CN112094926A

CN112094926A - Primer design and amplification method of mitochondrial genome complete sequence of gobiocypiocypris dabryi

Info

Publication number: CN112094926A
Application number: CN202011154521.4A
Authority: CN
Inventors: 阙延福; 汪鄂洲; 李伟涛; 朱滨; 邵科; 廖小林; 闫书详; 胡兴坤
Original assignee: Institute Of Water Engineering Ecology Chinese Academy Of Sciences
Current assignee: Institute Of Water Engineering Ecology Chinese Academy Of Sciences; Institute of Hydroecology MWR and CAS
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2020-12-18

Abstract

The invention relates to a primer design of a gobiocypiocyprisrario mitochondrial genome complete sequence and an amplification method thereof. Obtaining the complete sequence of the mitochondrial genome of the gobiocyprisrarus closely related species, performing multiple comparison on the sequences to obtain a relatively conserved region and a region with larger variation, and designing mutually nested primers in the relatively conserved region, namely, a downstream primer of a first pair of primers is arranged behind an upstream primer of a second pair of primers to ensure that amplified products are mutually overlapped. The obtained gobiocypiocypris mitochondrial genome complete sequence amplification primer can be used for the aspects of species identification, geographic population identification, germplasm resource assessment and the like.

Description

Primer design and amplification method of mitochondrial genome complete sequence of gobiocypiocypris dabryi

Technical Field

The invention belongs to the research field of fish mitochondrial genomes, and particularly relates to primer design and an amplification method of a gobiocypiocypobius (gobiocypobius) mitochondrial genome complete sequence.

Background

Gobiocyparidae (Acanthogobio guentheri Herzenstein) belongs to Cyprinoideae, Cyprinidae, gobiocyparidae, and pseudosciaena aurantiaca (Mylabris and Mylabris), and small fishes are the only species of gobiocaridae and are the specific fishes in China. The gobiocypris ventralis is higher in body and lateral in volume, the dorsal fin is higher in front, the abdomen is round, and the caudal peduncle is thick and short. The head is short and sharp, and the head length is usually shorter than the body height. The tip of the kiss is in a cone shape, and the length of the kiss is slightly shorter than the length of the back head of the eye. The lower part of the mouth is arc-shaped, the lip is simple and has no mastoid process. The posterior labial sulcus was interrupted. The last non-branching fin ray of the dorsal fin is a smooth hard thorn, the end part is soft, and the length of the fin is shorter than that of the head. The chest fin is long, and the end of the smaller individual can reach the starting point of the ventral fin. The ventral fin is located just below the dorsal fin starting point and ends short of the hip fin. The hip fin is short without hard prick, and the starting point is slightly closer to the ventral fin base than to the caudal fin base. Bifurcate caudal fin, two lobes, and pointed distal end. The anus is near the origin of the anal fin. The lateral line is complete and straight. The back of the body is brown or light brown, and a black stripe is arranged in the middle of the back. A row of black spots is arranged above the medial axis of the body. The dorsal fin and the caudal fin are gray black with black spots, and the other fins are gray white. It is described that the dry branches of yellow river from Qinghai-Hequgou to Gansu Lanzhou are distributed, and are important indigenous fish resources in the upstream of yellow river. The only three kinds of fishes with obvious scales on the body surfaces of the native fishes in the yellow river Qinghai section are the subfamily of Cyprinidae, the gobio rario ventralis and the gobio spinosa.

The fish mitochondrial genome consists of 37 coding genes of 13 coding protein genes, 22 tRNA genes and 2 rRNA genes (12S rRNA and 16S rRNA) and a section of main non-coding region (control region), and has the characteristics of simple structure, multiple copy numbers, strict maternal inheritance, almost no recombination, high coding efficiency, high evolution speed, difference in evolution speeds of different regions and the like. Due to these characteristics of the fish mitochondrial genome, it has been widely used in species identification, phylogeny, and population genetics research.

Due to the interference of artificial factors such as river damming, water pollution, over fishing and the like and the change of the living environment of the gobiocypris dabryi, the gobiocypris dabryi individuals tend to be low-aged and miniaturized, the fish resource amount is sharply reduced, and the genetic diversity is reduced. The subfamily of the Cyprinidae. At present, effective measures must be taken to protect and restore the rapidly declining natural population resources of gobiocypiocypobius. However, at present, research on gobiocypris dabryi mainly focuses on the fields of proteomics and resource biology, but the research on gobiocypris dabryi by adopting a molecular biology technology is not many, and no primer and method about mitochondrial genome complete sequence amplification are reported. Considering that the information content of the current gobiocyprisrario genetics, especially the mitochondrial genome, is limited, it is necessary to develop the complete sequence of the mitochondrial genome.

Disclosure of Invention

Aiming at the blank of the prior art, the invention provides a primer design of a gobiocypiocypogalus mitochondrial genome complete sequence and an amplification method thereof. The primer can be used for amplifying the mitochondrial genome of the gobiocypris rarus at one time, the complete genome sequence can be quickly obtained through sequencing and splicing, and the method has the advantages of simplicity, convenience, rapidness, high efficiency and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for designing a gobiocypris dabryi mitochondrial genome complete sequence amplification primer comprises the steps of obtaining a gobiocypris dabryi mitochondrial genome complete sequence, carrying out multiple comparison on the sequences to obtain a relatively conserved region and a region with larger variation, designing mutually nested primers in the relatively conserved region, namely, a downstream primer of a first pair of primers is arranged behind an upstream primer of a second pair of primers, and ensuring that amplified products are mutually overlapped.

According to the scheme, the closely related species of the gobiocypris rario are the thin-body rogoogobio (Romanogobio tenuicoprus), the gobiocypris (Sauroogobio dabryi), the gobiocypari (Gobio cynonocicepphalus) and the gobiocypari (pseudogonoobio variabili).

According to the scheme, the mitochondrial genome complete sequence of the gobiocypiocypris closely related species is downloaded from a Genbank database.

According to the scheme, the relatively conserved region is a conserved sequence region without mutation sites or with only 1 mutation site

According to the scheme, the amplified product sequences between the adjacent primer pairs have at least 100bp of overlapping regions. The downstream primer of the first pair of primers is nested behind the upstream primer of the second pair of primers, and the process is repeated in such a way, so that the splicing of the later-stage sequence is facilitated, the mutant sites possibly caused by the PCR and sequencing processes are avoided, and the success rate of PCR amplification and the accuracy of the amplified target sequence are ensured.

