CN107557479A - Microsatellite marker and application for Sepiella maindroni Parentage determination - Google Patents

Abstract

The invention discloses the microsatellite marker for Sepiella maindroni Parentage determination, also discloses that application of the microsatellite marker in Sepiella maindroni Parentage determination.Have the beneficial effect that：The microsatellite marker allele number for Sepiella maindroni Parentage determination that the present invention screens is more, and polymorphism is high, can be used for the population genetic variations of Sepiella maindroni, genetic thremmatology assessment and paternity test etc.；Nucleotides sequence is classified as SEQ ID No:1 19 microsatellite marker is used to identify Sepiella maindroni family, compensate for the limitation that genetic background is fuzzy, molecular labeling quantity is few in current Sepiella maindroni germplasm monitoring, and this method efficiency high, qualification cycle are short.

Description

Microsatellite marker and application for Sepiella maindroni Parentage determination

Technical field

The invention belongs to field of molecular biotechnology, the microsatellite of Sepiella maindroni Parentage determination is especially for Mark and application.

Background technology

Sepiella maindroni is commonly called as inkfish, Japanese sepiella maindroni de Rochebrune, is subordinate to Mollusca, Cephalopoda, Dibranchia, ten wrists Mesh, cuttlefish superfamily, Sepiidae, sepiella maindroni de Rochebrune category, it is distributed more widely in China coast, once it was China's " one of four big marine products ", had Higher economic value.With the breakthrough of Sepiella maindroni artificial breeding technique, the enhancement releasing of Sepiella maindroni and its The protection and repair in spawning ground progressively deploy.Do not focus on select index, breeding environment and nature in long-term breeding process The difference of environment, the problems such as causing cuttlefish sex premature, decline artificial farming Sepiella maindroni germ plasm resource, limit it and support Grow the development of industry.Microsatellite DNA (microsatellite) is also known as simple repeated sequence, is distributed because it has extensively, single The advantages that locus gene is in codominant inheritance, detection method fast and stable, there is obvious advantage in Parentage determination.

The content of the invention

It is an object of the invention to provide the microsatellite marker for Sepiella maindroni Parentage determination, the mark equipotential base Factor mesh is more, and polymorphism is high, can be used for the population genetic variations of Sepiella maindroni, and genetic thremmatology is assessed and parent-offspring's mirror It is fixed etc..

Another object of the present invention is to nucleotides sequence is classified as into SEQ ID No:1-19 microsatellite marker is used to identify Purposes in Sepiella maindroni family, microsatellite sequence SEQ ID No:1-19 allele numbers are more, and polymorphism is high, makes up The limitation that genetic background is fuzzy, molecular labeling quantity is few in current Sepiella maindroni germplasm monitoring, and this method efficiency high, Qualification cycle is short.

The nucleotide sequence SEQ ID No of microsatellite marker:1-19 is as follows：

SEQ ID No:1 is：attacgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaaa tctgtctgctgcaaagagcttgtcaaataaatttgttcatctttataagtgacgtttcaattgaactggcttcgatc atcatcatcatcatcatcatcatcatcatcatcatcatcatcatcatcatcatcatcaggatttgttcttttatttt gcgattttgaatacaagtggttctgttggaaactttgcagattaggatctcctgacaggctccagaactgacccaat catgtgaaaaggctgatggattgttcggtgaagatcgtcatggatgagttcacaaatattttctaaattttccggct ctctgctgatagcttttcgacttcaaccaatacatgactggccttgaaatgaggatctctctctcactcttccacac acacacacacacacacacacaaatatccagtttgtgacaaagctttatcctatgccttagaacttgtcaagatattt ggtaatgtaacaaaagtgacaatgaccctttgtacatgtagatttcatatttccttattcaaaaattgtggatgaga aaatatatatcacctgaaagcagttcccagcatacaatctggtacaactgtgcttcatcttggaacgtcctaattgc aatagaaaatgtgtaataaaaactgaaacaattactcaggactcatcaatcgtcgacctgcaggcatgcaagcttgg cactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccc cctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcg。

