

The 4 golden monkey samples of this example were collected from the national park of Shennong Ji, Hubei. After the golden monkey is anesthetized by an anesthetic blowing tube method, hindlimb venous blood is taken and transported by dry ice to 80 ℃ for storage for subsequent extraction of total RNA. Total RNA was extracted using QIAGENNeasy protective Animal Blood Kit (73224), and the quality of RNA was checked by agarose gel electrophoresis. Construction of transcriptome sequencing Library Using VAHTSTM mRNA-seq v2 Library Prep Kit for

(NR602-01) kit. Briefly, 1. mu.g of total RNA was taken from each sample, and then mRNA was isolated and purified using poly (A) beads, and fragmented by treatment with divalent cations at high temperature in VAHTS Frag/Prime Buffer (98 ℃ for 8 minutes). Purification is followed by first and second strand cDNA synthesis, followed by end filling, dATP endcapping and linker ligation. Agarose gel electrophoresis was performed to select the 150-and 200-bp fragment for magnetic bead purification, followed by amplification of the RCR library: 98Denaturation at 60 ℃ for 30s, conjugation at 72 ℃ for 30s, and cyclic amplification at 12 times. Finally, library quality was measured on-machine and finally quantified using Agilent 2100 Bioanalyzer (Agilent technologies) and qPCR.

II, transcriptome sequencing and data filtering:

mRNA libraries were sequenced using Illumina Hiseq 2500. Low-quality sequences with N contents of more than 30% or low-quality base contents of more than 10% in the raw data were first filtered out. Linker contamination was removed using the cutadapt version 1.9. And after low-quality data and joint pollution are filtered out from sequencing data, performing quality detection by using fastqc software to ensure that the data are effective. High-quality data are assembled by adopting Trinity 2.2.0 default parameters without reference genome transcripts, and each sample is independently assembled to obtain respective Trinity. Statistics on transcript assembly are shown in table 1.

Third, SSR site search and amplification primer design

The golden monkey's genomic reference sequence (Rox v1) and the corresponding annotation files were downloaded from a RefSeq database (http:// www.ncbi.nlm.nih.gov/RefSeq). Firstly, writing perl script to extract all exon sequences from the genome sequence file according to the intron information in the annotation file. Then, the software of Windows GMATA2.0 is adopted to carry out SSR search, 2-10 nucleotide repeating units are searched, and the number of times of repetition is more than or equal to 5 SSR sites. The results collectively searched for 5011 dinucleotide repeats, 2733 trinucleotide repeats, 217 tetranucleotide repeats, 60 pentanucleotide repeats, 42 hexanucleotide repeats and 2 octanucleotide repeats in the exon region. Primer design was performed using MGATA embedded Primer 3 with the following parameters: the length of the amplified product is between 120bp and 400 bp; the length of the product flanking sequence is 400 bp; the optimal annealing temperature Tm is 60 ℃; the maximum template length is 2000 bp. Results a total of successful primer 5279 pairs were designed, randomly selected 5 pairs are shown in table 2.

Fourth, screening of SSR markers and validation of polymorphisms

ePCR was performed using GMATA embedded ePCR program and genomic exon sequences extracted in the previous step as template. The ePCR process parameters were: the word size is 12, the continuous word size is 1, the maximum deletion maxindels is 1, the maximum mismatch max mismatch is 0, and the length of the amplification product is 100-1000 bp. Then, PCR primers with multiple amplified bands were filtered out to obtain 5253 pairs of SSR primers. The process aims to eliminate unqualified primers amplified to multiple bands in the candidate SSR primers and SSR sites sharing the same pair of primers so as to avoid interfering with subsequent SSR polymorphism verification. Then, ePCR amplification is carried out on all SSR primers by taking a transcript sequence assembled from each sample as a template. The numbers of SSR markers successfully amplified by the four samples are respectively as follows: 1520 samples S1 in total; 1502 samples S2; 888 samples S3; 755 samples S4; there were 1305 SSR sites that were successfully amplified in at least two samples. SSR sites with polymorphic products in all samples in the statistical results. Criteria for screening for well polymorphic primers were: the amplified product fragments differ in at least two different samples. As a result, a total of 17 pairs of SSR primers and amplification primers having polymorphisms were obtained (Table 3, Table 4). Product fragment length the more polymorphic fragments in all samples the better the SSR primer.

Fifth, verification of polymorphism of SSR primer in 9 golden monkeys

And in addition, 9 golden monkey sample transcriptome data are respectively assembled into transcripts, 17 pairs of polymorphic EST-SSR primers screened in the fourth step are subjected to ePCR amplification, and the amplification results are shown in a table 5. The results showed that 17 EST-SSR sites were phenotypically polymorphic to varying degrees in these golden monkey individuals. Two pairs of primers were randomly selected for PCR experiments on 9 samples, and the results of polyacrylamide gel electrophoresis are shown in FIG. 1. The method for screening SSR markers based on transcriptome data is accurate and effective, and the primers can be applied to genetic diversity detection and genetic relationship identification of golden monkeys.