A gobiocypiocypris mitochondrial genome complete sequence amplification primer group comprises twenty pairs of primers, wherein

A first pair of primers:

the upstream primer 1 is shown as SEQ ID CJ 01-F: 5'-CACTGAAGATGTTAAGATG-3', respectively;

the downstream primer 1 is shown as SEQ ID CJ 01-R: 5'-GTCTTCTCGGTGTAAGTGAG-3', respectively;

a second pair of primers:

the upstream primer 2 is shown as SEQ ID CJ 02-F: 5'-GGCTCTAAGGCGCGTACAC-3', respectively;

the downstream primer 2 is shown as SEQ ID CJ 02-R: 5'-TCCAGTGTGGGGTTAACGAT-3', respectively;

a third pair of primers:

the upstream primer 3 is shown as SEQ ID CJ 03-F: 5'-AGCACAAGTGTAAACCAGATCG-3', respectively;

the downstream primer 3 is shown as SEQ ID CJ 03-R: 5'-GAAGAGGATTTGAACCTCTGGT-3', respectively;

a fourth pair of primers:

the upstream primer 4 is shown as SEQ ID CJ 04-F: 5'-CTGTAACGCTACTTTTCCTAGT-3', respectively;

the downstream primer 4 is shown as SEQ ID CJ 04-R: 5'-TATAAGCCACTCTATCAAAGTG-3', respectively;

a fifth pair of primers:

the upstream primer 5 is shown as SEQ ID CJ 05-F: 5'-CTTGTATGGAAAAACTTCCT-3', respectively;

the downstream primer 5 is shown as SEQ ID CJ 05-R: 5'-GCTTTGAAGGCTCTTGGTCT-3', respectively;

a sixth pair of primers:

the upstream primer 6 is shown as SEQ ID CJ 06-F: 5'-GCCACAAAAATTAATACCCT-3', respectively;

the downstream primer 6 is shown as SEQ ID CJ 06-R: 5'-CCTAAAATTGATGAAACAC-3', respectively;

a seventh pair of primers:

the upstream primer 7 is shown as SEQ ID CJ 07-F: 5'-CTTTATAGTAATACCAATTCT-3', respectively;

the downstream primer 7 is shown as SEQ ID CJ 07-R: 5'-AAATAGGAACATAATTACTGC-3', respectively;

an eighth pair of primers:

the upstream primer 8 is shown as SEQ ID CJ 08-F: 5'-ACTTGAACAAAAATCCACTT-3', respectively;

the downstream primer 8 is shown as SEQ ID CJ 08-R: 5'-CCACAGATTTCAGAGCATTG-3', respectively;

a ninth pair of primers:

the upstream primer 9 is shown as SEQ ID CJ 09-F: 5'-ACCTAACAATTAAAGCCATAG-3', respectively;

the downstream primer 9 is shown as SEQ ID CJ 09-R: 5'-ATGCATGTGCTTGGTGTGCCAT-3', respectively;

a tenth pair of primers:

the upstream primer 10 is shown as SEQ ID CJ 10-F: 5'-GCCAGTACTAATTATTATCG-3', respectively;

the downstream primer 10 is shown as SEQ ID CJ 10-R: 5'-ATGGATACGTAAAGGAATAGT-3', respectively;

an eleventh pair of primers:

the upstream primer 11 is shown as SEQ ID CJ 11-F: 5'-CAATACCACTTCACATCTGAAC-3', respectively;

the downstream primer 11 is shown as SEQ ID CJ 11-R: 5'-AGGTTTAGGTTTTGTAGGCG-3', respectively;

a twelfth pair of primers:

the upstream primer 12 is shown as SEQ ID CJ 12-F: 5'-CTTGGCCTAATAGGACTAGC-3', respectively;

the downstream primer 12 is shown as SEQ ID CJ 12-R: 5'-GGTATTTTTACTAGAAATGC-3', respectively;

a thirteenth pair of primers:

the upstream primer 13 is shown as SEQ ID CJ 13-F: 5'-CATTCCAACCCTAATTATTAT-3', respectively;

the downstream primer 13 is shown as SEQ ID CJ 13-R: 5'-GAGGAGGATATAATTAGTGTT-3', respectively;

a fourteenth pair of primers:

the upstream primer 14 is shown as SEQ ID CJ 14-F: 5'-GTACTGCTAATCCTTATGCAC-3', respectively;

the downstream primer 14 is shown as SEQ ID CJ 14-R: 5'-GATGTTGAGAAAGCTACAATT-3', respectively;

a fifteenth pair of primers:

the upstream primer 15 is shown as SEQ ID CJ 15-F: 5'-AAGTCGGCCCAATTTGGCCT-3', respectively;

the downstream primer 15 is shown as SEQ ID CJ 15-R: 5'-ATGGTTAGGTATGTTTTAATTAT-3', respectively;

a sixteenth pair of primers:

the upstream primer 16 is shown as SEQ ID CJ 16-F: 5'-GGAAGCATTATTGCAGGACTTAT-3', respectively;

the downstream primer 16 is shown as SEQ ID CJ 16-R: 5'-CGTTGGCGTGCATGTTACGGAT-3', respectively;

a seventeenth pair of primers:

the upstream primer 17 is shown as SEQ ID CJ 17-F: 5'-ATCGCTAATGACGCACTAGTTG-3', respectively;

the downstream primer 17 is shown as SEQ ID CJ 17-R: 5'-ATATGGGTACTATTGCATAATGT-3', respectively;

an eighteenth primer pair:

the upstream primer 18 is shown as SEQ ID CJ 18-F: 5'-ACTATCTTCTGATAGTAACATGT-3', respectively;

the downstream primer 18 is shown as SEQ ID CJ 18-R: 5'-GCGGAGACTTGCATGTGTAAGT-3', respectively;

a nineteenth pair of primers:

the upstream primer 19 is shown as SEQ ID CJ 19-F: 5'-ACATAAGCTTCTGACTTCTC-3', respectively;

the downstream primer 19 is shown as SEQ ID CJ 19-R: 5'-AGATGAGTGATCCGATGGAT-3', respectively;

a twentieth primer pair:

the upstream primer 20 is shown as SEQ ID CJ 20-F: 5'-CTCTCGTAGCAGATATACTT-3', respectively;

the downstream primer 20 is shown as SEQ ID CJ 20-R: 5'-CGTGAAAGAGATAACCAGAT-3', respectively;

an amplification method of a gobiocypiocypiocypiocyprocypiocyprocypiocyprocypiocypiocyprocypiocyprocyprocyprocyprochenogenome complete sequence amplification primer comprises the following steps:

(1) extracting total DNA of the mitochondrial genome of the gobiocypiocypris ventralis by adopting a phenol/chloroform method;

(2) taking total DNA of the mitochondrial genome of the gobiocypris dabryanus as a template, and respectively carrying out PCR amplification by adopting each pair of primers in the complete sequence primer group of the mitochondrial genome of the gobiocypris dabryanus;

(3) and detecting the amplified product through gel electrophoresis, sending the single PCR product with clear target band to a sequencing company for bidirectional sequencing to obtain the sequence of the amplified product, and splicing the sequence to obtain the original DNA sequence.

According to the scheme, the reaction system for PCR amplification comprises the following components: 20. mu.L of Taq enzyme PCR preliminary mixture (composition: 0.1U/. mu.L Taq DNA Polymerase, 2 XPCR reaction buffer, 3mM MgCl₂0.4mM dNTPs), 2. mu.L of each primer pair (1. mu.L of each of the upstream and downstream primers), 2. mu.L of DNA template, and 40. mu.L of sterile double distilled water; the PCR amplification conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 56 deg.C for 45s, extension at 72 deg.C for 2min, extension at 72 deg.C for 10min after 35 cycles, and storage at 4 deg.C.

According to the above protocol, 1% agarose was used for the gel electrophoresis.

According to the scheme, the step (3) is as follows: comparing the sequencing result with the homologous sequence on GenBank to confirm that the sequencing sequence is highly similar to the closely related species; then, splicing sequences of the overlapped parts of the sequencing results of the forward primers and the reverse primers in the adjacent primers, repeating the sequence splicing, and sequentially splicing the sequences obtained by the multiple groups of primers; finally, splicing a plurality of sequences to obtain the complete sequence of the mitochondrial genome of the gobiocypris rhinogobiocephalus.

