CN108531609A

CN108531609A - One group of Rhinopithecus roxellana EST-SSR primers and kit based on transcript profile sequencing exploitation

Info

Publication number: CN108531609A
Application number: CN201810106809.0A
Authority: CN
Inventors: 刘学端; 张杜; 梁伊丽; 胡琪; 刘宏伟; 郭雪; 尹华群; 高飞
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2018-02-02
Filing date: 2018-02-02
Publication date: 2018-09-14
Anticipated expiration: 2038-02-02
Also published as: CN108531609B

Abstract

The invention discloses one group of Rhinopithecus roxellana EST SSR primers and kit based on transcript profile sequencing exploitation.The present invention provides 17 couples of Rhinopithecus roxellana EST SSR label primers such as SEQ ID NO:1~SEQ ID NO:Shown in 34, these EST SSR markers have polymorphism in 9 Rhinopithecus roxellana individuals, and different golden monkey individuals can be identified by being used in combination, and be the SSR marker being stabilized, be can be used for the analysis of Rhinopithecus roxellana Genetic Diversity of Germplasm, Relationship iden- tification etc..

Description

One group of Rhinopithecus roxellana EST-SSR primers and kit based on transcript profile sequencing exploitation

Technical field

The present invention relates to one group of Rhinopithecus roxellana EST-SSR primers and kit based on transcript profile sequencing exploitation, belong to point Sub- biological molecule labelling technique field.

Background technology

Microsatellite marker (microsatellite), the short tandem repeat that is otherwise known as (short tandem Repeats, STRs) or simple repeated sequence (simple sequence repeats), it is to be uniformly distributed in eukaryotic gene Simple repeated sequence in group is made of the tandem repeat of 2~6 nucleotide, since the number of repetition of recurring unit exists It is abundant in variability and quantity between individual, thus microsatellite marker be widely used it is general.Microsatellite marker codominance The characteristics of enable it to for studying allele, distinguish the homozygote or heterozygote of diploid (or polyploid), this be AFLP, RAPD codominances label can not be accomplished.In addition, the primer amplified of microsatellite has good repeatability and fidelity Property, facilitate the exchange of each laboratory monitoring.Currently, microsatellite marker be widely applied to gene linkage and genetic map construction, Genetic diversity Journal of Sex Research, pedigree and development research, disease detection and cultivar identification, parent's analysis and individual, pure lines are examined.

SSR marker is divided into genome SSR marker and EST SSR marker (EST-SSR).Compared to genome SSR Label, EST-SSR labels conservative in Spherical scanning is more preferable, more efficient.And the SSR marker based on expression data is base The expressed sequence in one period of Mr. Yu, it is directly related with gene function and trait phenotypes, thus have important researching value.

Rhinopithecus roxellana (Rhinopithecus roxellana), is under the jurisdiction of Primates (Primates), monkey section (Cercopithecidae), colobinae (Colobinae), Rhinopithecus (Rhinopithecus), are China endemic species, are China I grades saves the wild animals, by International Union for Conservation of Nature and Natural Resources (International Union for Conservation of Nature, IUCN) register be classified as easily danger (IUCN, 2011).Rhinopithecus roxellana is only distributed in several at present Mutually isolated area (Northern Sichuan Province and 3 SOUTH OF GANSU, Qinling Mountains in Shaanxi and Shennongjia, Hubei Province areas), studies Rhinopithecus roxellana Genetic diversity and population structure have a very important significance the protection of China's rare and en-damaged resources.Currently used for wearing The SSR marker of golden monkey Diversity Detection this is being belonged to by SSR marker mostly from other nearly edge primates Or the method for the transfer characteristic of equal species, it is easy to shift failure because of the mutation of flanking sequence.Although Rhinopithecus roxellana Although genome sequencing has been completed, the exclusive SSR marker of golden monkey simple and practicable can be used for river spun gold as a kind of The important way of monkey genetic diversity and Relationship iden- tification, is not developed yet at present.It is exclusive to develop one group of Rhinopithecus roxellana SSR label primer and reagent it is already extremely urgent.