TABLE 1 results of separate assembly of 4 Simian sample transcriptomes in example 1

TABLE 2 part of candidate SSR markers identified based on exon sequences of golden monkey genome and corresponding amplification primers in example 1

TABLE 3 partial results of screening validation of candidate SSR markers in example 1 based on Simian Sichuan-Reptilia-transcriptome sequencing

TABLE 4 17 pairs of SSR with polymorphisms and amplification primer sequences screened in example 1

TABLE 5

Sequence listing

<110> university of south-middle school

<120> a set of golden monkeys EST-SSR primers and kit developed based on transcriptome sequencing

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atttgggaga agggcagagt 20

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<211>21

<212>DNA

<213> Unknown (Unknown)

<400>2

agcaactcac acacacacac a 21

<210>3

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>3

atttgggaga agggcagagt 20

<210>4

<211>22

<212>DNA

<213> Unknown (Unknown)

<400>4

ggcccacatc tgtacataac aa 22

<210>5

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>5

caattcccct ctcctcttcc 20

<210>6

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>6

caggggctgg agtttgatta 20

<210>7

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>7

ccaaaagaaa accccatcaa 20

<210>8

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>8

ctgggtgtga gcctgtaatg 20

<210>9

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>9

cctggtagct caacctcctg 20

<210>10

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>10

cacgcccatc tttaccattt 20

<210>11

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>11

gagccccatg acttttctca 20

<210>12

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>12

gaagccatga gaatggagga 20

<210>13

<211>21

<212>DNA

<213> Unknown (Unknown)

<400>13

cagcttatcc aaagctctcc a 21

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<211>20

<212>DNA

<213> Unknown (Unknown)

<400>14

gtccctccct tccactcttc 20

<210>15

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>15

ggtaaccaca ccaggtcagc 20

<210>16

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>16

cccagtgaga agacctttgc 20

<210>17

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>17

gattcccctg aatccctacc 20

<210>18

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>18

ggtagtcgaa gccgtagctg 20

<210>19

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>19

ttttggggtg tctctgtgtg 20

<210>20

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>20

tcttgggcct acctgaattg 20

<210>21

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>21

caggcccttc ttcctctagc 20

<210>22

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>22

gaagaaatgg gcacctttga 20

<210>23

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>23

aaatatctgg ggagggaagg 20

<210>24

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>24

ccatcccctt tgcttttaca 20

<210>25

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>25

aggaccactt agcccaacct 20

<210>26

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>26

aaatgccacg tctgctcttc 20

<210>27

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>27

ggggctgtct gaaaactgtg 20

<210>28

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>28

ttcctttggg gatatgatgc 20

<210>29

<211>22

<212>DNA

<213> Unknown (Unknown)

<400>29

agctttgtgt gaaaaccagt ca 22

<210>30

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>30

gggttttaga aaggcagcaa 20

<210>31

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>31

gtatggtggg ccctaggaaa 20

<210>32

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>32

ctctgggact cctgtgcttc 20

<210>33

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>33

caagcctggt gaagaggaag 20

<210>34

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>34

caggtgatct tggggagaga 20

<210>35

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>35

ggttgggagt tcaagaccag 20

<210>36

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>36

ccgaagacct taagcccaaa 20

<210>37

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>37

gggagcatct tctgtgtcaa 20

<210>38

<211>21

<212>DNA

<213> Unknown (Unknown)

<400>38

tgtacagcat gtcggtctga a 21

<210>39

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>39

ttagcggtca ctgccttagc 20

<210>40

<211>19

<212>DNA

<213> Unknown (Unknown)

<400>40

gactccccat gctcctctc 19

<210>41

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>41

aagaggctga ggttgtggaa 20

<210>42

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>42

tcagcacaag tccgtcagtc 20

<210>43

<211>20

<212>DNA

<213> Unknown (Unknown)

<400>43

catgactctc ctggtccaca 20

<210>44

<211>21

<212>DNA

<213> Unknown (Unknown)

<400>44

cccaactctc tgcattattc g 21

Claims (1)

1. A set of golden monkey EST-SSR primer group developed based on transcriptome sequencing is characterized in that the primer group has 17 pairs in total and respectively comprises:

the 1 st pair of primers has the sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

the 2 nd pair of primers has the sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4;

the 3 rd pair of primers has the sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6;

the 4 th pair of primers has the sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the 5 th pair of primers has the sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the 6 th pair of primers has the sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12;

the 7 th pair of primers has the sequences shown as SEQ ID NO. 13 and SEQ ID NO. 14;

the 8 th pair of primers has the sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16;

the 9 th pair of primers has the sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18;

the 10 th pair of primers has the sequences shown as SEQ ID NO. 19 and SEQ ID NO. 20;

the 11 th pair of primers has the sequences shown as SEQ ID NO. 21 and SEQ ID NO. 22;

the 12 th pair of primers has the sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24;

the 13 th pair of primers has the sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26;

14 th pair of primers, the sequences of which are shown as SEQ ID NO. 27 and SEQ ID NO. 28;

15 th pair of primers, the sequences of which are shown as SEQ ID NO. 29 and SEQ ID NO. 30;

the 16 th pair of primers has the sequences shown as SEQ ID NO. 31 and SEQ ID NO. 32;

the 17 th pair of primers has the sequences shown in SEQ ID NO. 33 and SEQ ID NO. 34.

Images