The invention can amplify the mitochondrial genome of the gobiocypris dabryanus at one time by designing the amplification primer of the complete sequence of the mitochondrial genome, can quickly obtain the complete sequence of the mitochondrial genome of the gobiocypris dabryanus by sequencing and splicing, has the characteristics of simplicity, convenience, rapidness and high efficiency, and provides important molecular means and data accumulation for species identification, group inheritance, germplasm resource protection and sustainable development and utilization of the gobiocypris dabryanus and phylogenetic research of the gobiocypris dabryanus.

The invention has the beneficial effects that:

(1) according to the gobiocypris dabrygii mitochondrial genome complete sequence amplification primer design method provided by the invention, the mitochondrial complete genome sequences of a plurality of gobiocypris dabrygii kindred closely related species are downloaded during primer design to carry out multiple comparison, and a proper primer pair is designed by selecting a conserved sequence region without mutation sites or with only 1 mutation site, so that the success rate of PCR amplification is ensured. The relative conservation rate of the common conserved sequence region (without mutation sites or with only 1 mutation site) of the four reference sequences is as high as 93.8 percent, and the sequence length containing 2 or more than 2 variation points is only 6.2 percent; in the design process of the primers, a mode of mutually nesting the primer pairs is adopted, namely, the downstream primer of the first pair of primers is nested behind the upstream primer of the second pair of primers, and the process is repeated in this way, so that the sequences amplified between the adjacent primer pairs are spliced in a nested manner, and the amplified sequences are connected end to end. The method is beneficial to the later-stage splicing of the sequence, avoids mutation sites possibly caused by PCR amplification and sequencing processes, and ensures the accuracy of the amplified gobiocypiocypris mitochondrial whole genome sequence; further designing and obtaining a primer group capable of amplifying and obtaining the mitochondrial whole genome sequence of the gobiocypris dabryi based on the design method;

(2) the gobiocypiocypris mitochondrial genome complete sequence amplification primer group (20 pairs of primer sequences) provided by the invention can amplify, sequence and splice the gobiocypiocypiocypris mitochondrial complete genome sequence at one time, and has the advantages of strong specificity, stability, convenience and high efficiency; the primer quantity is small, time and labor are saved, the reagent cost is saved, and the reliability of the obtained original sequence is high;

(3) the method for the gobiocypiocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyprocyproc.

Drawings

FIG. 1 is a schematic diagram of mutual "nesting" of the design principles of the primer of gobiocypris ventralis.

FIG. 2 is the agarose gel electrophoresis pattern of the gobiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocyp. Wherein, the lane M is a DL2000 Marker, and the lanes 1 to 20 are electrophoresis pattern results of the primer pairs 1 to 20 respectively.

FIG. 3 is a visual annotation chart of the complete sequence of the mitochondrial genome of gobiocypiocypris (annotated based on MitoFish software).

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, belong to the scope of the present invention.

Example 1:

(1) collection, identification and preservation of gobiocypris dabryi sample

In the embodiment, the gobio ventralis samples are collected from natural water areas of Yangtze river section of the yellow river in 2017 in 6-7 months, the collected samples are brought back to a laboratory for information registration, and the gobio ventralis is determined through morphological observation comparison and anatomical identification. The gobiocypris rarus sample after species identification is soaked in 100% absolute ethyl alcohol and stored in a refrigerator at 4 ℃ for later use.

(2) Extraction of mitochondrial genome of gobiocypris robio

Mitochondrial genome DNA of the gobiocypris rarus sample to be detected is extracted by a phenol/chloroform method, and the extracted total mitochondrial genome DNA is stored at the temperature of minus 20 ℃ for later use.

(3) Design and synthesis of gobiocypiocypogalus mitochondrial gene complete sequence primer

Downloading mitochondrial genome complete sequences of allied species of gobiocypris spinosus (Romanogobio tenuicoprus), gobiocypiocypris (Saurogiobium dabryi), gobiocypobio (Gobio cynocephalus), and gobiocypobio (Pseudogobiocephalus), from a Genbank database, finding out relative conserved regions and regions with larger variation of the mitochondrial complete genomes of a plurality of allied species by comparing 4 allied species mitochondrial complete genome sequences, designing the relative conserved regions, and forming different primer pairs to be nested with each other, namely, after the downstream primer of the first pair of primers is nested in the upstream primer of the second pair of primers, repeating the steps to form the head-to-tail connection of amplified sequences. The method ensures that the target sequence amplified between the adjacent primer pairs has at least a 100bp overlapping region, is beneficial to splicing the later sequence, avoids mutation sites possibly caused by PCR and sequencing processes, and ensures the success rate of PCR amplification and the accuracy of the amplified target sequence. The primers synthesized were as follows:

a first pair of primers:

a second pair of primers:

a third pair of primers:

a fourth pair of primers:

a fifth pair of primers:

a sixth pair of primers:

a seventh pair of primers:

an eighth pair of primers:

a ninth pair of primers:

a tenth pair of primers:

an eleventh pair of primers:

a twelfth pair of primers:

a thirteenth pair of primers:

a fourteenth pair of primers:

a fifteenth pair of primers:

a sixteenth pair of primers:

a seventeenth pair of primers:

an eighteenth primer pair:

a nineteenth pair of primers:

a twentieth primer pair:

(4) PCR amplification of mitochondrial genome of gobiocypris dabryanus

The PCR amplification reaction system comprises: mix 20. mu.L (0.1U/. mu.L of Taq DNA Polymerase, 2 XPCR reaction buffer, 3mM MgCl₂0.4mM dNTPs), 2. mu.L of each primer pair (1. mu.L of each of the upstream and downstream primers), 2. mu.L of DNA template, and 40. mu.L of sterile double distilled water; the PCR amplification conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 56 deg.C for 45s, extension at 72 deg.C for 2min, extension at 72 deg.C for 10min after 35 cycles, and storage at 4 deg.C.

(5) Sequencing of PCR products

Sending the PCR product with clear and single target band obtained by PCR amplification in the step (4) to a sequencing company for bidirectional sequencing to obtain a sequencing result, comparing the sequencing result with a homologous sequence on a GenBank to ensure high similarity of the sequencing sequence and closely related species, wherein the sequencing result is a gobiocypiocypris mitochondrial sequence; comparing the sequencing results by using a Contig Express program in Vector NTI software, automatically finding out the parts of the two adjacent groups of primers with the overlapped sequencing results of the forward and reverse primers by the software for sequence splicing (shown in figure 1), repeatedly and sequentially splicing the sequences obtained by the multiple groups of primers in this way, and finally splicing the multiple sequences end to obtain the gobiocypris entrance mitochondrial genome complete sequence. The sequencing result is shown in a sequence table SEQ ID NO. 41.