Invention content

For the current present situation being badly in need of but lack the exclusive SSR marker of Rhinopithecus roxellana, primary and foremost purpose of the invention there is provided One group of exploitation Rhinopithecus roxellana EST-SSR labeled primer based on transcript profile sequencing.The present invention is from the exon sequence of genome The sites EST-SSR are searched for, and design corresponding PCR amplification primer, the ePCR verifications of exon is first passed through, finally utilizes 4 river gold Silk monkey individual transcript profile sequencing data carries out carrying out ePCR without the transcript that ginseng assembling obtains, and screening, which obtains 17 pairs, has polymorphism EST-SSR label and its primer.It is marked using these EST-SSR and successfully verifies its polymorphism in 9 Rhinopithecus roxellana individuals. The present invention provides a kind of quick approach of precise and high efficiency for the genetic diversity and affiliation for studying Rhinopithecus roxellana.

One group of Rhinopithecus roxellana EST-SSR primer pair based on transcript profile sequencing exploitation, the primer share 17 pairs, respectively For：

1st pair of primer, sequence such as SEQ ID NO:1 and SEQ ID NO:Shown in 2；

2nd pair of primer, sequence such as SEQ ID NO:3 and SEQ ID NO:Shown in 4；

3rd pair of primer, sequence such as SEQ ID NO:5 and SEQ ID NO:Shown in 6；

4th pair of primer, sequence such as SEQ ID NO:7 and SEQ ID NO:Shown in 8；

5th pair of primer, sequence such as SEQ ID NO:9 and SEQ ID NO:Shown in 10；

6th pair of primer, sequence such as SEQ ID NO:11 and SEQ ID NO:Shown in 12；

7th pair of primer, sequence such as SEQ ID NO:13 and SEQ ID NO:Shown in 14；

8th pair of primer, sequence such as SEQ ID NO:15 and SEQ ID NO:Shown in 16；

9th pair of primer, sequence such as SEQ ID NO:17 and SEQ ID NO:Shown in 18；

10th pair of primer, sequence such as SEQ ID NO:19 and SEQ ID NO:Shown in 20；

11st pair of primer, sequence such as SEQ ID NO:21 and SEQ ID NO:Shown in 22；

12nd pair of primer, sequence such as SEQ ID NO:23 and SEQ ID NO:Shown in 24；

13rd pair of primer, sequence such as SEQ ID NO:25 and SEQ ID NO:Shown in 26；

14th pair of primer, sequence such as SEQ ID NO:27 and SEQ ID NO:Shown in 28；

15th pair of primer, sequence such as SEQ ID NO:29 and SEQ ID NO:Shown in 30；

16th pair of primer, sequence such as SEQ ID NO:31 and SEQ ID NO:Shown in 32；

17th pair of primer, sequence such as SEQ ID NO:33 and SEQ ID NO:Shown in 34.

Second object of the present invention is to provide a kind of research Rhinopithecus roxellana genetic diversity and affiliation kit, packet Include above-mentioned one or more pairs of primers.

The primer or kit of the present invention uses one or more pairs of primers when in use.

The advantageous effects that the present invention is brought have：The present invention provides one group of river based on transcript profile sequencing exploitation for the first time The exclusive EST-SSR labeled primers of golden monkey and kit, genetic diversity and Relationship iden- tification to study golden monkey provide A kind of mode of precise and high efficiency.

Description of the drawings

Fig. 1 is the electrophoresis result for two pairs of primers that present invention screening obtains.

Specific implementation mode

The content of present invention is further elaborated by the following examples, but model is protected not as to the claims in the present invention The restriction enclosed.The operation of all kits carries out to specifications, at transcript profile Library development flow not specified (NS) according to normal process into Row.