Sequence listing

<110> institute of Water engineering ecology of national academy of sciences in Water conservancy department

Primer design and amplification method of <120> gobiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiochliogenome complete sequence

<160> 41

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Forward primer (primer-F)

<400> 1

cactgaagat gttaagatg 19

<210> 2

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 2

gtcttctcgg tgtaagtgag 20

<210> 3

<211> 19

<212> DNA

<213> Forward primer (primer-F)

<400> 3

ggctctaagg cgcgtacac 19

<210> 4

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 4

tccagtgtgg ggttaacgat 20

<210> 5

<211> 22

<212> DNA

<213> Forward primer (primer-F)

<400> 5

agcacaagtg taaaccagat cg 22

<210> 6

<211> 22

<212> DNA

<213> reverse primer (primer-R)

<400> 6

gaagaggatt tgaacctctg gt 22

<210> 7

<211> 22

<212> DNA

<213> Forward primer (primer-F)

<400> 7

ctgtaacgct acttttccta gt 22

<210> 8

<211> 22

<212> DNA

<213> reverse primer (primer-R)

<400> 8

tataagccac tctatcaaag tg 22

<210> 9

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 9

cttgtatgga aaaacttcct 20

<210> 10

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 10

gctttgaagg ctcttggtct 20

<210> 11

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 11

gccacaaaaa ttaataccct 20

<210> 12

<211> 19

<212> DNA

<213> reverse primer (primer-R)

<400> 12

cctaaaattg atgaaacac 19

<210> 13

<211> 21

<212> DNA

<213> Forward primer (primer-F)

<400> 13

ctttatagta ataccaattc t 21

<210> 14

<211> 21

<212> DNA

<213> reverse primer (primer-R)

<400> 14

aaataggaac ataattactg c 21

<210> 15

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 15

acttgaacaa aaatccactt 20

<210> 16

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 16

ccacagattt cagagcattg 20

<210> 17

<211> 21

<212> DNA

<213> Forward primer (primer-F)

<400> 17

acctaacaat taaagccata g 21

<210> 18

<211> 22

<212> DNA

<213> reverse primer (primer-R)

<400> 18

atgcatgtgc ttggtgtgcc at 22

<210> 19

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 19

gccagtacta attattatcg 20

<210> 20

<211> 21

<212> DNA

<213> reverse primer (primer-R)

<400> 20

atggatacgt aaaggaatag t 21

<210> 21

<211> 22

<212> DNA

<213> Forward primer (primer-F)

<400> 21

caataccact tcacatctga ac 22

<210> 22

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 22

aggtttaggtt ttgtaggcg 20

<210> 23

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 23

cttggcctaa taggactagc 20

<210> 24

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 24

ggtattttta ctagaaatgc 20

<210> 25

<211> 21

<212> DNA

<213> Forward primer (primer-F)

<400> 25

cattccaacc ctaattatta t 21

<210> 26

<211> 21

<212> DNA

<213> reverse primer (primer-R)

<400> 26

gaggaggata taattagtgt t 21

<210> 27

<211> 21

<212> DNA

<213> Forward primer (primer-F)

<400> 27

gtactgctaa tccttatgca c 21

<210> 28

<211> 21

<212> DNA

<213> reverse primer (primer-R)

<400> 28

gatgttgaga aagctacaat t 21

<210> 29

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 29

aagtcggccc aatttggcct 20

<210> 30

<211> 23

<212> DNA

<213> reverse primer (primer-R)

<400> 30

atggttaggt atgttttaat tat 23

<210> 31

<211> 23

<212> DNA

<213> Forward primer (primer-F)

<400> 31

ggaagcatta ttgcaggact tat 23

<210> 32

<211> 22

<212> DNA

<213> reverse primer (primer-R)

<400> 32

cgttggcgtg catgttacgg at 22

<210> 33

<211> 22

<212> DNA

<213> Forward primer (primer-F)

<400> 33

atcgctaatg acgcactagt tg 22

<210> 34

<211> 23

<212> DNA

<213> reverse primer (primer-R)

<400> 34

atatgggtac tattgcataa tgt 23

<210> 35

<211> 23

<212> DNA

<213> Forward primer (primer-F)

<400> 35

actatcttct gatagtaaca tgt 23

<210> 36

<211> 22

<212> DNA

<213> reverse primer (primer-R)

<400> 36

gcggagactt gcatgtgtaa gt 22

<210> 37

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 37

acataagctt ctgacttctc 20

<210> 38

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 38

agatgagtga tccgatggat 20

<210> 39

<211> 20

<212> DNA

<213> Forward primer (primer-F)

<400> 39

ctctcgtagc agatatactt 20

<210> 40

<211> 20

<212> DNA

<213> reverse primer (primer-R)

<400> 40

cgtgaaagag ataaccagat 20

<210> 41

<211> 16604

<212> DNA

<213> Sequencing results (Sequencing results)