Embodiment 1

One, the extraction of total serum IgE and transcript profile library construction

4 Rhinopithecus roxellana samples of the present embodiment pick up from Shennongjia, Hubei Province national park.The method that cylinder is blown with anesthesia will be golden After silk monkey anesthesia, takes hind leg vein blood dry ice to transport -80 DEG C and preserve for subsequent extracted total serum IgE.Total serum IgE uses QIAGEN RNeasy Protect Animal Blood Kit (73224) are extracted, and agarose gel electrophoresis is used in combination to check the matter of RNA Amount.Transcript profile sequencing library structure uses VAHTSTM mRNA-seq v2 Library Prep Kit for (NR602-01) kit.It is exactly that each sample takes 1 μ g total serum IgEs, is then purified with polyadenylic acid Beads enrichment in simple terms MRNA is obtained, (98 DEG C 8 points of fragmentation is carried out in the processing of VAHTS Frag/Prime Buffer high temperatures with bivalent cation Clock).The first chains of cDNA and the synthesis of the second chain are carried out after purifying, then carry out end-filling, dATP adds end to be connected with connector.Fine jade Sepharose electrophoresis selects 150-200bp segments to carry out magnetic beads for purifying, carries out RCR amplified libraries later：98 DEG C of 10s denaturation, 60 DEG C 30s is combined, and 72 DEG C of 30s extend, cyclic amplification 12 times.Finally, Library Quality 2100 Bioanalyzer of Agilent Detection is quantified with final before (Agilent Technologies) and qPCR carry out machine.

Two, transcript profile sequencing and data filtering：

The libraries mRNA are sequenced using Illumina Hiseq2500.N content is more than 30% or low-quality in initial data The low quality sequence that base contents are more than 10% is measured to be filtered first.Connector pollution is gone using cutadapt1.9 versions It removes.Sequencing data carries out quality testing, it is ensured that data after filtering out low quality data and connector pollution with fastqc softwares Effectively.Quality data using Trinity 2.2.0 default parameters assemble without reference gene group transcript, each sample list Solely assembling, obtains respective Trinity.fasta files.Row statistics such as table 1 are put into transcriptome.

Three, the search of the sites SSR and amplimer design

It is downloaded from RefSeq databases under the genome reference sequences (Rox v1) of golden monkey and corresponding comment file (http://www.ncbi.nlm.nih.gov/refseq).It first writes in perl script foundation comment file and includes sub-information, from All exon sequences are extracted in genome sequence file.Then Windows editions GMATA2.0 softwares are used to carry out SSR search, 2~10 nucleotide repeating units are searched, and number of repetition is more than or equal to 5 sites SSR.As a result it searches altogether outer aobvious Sub-district dinucleotides repeat 5011, Trinucleotide repeats 2733, tetranucleotide repeat 217, pentanucleotide repeat 60, Hexanucleotide repeats 42 and eight nucleotide repeat 2.Design of primers, setting ginseng are carried out using the Primer 3 that MGATA is embedded Number is：Amplified production length is between 120bp~400bp；Product flanking sequence length is 400bp；Optimum annealing temperature Tm is 60℃；Maximum template length is 2000bp.As a result it is right to have successful design primer 5279 altogether, selects 5 pairs to be illustrated in table 2 at random.

Four, the verification of the screening of SSR marker and polymorphism

EPCR programs are embedded in first with GMATA, the genomic exon sequences of the above onestep extraction are carried out as template ePCR.EPCR procedure parameters are：Word length word size are 12, and continuous word length contiguous word are 1, maximum missing max Indels is 1, and maximum mispairing max mismatch are 0, and amplified production length is 100-1000bp.Then there will be a plurality of amplification The PCR primer of band filters out, and obtains 5253 pairs of SSR primers.This process purpose is, amplification in candidate SSR primers is arrived more The unqualified primer of a band and the shared sites SSR with pair of primers exclude, in order to avoid follow-up SSR polymorphisms is interfered to verify. Next by all SSR primers, ePCR amplifications are carried out using the transcript sequence that each sample assembles respectively as template.Four The succeed SSR marker number of amplification of sample is respectively：Sample S1 totally 1520；Sample S2 totally 1502；Sample S3 totally 888 It is a；Sample S4 totally 755；The sites SSR of Successful amplification have 1305 at least in two samples.In all samples in statistical result The sites SSR with polymorphism product in this.Screening the good primer of polymorphism standard be：At least in two different samples Amplified production segment is variant.As a result totally 17 couples of SSR and amplimer (table 3, table 4) with polymorphism are obtained.Product segment The more SSR primers of length polymorphic bands in all samples are better.