<400> 41

gctagcgtag cttaatacaa agcatgacac tgaagatgtt aagatgggcc ctagtaagct 60

ccgcatgcac aaaggcatgg tcctgacctt attatcagct ttaacccaac ttacacatgc 120

aagtctccgc agtcccgtga atatgccctc aatcccctgc ccggggacga ggagcgggca 180

tcaggcacaa aatttagccc aagacgccta gccaagccac acccccaagg gaattcagca 240

gtgataaaca ttaagccata agtgaaaact tgactcagtc agggttaaga gggccggtaa 300

aactcgtgcc agccaccgcg gttaaacgag aggccctagt tgatattatc acggcgtaaa 360

gggtggttaa gggatgcaga ataataaagc caaatggccc tttggccgtc atacgcttct 420

aggtgtccga agcccaatta tacgaaggta gctttaataa aacccacctg accccacgaa 480

agctaagaaa caaactggga ttagataccc cactatgctc agctataaac ctagacgtcc 540

acatacaatt agacgtccgc ccgggtacta cgagcatcag cttgaaaccc aaaggacctg 600

acggtgcctt agacccccct agaggagcct gttctagaac cgataacccc cgttaaacct 660

caccacttct agccacccca gcctatatac cgccgtcgcc agcttaccct gtgaaggcaa 720

taaaagtaag caaaatgggc acagcccaga acgtcaggtc gaggtgtagc gtacgaagcg 780

ggaagaaatg ggctacattt tctactaata gaacactacg aatatgcaac atgaaatagt 840

gcttgaagga ggatttagta gtaaaaagga aacagagtgt ccttttgaac ccggctctaa 900

ggcgcgtaca caccgcccgt cactctcccc tgtcaatacg cagtaaagat ccataacacc 960

aaagcactga caaggggagg caagtcgtaa catggtaagt gtaccggaag gtgcacttgg 1020

attaaaccca gggcgtggct gagttagtta agcatctcac ttacaccgag aaaacgtcca 1080

tgcaaattgg atcaccctga gccagacagc tagcctgatt accaatataa cacaacaatg 1140

tttataacaa aacacgactt aacgctaaaa attaaaccat ttttttacct gagtatggga 1200

gacagaaaag gttcaaccaa gcgcgataga aaaagtaccg caagggaaag ctgaaagaga 1260

aatgaaataa accatataag caccagaaaa caaagactaa accttgtacc ttttgcatca 1320

tgatttagcc agaaccctca agcgaagaga cctttagttt gaaaccccga aaccaggtga 1380

gctaccccga gacagcctat ataacttagg gctaacccgt ctctgtggca aaagagtggg 1440

aagagctccg ggtagaagtg acagacctac cgaacctggt gatagctggt tgcctaagaa 1500

atggatagaa gttcagcccc gtgcccccca aaccaagacg cattttatta aggtagtttt 1560

taagggaaac acacgggagt tagttagagg gggtacagcc cctccaacaa aggatacaac 1620

cttatttaag gaggataaag atcacaatac ttaaaacaca ctgttctagt gggcctaaaa 1680

gcagccatct aagcagagag cgttaaagct cagacagagt gaagtttatt atactgataa 1740

aaaatcttat tcccctagta atattaggct accccatgcc cgcatgggag agattatgct 1800

aaaatgagta acaagaagac ctgttcttct ccaagcacaa gtgtaaacca gatcggacaa 1860

gccgctggaa aataacgaac ccaaccaaag agagaattgt ggacaataaa aagaccaaga 1920

aaaccccgca actaattgat cgttaacccc acactggagt gctatttaaa aggaaagact 1980

aaaagaaagg gaaggaactc ggcaaacaca agcctcgcct gtttaccaaa aacatcgcct 2040

cctgcaatca gtaagtatag gaggtccagc ctgcccagtg actacgggtt caacggccgc 2100

ggtattttga ccgtgcaaag gtagcgcaat cacttgtctt ttaaatagag acctgtatga 2160

atggccaaac gagggcttaa ctgtctcccc cttcaagtca gtgaaattga tctatccgtg 2220

cagaagcggg tgtaataata caagacgaga agaccctttg gagcttaagg tacaaagtta 2280

agccacgtca aacaactccg taaaaagtaa aaacttagtg gaatatgaaa ttttaccttc 2340

ggttggggcg accgcggagg aaaatatagc ctccgagtgg aatgggataa acccctaaaa 2400

ccatgagaaa catctctaag ccgcagaaca tctgaccaaa aatgatccgg ccaaccggcc 2460

gatcaacgaa ccaagttacc ctagggataa cagcgcaatc ctctcccaga gtccatatcg 2520

acgagggggt ttacgacctc gatgttggat caggacatcc taatggtgca gccgctatta 2580

agggttcgtt tgttcaacga ttaaagtcct acgtgatctg agttcagacc ggagtaatcc 2640

aggtcagttt ctatctgtaa cgctactttt cctagtacga aaggatcgga aaaggggggc 2700

ctatacttaa agcacgcccc acccctaatt gatgaaagca aataaattag ataaagggag 2760

ggccaaaacc cctaccgccc aaaataaggg catactgggg tggcagagca tggtaaattg 2820

cgaaaggcct aagcccttta caccagaggt tcaaatcctc ttcccagttt atgctaaacg 2880

gtttaataaa tcacttaatc aaccccttag cctacattgt ccctgtcctt ttagcagtag 2940

cctttcttac gctactcgag cgaaaggtcc taggatatat acaactacga aaagggccta 3000

acgtcgtagg gccttacggg ctattacaac cgattgctga cggggtaaaa ctatttatta 3060

aagaacccgt gcgcccctcc acatcctccc catttctatt tttagcagcc cctattcttg 3120

cactaaccct tgccatgacg ctatgagcgc ccatacctat accctaccca gtaattgacc 3180

taaacctggg aattctattt atcctggccc tctcaagcct cgcggtatac tccattttag 3240

gctcagggtg agcatcgaat tcaaaatatg cactaattgg agccctgcga gcggtggccc 3300

agacaatttc atatgaggta agcctgggac taattctact ctccgtaatt attttttccg 3360

gagggtatac cctgcaaaca tttaatacag ctcaagaaag catctgactt ttagcccccg 3420

catgacccct agctgcaatg tgatatattt caacactagc tgaaacaaac cgggcacctt 3480

tcgacctaac agagggggaa tcagaactag tttctgggtt caacgtagag tacgcagggg 3540

gcccgtttgc cctctttttc ctcgcagagt atgctaacat tttattaata aataccttat 3600

cagctgttct attcttaggg gcatcccact tccccaacat acctgaatta acaacaatta 3660

gcctaatagt taaagctgcg tttttatcag tcgttttcct ttgagtccgg gcatcctacc 3720

cacgattccg atatgaccag cttatacatc ttgtatggaa aaacttcctc ccactaacgc 3780

tggccctagt actatgacat attgccctac cggttgcaat agcaggcctt cctccgcaac 3840

tatagtctag gaactgtgcc cgaatgctta gggaccactt tgatagagtg gctgataggg 3900

gctaaaatcc cctcagttct tagaaagaag ggggtcgaac ccatgcccaa gagatcaaaa 3960

ctcttagtgc ttcctctaca ccactttcta agatggggtc agctaattaa gctttcgggc 4020

ccataccccg aacatgacgg ttgaagtccc tcctccatca atgaacccct acgtactaat 4080

aattctatta tccagcctag ggttagggac caccctaacc tttgccagct ctcactgact 4140

actagcttga atggggttag aaattaatac cctagcaatc gtgcccttaa tggcacagcg 4200

ccaccaccca cgtgcagtag aggccactac gaaatacttc ctcacccaag ccaccgcagc 4260

agccataatt ctatttgcga gcacaacgaa cgcctgaatt accggtgagt gggatataaa 4320

taatatgtca gatccaattg ccagtacaat aattattacc gccctcgcgc ttaaaattgg 4380

actagcacca atacacttct gaataccaga ggttctgcaa ggcctggacc tactaaccgg 4440

cctaatttta tcaacctggc agaagctggc ccccctcgcc cttattattc agacagccca 4500

ggccattgac cccctcctgc tgacctcact tggacttatg tccacgttag ccgggggatg 4560

gggaggactg aatcaaaccc agctccgaaa aattctagcc tactcctcta ttgcacacat 4620

gggttgaata attattgtcc ttcaatatgc cccacagctc acactcctcg cactagggac 4680

ttacatctta ataacttcgg cagcatttct tacactgaaa tcatcctccg ccacgaaaat 4740

taatacccta gcgatagcct gatcaaacag ccctgtttta acagcgacga ctgccctagt 4800

tctactgtca ctgggaggac tccccccact aacaggattt ataccaaaat gattggttct 4860

acaggaactg gcaaaacagg attttcccct tactgccacg gttatagccc tagctgccct 4920

ccttagcctg tatttttatc tgcggctctg ctacgcaatg acactcaccg tgtcccccaa 4980

tgcagccaac tcagttgccc cctgacgggt ccaagcaacc caaagttccc ttccattagc 5040