Five, the verification of SSR primers polymorphism in 9 Rhinopithecus roxellanas

In addition 9 golden monkey sample transcript profile data is taken to be assembled into transcript respectively, it is polymorphic with 17 couple that the 4th step is screened Property EST-SSR primers carry out ePCR amplifications, amplification such as table 5.As a result show 17 sites EST-SSR in these golden monkeys Same phenotype goes out different degrees of polymorphism in body.Random 9 samples of wherein two pairs of primer pairs of selecting carry out PCR experiments, and poly- third Acrylamide Gel electrophoresis results such as Fig. 1.Illustrate that the method provided by the invention based on transcript profile data screening SSR marker accurately has Effect, primer can apply in the Diversity Detection and Relationship iden- tification of Rhinopithecus roxellana.

The result that 4 Rhinopithecus roxellana sample transcript profiles assemble respectively in 1 embodiment 1 of table

Part candidate's SSR marker and correspondence based on the identification of Rhinopithecus roxellana genomic exon sequences in 2 embodiment 1 of table Amplimer

The part knot that screening verification is carried out to candidate SSR marker is sequenced in 3 embodiment 1 of table based on Rhinopithecus roxellana transcript profile Fruit

17 couple screened in 4 embodiment 1 of table has the SSR and amplimer sequence of polymorphism

Table 5

Sequence table

<110>Central South University

<120>One group of Rhinopithecus roxellana EST-SSR primers and kit based on transcript profile sequencing exploitation

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213>Unknown (Unknown)

<400> 1

atttgggaga agggcagagt 20

<210> 2

<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

<210> 14

<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. one group of Rhinopithecus roxellana EST-SSR primer based on transcript profile sequencing exploitation, which is characterized in that the primer shares 17 It is right, respectively：

1st pair of primer, sequence such as SEQ ID NO:1 and SEQ ID NO:Shown in 2；

2nd pair of primer, sequence such as SEQ ID NO:3 and SEQ ID NO:Shown in 4；

3rd pair of primer, sequence such as SEQ ID NO:5 and SEQ ID NO:Shown in 6；

4th pair of primer, sequence such as SEQ ID NO:7 and SEQ ID NO:Shown in 8；

5th pair of primer, sequence such as SEQ ID NO:9 and SEQ ID NO:Shown in 10；

6th pair of primer, sequence such as SEQ ID NO:11 and SEQ ID NO:Shown in 12；

7th pair of primer, sequence such as SEQ ID NO:13 and SEQ ID NO:Shown in 14；

8th pair of primer, sequence such as SEQ ID NO:15 and SEQ ID NO:Shown in 16；

9th pair of primer, sequence such as SEQ ID NO:17 and SEQ ID NO:Shown in 18；

10th pair of primer, sequence such as SEQ ID NO:19 and SEQ ID NO:Shown in 20；

11st pair of primer, sequence such as SEQ ID NO:21 and SEQ ID NO:Shown in 22；

12nd pair of primer, sequence such as SEQ ID NO:23 and SEQ ID NO:Shown in 24；

13rd pair of primer, sequence such as SEQ ID NO:25 and SEQ ID NO:Shown in 26；

14th pair of primer, sequence such as SEQ ID NO:27 and SEQ ID NO:Shown in 28；

15th pair of primer, sequence such as SEQ ID NO:29 and SEQ ID NO:Shown in 30；

16th pair of primer, sequence such as SEQ ID NO:31 and SEQ ID NO:Shown in 32；

17th pair of primer, sequence such as SEQ ID NO:33 and SEQ ID NO:Shown in 34.

2. a kind of kit of identification Rhinopithecus roxellana affiliation and genetic diversity, which is characterized in that it includes that right such as is wanted Seek one or more pairs of primers described in 1.