cctctccact acacttgcac tcggtctttt gcccgtaacc ccagctattt taatgctagc 5100

cacctaggga cttaggatag cattagacca agagccttca aagctctaag cagaagtgag 5160

aatcttctag tccctggata agacctacag gagtctatct tgcattttct gattgcaaat 5220

caaatgtttt tgttaaacta aggccttact agatgggaag gcctcgatcc tacaaactct 5280

tagttaacag ctaagcgctc aagccagcga gcatccatct actttcccgc cgttagccta 5340

gtaaggcggg aaagccccgg tagggtatta atctacgtct ttggatttgc aatccaacat 5400

gcttcttcac cacggggctg gtgataggga gaggacttaa acctctgtct tcggggctac 5460

aacccaccgc ctgaatactc ggctacccta cctgtggcaa ttacgcgctg attcttttct 5520

acaaaccaca aagacattgg taccctttat cttgtatttg gtgcctgagc tggaatagtg 5580

gggactgctt taagcctcct cattcgagct gagctgagtc aacctggctc acttctaggt 5640

gatgatcaaa tttataatgt aatcgtcact gcccacgcct tcgtaataat tttctttata 5700

gtaataccga ttctcattgg aggatttgga aactggcttg taccactaat gattggggcc 5760

ccggacatgg cattcccgcg aataaataac ataagcttct gacttctccc cccatcattt 5820

cttcttctac tagcctcctc cggagtcgag gccggggctg ggacagggtg aacagtttat 5880

ccaccacttt caggcaacct tgctcatgct ggagcctcag tagacctcac cattttctcg 5940

ctccatctag caggtgtttc atcaatttta ggggctatta attttattac cacctctatc 6000

aatatgaaac ccccagccat ttcccagtac caaactcccc tctttgtgtg agccgtgctt 6060

gtaacagccg tacttctact tctttcacta ccagttctgg ctgccgggat tacgatgctt 6120

ctcacggacc gaaatcttaa cactacattc tttgacccgg cagggggagg agacccaatt 6180

ctttaccaac acctattctg attcttcggc cacccagaag tatatattct tattttacca 6240

ggatttggca tcatttcaca tgttgtagcc tactactcag gcaaaaaaga gccatttggg 6300

tacatgggaa tagtatgggc tataatggct attggtctcc taggctttat tgtgtgggca 6360

caccatatgt tcaccgtagg aatagacgta gacacccgtg cctacttcac atccgcaaca 6420

ataattattg ctattccaac aggggtaaaa gtatttagct gacttgctac actacatgga 6480

ggctctgtta aatgagacac gcctatgctg tgagcccttg ggttcatctt cctttttacc 6540

gtgggggggc taacaggaat tgtcctagcc aattcttcac tagatattgt cctccacgac 6600

acatactacg tagttgcaca ctttcactat gtattatcaa taggggccgt atttgctatc 6660

atagcagcct tcgtccactg attcccccta ttttcaggat acaccctaaa tgacacttga 6720

acaaaaatcc acttcggtgt aatgtttatt ggtgtaaatc tcacattctt ccctcaacat 6780

ttcctaggtc tagcaggaat gccacgacga tactctgatt accccgacgc ctacgccctg 6840

tgaaatacag tgtcatccat cggatcactc atctccctgg tggcagtaat tatatttcta 6900

tttattctgt gagaggcctt cgccgccaaa cgggaagttt cctcagtaga actaaccatg 6960

acgaacgtag aatgactgca cggctgccct cccccttatc atacatttga ggagccagcg 7020

ttcgtacgag ttcaatcaaa ctaacgagaa aggaaggaat tgaaccccca tgtactggtt 7080

tcaagccagt cacataacca ctctgtcact ttcttctgaa gacattagta aaatgcaaat 7140

tacatcacct tgtcaaggtg aaattgcagg ttaaatccct gtatgtctta agcccaaggc 7200

ttaatggcac atcccacaca actaggattc caagacgcgg catcacccgt tatagaagaa 7260

cttcttcact tccacgacca tgctctgata attgtatttt taattagtac cctagtactt 7320

tacattatta ttgcaatggt ttcaaccaaa cttaccaaca agtacatctt ggactcccaa 7380

gaaatcgaaa ttgtatggac cgttctacca gccgtcattt tagtcttgat tgcactgcca 7440

tcccttcgaa tcttatatct tatagacgaa attaatgacc cccacctaac aattaaagcc 7500

ataggacacc aatgatactg aagttacgaa tacacggact atgaagacct cggattcgat 7560

tcctatataa ttccaactca agacctcaca ccgggtcaat tccgattact agagacagac 7620

caccgaatag ttgttccaat agaatcacct attcgtgtac tagtatccgc tgaagacgtt 7680

ttacactcat gagccgtccc atcgctagga gtaaaaatag acgcagtacc aggacgacta 7740

aaccaaactg cctttattgc ctcccgccca gggctcttct atgggcaatg ttctgaaatc 7800

tgtggtgcca atcacagctt tatgccaatt gtagttgaag ccgttccact agaacacttt 7860

gaaagctgat cctcattaat actagaagac gcctcactag aaagctaatt attggacaaa 7920

gcgttggcct tttaagccaa agtttggtga ctaccgacca cctctagtga aatgccccaa 7980

ttaaacccca acccttgatt tgctattcta gtgttttcat gaattgtatt tcttaccgtc 8040

atccccacca aaattctaag ccatacggca ccaaatgaac ccgtgcctat gagtgaagaa 8100

aaacacaaaa ctgagccttg agattgacca tgataataag cttttttgac cagtttgcaa 8160

gcccctcctt cctaggaatt ccccttattg ccgtcgcaat cgcactcccc tgaatactat 8220

ttccaacacc cccatcccga tgactaaaca accgactaat tacccttcaa acatgattca 8280

ttaaccgatt caccaaccaa ctactgttac ccctgaacac aggggggcac aaatgggcct 8340

tattatttgc gtccctgata gtctttttaa ttactattaa tatactaggc cttcttccat 8400

acaccttcac accaacaact caattatctt taaacatagg gtttgctgta cccttgtgac 8460

ttgctacagt aattattggg atgcgcaatc aaccaaccgt tgcccttgga catcttcttc 8520

cagaaggaac cccaatccca cttattccag tactaattat tatcgaaaca attagtctgt 8580

ttattcggcc attggcgcta ggggtacgac tcacagccaa tttgactgca ggtcatctac 8640

tcattcaact aattgctacc gccgtatttg tattaatgcc tataatacca acagtggcaa 8700

tcttaacggc cgccgttcta ttccttctta cacttctaga agttgcagtt gcaataattc 8760

aagcttatgt atttgtgctt ctactaagcc tctacctgca agaaaacgtc taatggcaca 8820

ccaagcacat gcatatcata tggtcgaccc cagcccatga ccactaaccg gagccgtcgg 8880

tgcactacta ataacatccg gcctagcaat ctggtttcac ttccactcaa caacgttaat 8940

aacccttgga ttaatccttc ttcttcttac cataatccag tgatgacgtg atattattcg 9000

ggaaggcacc ttccaaggac atcacacacc cccagtacaa aaaggcctac gctatggtat 9060

aattttattt attacatcag aggtgttttt cttccttggg ttcttctgag ctttctatca 9120

ctcaagctta gccccaacac ccgaactagg gggatgttga ccaccaactg gaattaccac 9180

actagacccc tttgaagtac cccttctaaa tacggcagta ttgttagcat caggggtaac 9240

ggttacatga gcccaccaca gcattatgga aggtgagcga aaacaagcta ttcagtcgct 9300

agggctcaca attttattag gactctactt cactgcccta caagccatag aatattatga 9360

ggcacctttc acaattgcag acggagtcta tggctctaca ttcttcgtag ctacaggatt 9420

ccacggacta cacgttatta ttgggtcaac cttcctggcc gtttgcctcc tccgacaaat 9480

ccaataccac ttcacatctg aacaccactt tggctttgag gccgctgcct gatactgaca 9540

ttttgttgac gtagtgtgac tattccttta cgtatctatt tactgatgag gctcatatct 9600

ttctagtatt aaattagtac aagtgacttc caatcattta gtcttggtta aaccccaggg 9660

aaagataatg aacctaatta cgactatttt tcttattaca gtagcgttat catcagtttt 9720

agcaattgta tccttctgac taccacaaat gaccccggat gcagagaaac tctccccata 9780

cgagtgtggg tttgaccctc taggatctgc ccgactacca ttctccttac gattctttct 9840

agttgccatt ctattcctct tatttgatct agagattgcc ctccttctcc ccttaccctg 9900

aggagatcaa cttcacaacc ccacaggaac attcttttga gcaaccgcag tcctgatttt 9960

actaacccta gggttggttt atgaatggac ccaaggaggc ttagaatgag cagagtaagg 10020

gagttagtcc aaaataagac ctctgatttc ggctcagaaa attgtggttt aagtccacga 10080

cccccttatg acaccagtac atttcagctt cagttcagca tttattcttg gcctaatagg 10140

actagcattt caccgtaccc acctgctatc agcgcttcta tgcttagagg gcataatact 10200

atccctattt attgcactag ccctatgagc gctacaattc gagtctacta gcttctccac 10260

tgcccccatg cttcttctgg ccttctcggc ttgtgaagca agcacaggac tagcacttct 10320

agtagccacg gcccgaaccc atgggacaga ccgcctacag aacttaaacc tcctacaatg 10380

ttaaaagtat taatcccgac aatcataata tttccaacaa tctgactaat ttccccaaaa 10440

tggttatgga cagccacaat cacccacagc ctttcgattg ccctcattag cctcacatgg 10500

ctaaaatgaa catccgagac agggtgggcc acctctaata catacctggc tacagacccc 10560

ttatcaaccc ccctgctagt actaacctgc tggcttcttc cacttataat tttagccagc 10620

caaaaccaca ttaaccccga acctattaac cgacaacgcc tttatattat gcttcttaca 10680

tccttacaag ctttcctaat cttggccttc ggtgctactg agattatcat gttctacatc 10740

atgtttgagg ccacactcat cccgacctta attattatta ctcggtgagg taaccaaacc 10800

gagcgactta atgccggcac atactttctg ttctacacac tagcaggctc tttgccgctc 10860

cttgtcgcgc tactactcct acagcagtcc acggggaccc tctccatatt agtaatccaa 10920

tattcgcaac cgctcctatt aaactcctgg ggccataaaa tctgatgagc cgcctgtcta 10980

gtcgcatttc tagtaaaaat accgctctac ggcgttcacc tatgactccc aaaagcacat 11040

gtagaagctc cagtagcagg gtctatagta ctagcagccg tgctccttaa actaggaggg 11100

tatggtataa tacggataat aattatacta gacccgctct caaaacaact ggtctacccc 11160

ttcattatcc tagcattatg gggtattatt atgaccgggt ctatttgcct acgacaaaca 11220

gaccttaaat gcctaatcgc ctattcatca gtcagccata tgggacttgt agcaggtggc 11280

atcctaattc aaaccccgtg aggtttttca ggagcaatta tccttataat cgcccacggg 11340

ttagtatcct caatactttt ctgcctagct aacacggcct acgaacggac acacagtcgt 11400

accatagtcc ttgcccgagg actacagata atttttccac taacagcagt ttgatgattc 11460

attgccaatc tggctaacct ggccctccct ccgctaccta atttaatagg agaacttata 11520

attatcacaa ccctgtttaa ctgatcccca tgaactatcg cactgacagg aacgggcaca 11580

ttaattacag ccggctactc cctctactta tttctaataa ctcagcgagg cccaacacca 11640

ggccacatca tgaacctccc accgttccac acccgggaac acctattgat agcccttcac 11700

ctgatccccg taattctcct tgtagcaaaa ccggaactta tgtgaggatg atgttactag 11760

taagtatagt ttaactaaaa tattagattg tgattctaaa gacaggggtt aaagtcccct 11820

tactcaccaa ggaagaacag aaaccagtaa gtactgctaa tccttatgca ccgcggttaa 11880

actccgcggc ttccttacgc ttctgaagga taacagctca tccattggtc ttaggaacca 11940

aaaactcttg gtgcaaatcc aagtggaagc tatgaattca ataacactaa ttatatcctc 12000

ctcacttatt ttagttatca caatcctcat cgtcccgcta ctaataacat taaacccaaa 12060

gccccaacac cctaattgag caaacgcaca tgttaagact gctgtcagca ccgcattctt 12120

tattagtctt cttccactta taatcttttt agatcaggga gccgagagca ttactacaaa 12180

ctgacactga ataaacacac acatatttga cacgaacatt agctttaaat ttgatcacta 12240

ctcccttatt ttcaccccta ttgccctgta cgtcacctga tcaattttag agtttgcgct 12300

atggtatata cactcggacc ctaatataaa tcggtttttc aagtatctac tactattttt 12360

agtagctatg atcacccttg tcaccgctaa taatatattt caactattta ttgggtgaga 12420

gggggttggg attatgtcct ttttattgat cgggtgatga tatggccggg cggacgctaa 12480

tacagcagcc cttcaggctg tcatctacaa ccgggtaggg gatattggat taatcctcag 12540

catggcctga ttcgcgatga atttgaactc ctgagaaatt caacaaatct tctttctgtc 12600

aaaaaatttt gacatgacgg tcccgttaat tggattaatt ctagcagcaa caggaaagtc 12660

ggcccaattt ggccttcatc cgtgactacc ttctgccatg gagggcccta cgccagtatc 12720

tgccctactc cattccagca caatagtagt tgccggaatt ttcctactaa ttcgccttca 12780

cccccttata gagggtaacg aaactgcatt gacaatttgt ctttgcctcg gcgcactaac 12840

aacactattt acagccacct gcgccctaac ccaaaatgac atcaagaaaa ttgtagcttt 12900

ctcaacatca agtcagctgg gtctgataat agttacaatt gggcttaatc agccacagct 12960

ggcatttctt cacatctgta cacacgcctt tttcaaggcc atgttattcc tgtgctcggg 13020

ctcaattatt cacagcctaa atgacgaaca agacatccga aaaatagggg gcctccacaa 13080

cttgataccc gctacttcaa cctaccttac aattggcagt ctagcactaa caggaactcc 13140

attcctcgcc gggttttttt caaaagacgc cattattgaa gccctaaaca cctctcacct 13200

taacgcctgg gccctaactc ttacactaat tgccacatca ttcaccgcag tctacagctt 13260

ccgagtcgta ttctttgtaa ctatagggtc cccacggttc ctcccactat ccccgattaa 13320

tgagaacaac ccattagtaa ttaaccctat caagcgactt gcttgaggaa gcattgttgc 13380

gggacttatt attacatcca acttcctccc ctcaaaaaca cccattatga ctataccctt 13440

ggccctaaaa atagcggccc tcatggttac cattgtcgga ctcctggtag ccatggaatt 13500

aacggccata accaacaaac agattaaaat tacccccaca atgcacctgc acaatttttc 13560

aaacatactg ggatactttc catcattaat tcatcgacta cccccgaaac ttaacctggc 13620

cttaggacaa tcaatcgcca ctaagctcga ccaaacatgg ttcgagatct cagggccaaa 13680

agggttggca cttacacaaa tgataatatc aaaagtcaca agtggcatcc aacgaggaat 13740

aattaaaaca tacctaacca tcttcctgtt aaccttaacc ctggccgtcc ttttaacaat 13800

tatttaaaca gcccgaaggg tgccacgact tagccctcga gttagctcca acactacaag 13860

aagtgttaaa agcaacaccc aagcacaaag aaccagcata gccccaccaa aagaatatat 13920

aacagctaca ccactaacgt caccacgtag tattgaaaac tctttcagcc catcgataat 13980

tacccaagaa ccttcatatc acccacccca aagcatacta cctgctaaag tcacgcctaa 14040

taaataaacc aacacgtacc ctgcaacaga tcgacttcct caggcttcag gaaagggctc 14100

agcagccaaa gccgctgaat aagcaaacac cactagtatg ccccctagat aaattaaaaa 14160

aagaaccaaa gataagaaag accccccaca cccaactaaa accccgcacc ccactcccgc 14220

tgctaccact aaccccaaag cagcaaaata gggagtggga tttgaagcaa cagcaattaa 14280

ccccacaatt aaggccacca ataacataaa cataaaatag gtcataattc ttgctcggac 14340

tttaaccgag accagtgact tgaagaacca ccgttgtagt tcaactacaa gaactaatgg 14400

caagcctacg aaaaacccac ccgctaataa aaatcgctaa tgacgcacta gttgacctac 14460

caacaccatc taacatttca gtatgatgaa actttggctc cctactggga ctctgcctaa 14520

ttacgcaaat cttaacagga ttattcctgg ctatacacta cacctcagac atttcaactg 14580

cattctcgtc agttgcccac atctgccgag acgtaaacta cggctgattt atccgtaaca 14640

tgcacgccaa cggagcatca ttctttttca tctgcattta tatgcacgtt gcccgaggcc 14700

tttattacgg atcctacctc tacaaagaaa cctgaaacat cggggtagtt ctactcctgc 14760

tggtcataat gacagccttc gttggctatg tcctaccatg aggtcaaatg tcattctgag 14820

gggccacagt aattactaat cttctatcag cagtccctta tatgggggat acccttgtcc 14880

aatgaatttg aggcggtttt tcagtagata acgcaacact aacacggttc ttcgcctttc 14940

actttctcct accatttgtg attaccgctg caaccgtcct ccacctgcta tttttacacg 15000

aaacgggatc aaataaccca gccggactaa actctgatgc agacaagatc tccttccatc 15060

cgtacttctc ttacaaggac cttcttggtt ttgttctaat gcttctagct ctcacatcat 15120

tagcgctgtt ttcccccaac ctattagggg atccagacaa tttcaccccc gcaaacccaa 15180

tagttacccc tccccacatc aaacccgagt gatacttcct atttgcttac gccattctgc 15240

gatccatccc caataaatta ggaggcgtcc ttgcactatt atcttctatt ctgattctca 15300

tagtggtccc aattttacac acctcaaaac aacgaggtct aacattccgc cccctgacac 15360

aattcttatt ctgaactctc gtagcagata tacttattct aacgtgaatc ggaggcatgc 15420

ccgtagaaca cccatatgtt gtcatcggcc aagtcgcttc aattctgtat tttgcacttt 15480

tccttatcct cgtcccagtg gcaggctgat tggaaaataa agcattaaaa tgagcttgcc 15540

ctagtagctt agcctaaaag catcggtctt gtaatccgaa gatcggaggt taaattcctc 15600

cctagcgccc agaaaaggga gattttaact cccacccctg gctcccaaag ccagaattct 15660

aaattaaact atcttctgat agtaacatgt atggtatagt acataatatg cataatatta 15720

cattatgcaa tagtacccat atatgtatta tcaccattaa attattttaa ccccaaagca 15780

ggtactatcg tcctaaacgt acataagcta aatattaaaa ctcagaaata atttatatta 15840

acctgggaaa tagattaatc cctaaaaaat ggaactcaca ttttccctcg aaatactcaa 15900

ctaaggttta tcttaaaaat attaatgtag taagagacca ccaactggtt gaaataaagg 15960

catactatta atgatagaat cagggacaca aaccgtaggt gtggtatttt atgaattatt 16020

ccttgtatct ggttatctct ttcacgtact taactgtttt tcccacactt atctcattgt 16080

tcctgcatac ggttaatggt gtaattacat actcctcgtt acccaacatg ccgggcattc 16140

ttttatatgc atagggttct cttttttggt ttcctttcaa ttacatttca gagtgcaggc 16200

gcaacaaata tatcaaggtt gtactcttcc cttggtaaca gttaaagtag gttaattatt 16260

aaaagacata acttaagaat tacatattaa taactcaagt gcataacata tctattcctt 16320

cttcaactta cccttatata tatgcccctc ttttggtttc tgcgcgacaa acccccctac 16380

cccctacgct cagcaaatcc tgttatcctt gtcaaacccc gaaaccaagg aaggttcgag 16440

aacgtgcaga ctaacaagtt gggatatgaa ttagccatcg cattatatat atatacatgc 16500

atgccgcgct agcccatcgc gtaaatcacc taaatattag cccgaataac tcgattgaaa 16560

tttttgggaa tttcttaatg ctaaaaaatt caacatttta taat 16604

Claims

1. The design method of the gobiocypogalus mitochondrial genome complete sequence primer is characterized by comprising the following steps: obtaining the complete sequence of the mitochondrial genome of the gobiocyprisrarus closely related species, performing multiple comparison on the sequences to obtain a relatively conserved region and a region with larger variation, and designing mutually nested primers in the relatively conserved region, namely, a downstream primer of a first pair of primers is arranged behind an upstream primer of a second pair of primers to ensure that amplified products are mutually overlapped.

2. The design method according to claim 1, wherein: the closely related species of the gobiocypris rario are the thin-body rogoogobio (Romanogobio tenuicoprus), the gobiocypio (Sauroogobio dabryi), the gobiocypio (Gobio cynocephalus), and the gobiocypio (Pseudonogobio variabili).

3. The design method according to claim 1, wherein: the mitochondrial genome complete sequence of the rhinogobio ventralis kindred species is downloaded from a Genbank database.

4. The design method according to claim 1, wherein: the relatively conserved region is a conserved sequence region without mutation sites or with only 1 mutation site.

5. The design method according to claim 1, wherein: the amplified product sequences between adjacent primer pairs have at least 100bp overlapping regions.

6. The gobiocypiocypogalus mitochondrial genome complete sequence primer set is characterized by comprising twenty pairs of primers, wherein

A first pair of primers:

a second pair of primers:

a third pair of primers:

a fourth pair of primers:

a fifth pair of primers:

a sixth pair of primers:

a seventh pair of primers:

an eighth pair of primers:

a ninth pair of primers:

a tenth pair of primers:

an eleventh pair of primers:

a twelfth pair of primers:

a thirteenth pair of primers:

a fourteenth pair of primers:

a fifteenth pair of primers:

a sixteenth pair of primers:

a seventeenth pair of primers:

an eighteenth primer pair:

a nineteenth pair of primers:

a twentieth primer pair:

the downstream primer 20 is shown as SEQ ID CJ 20-R: 5'-CGTGAAAGAGATAACCAGAT-3' are provided.

7. The primer set as claimed in claim 5, which is used in the amplification method of the complete sequence of mitochondrial genome of gobiocypiocypiocypris, and comprises the following steps:

(2) using total DNA of the mitochondrial genome of gobiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypiocypioc;

(3) and detecting the amplified product through gel electrophoresis, sending the single PCR product with clear target band to a sequencing company for bidirectional sequencing, and splicing to obtain an original DNA sequence.

8. The method of claim 7, wherein: the PCR amplification reaction system comprises: 20. mu.L of Taq enzyme PCR preliminary mixture, 2. mu.L of each pair of primers (1. mu.L of each of the upstream and downstream primers), 2. mu.L of DNA template and 40. mu.L of sterilized double distilled water, the composition of the Taq enzyme PCR preliminary mixture being 0.1U/. mu.L of Taq DNApolymerase (DNA polymerase), 2 XPCR reaction buffer, 3mM MgCl₂，0.4mM dNTPs。

9. The method of claim 7, wherein: the PCR amplification conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 56 deg.C for 45s, extension at 72 deg.C for 2min, extension at 72 deg.C for 10min after 35 cycles, and storing at 4 deg.C; 1% agarose was used for the gel electrophoresis.

10. The method of claim 7, wherein: the step (4) is as follows: comparing the sequencing result with the homologous sequence on GenBank to confirm that the sequencing sequence is highly similar to the closely related species; and then comparing sequencing results, splicing sequences of the overlapped parts of the sequencing results of the forward primers and the reverse primers in one group of primers, repeatedly splicing the sequences obtained by the multiple groups of primers in sequence, and finally splicing the multiple sequences end to obtain the complete sequence of the mitochondrial genome of the gobiocypiocypris dabryi.