SEQ ID No:2 are：cgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaac tgccagccgggtcacagttaggcattcaaacaccaactcgacattcaacgccagtctgaagacttggcattcaaacg cgagtcgggtcggcacatcaaagaaagtttggcatcgcagaagacaatcagacccggacatattttcgcgaacaagt cggcacaaaaggattctcaagtcaaagatagccacacccgtacacaccaggatctgaccaagtcgagaaccaagtac agaaactcaaaaccggatcttcgcacacagagaacaaccgccgttctccgggttacacacgcctggcatatctcgtt tcacacacacacacacacacacacacacacacacacacgcacacacacacacatatgtatacacacacatatctcta cttatataaagttgctgtctctgtcattctgtttttctgtctgtttccattactcaggactcatcaatcgtcgacct gcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaat cgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagtt gcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatat ggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcg ccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagag gttttcaccgtcatcaccgaaacgcgcgagaacgaaagggcctcgtgatacgcctattt。

SEQ ID No:3 are：acgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaa gtatgcacattacgggaatagcttgcaattcttcacacacacacacacacacacacacacacacacacacacacaca cacactctctctctctctctccctctctctctctctctctgtgaaacacacatacaggaatacacatacgtgtatcg gcgttggttgataatgatgttttatttgacctgcgatatacttttatattcgacgaattttgcgtagcagtaaatat atactatttacatcattcgaactgttcttcccatctttgtcgtcatattcagaacaacgtttcggttgttgtacgct ccagccttcttcggttggttggaatgtcgaactgaatcctttatatatctcattgaagtagtctgttctagttccac gtgccatgccttttctgggtgtcaattaccagttatctcctgttactcaggactcatcaatcgtcgacctgcaggca tgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagc ctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcac tctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgac gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttca ccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgccta。

SEQ ID No:4 are：tacgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagta actcacaccgtcttttgcaagtgaacgttctcgcattcgcgcccgcaatccattctcgaacacgcagcgcaccacgt gttgtttttttttgatggacgcggacgcacatcatcatcaacggtagtcgacagttcgttctgcttttcatttccgc tacaataaggcatgtttgtgcgaataacatgcacgctctctctctctctctcactcatacacactctctctctctca ctcactcacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacac acacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacaca cacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacaatcgcagta ccccaggtcaagtgagtcagccacttactcaggactcatcactcgtcgacgtgcacgcatgcgagcgtgacactgtg cgtcgtttacaacgtcgtgactggaaaaccgtgcgtaccaacttatcgcgtgcagcacatcccttcgcagctggcgt atagcgagagccgcacgatcgccttccacagtgcgcagcgtgatgcgaatgcgctgatgcggatttctctcacgcat ctgtgcggtattcacaccgcatatggtgcactctcagtacaatctgctctgatgcgcatagataagccagaccagaa tcagcaacaccgctgacgcgccctgacgctgtctgtctgcatcgcatcgcggctgtacgttctgactgcatgttcga gttcacg。

SEQ ID No:5 are：cgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaaa gcggataaaattatatttatgtctgtatacccctcattaggggtattggttgatctggtctgtgtttgtttttgtgt tttttttattgtttgtgtgttttctgtaagatacagccacttgctcaccgccgggaagtcgcctctctgtgcctctt ttataagtactgtcacggcttctgttcggatgagctccgcactatggtccctagcgtgtacagattcaagcgcaata ctcgtctagcagcgcgatctcatcaatttacagtcaagctggctagatgtaaccgcagtttctatgctaacagtttc ttttcccgcacctccagattgtggaactcctcccggccttttgctttcctgacggcttcaacctgcaaaggttcaag tgcaacgtcaatcgttacctgctctcttcgtaacccagtatacccttccttctgtaacccctagcatgcaagtggcg ctcctgccttgcttggggtgactgattactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactgg ccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttc gccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcg cctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgct ctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccg gcatccgcttacagacagctgtgaccgtctccgggagctgcatgtgt。

SEQ ID No:6 are：gattacgaattcgagctcggtacccggggatcctctagagattgatgagtcctga gtaaacataaaaatacataaacacacaattcatcttttcgcctccgatctttatcagatcagtgagcggaatgttcc gttgttctttcgaaacattatatttttatcaactcttttccaattgtcaataatttataagtttgtcgggatcatat acagaaatttatcacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacac acacacacacacacacacacacacacacacatatatataaatatatatgcaaggctcaaatataagtaaacactgta ataacatttacggtaatgccagattactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactggcc gtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgc cagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcc tgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctct gatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggc atccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcg cgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggt ggcacttttcggggaaatgtgcgcggaacccctatttgtat。

SEQ ID No:7 are：attacgaattcgagctcggtacccggggatcctctagagattgatgagtcctgag taaaaaagtaagtcattagaatgacactacgcaggtgacttttgaatctgtgatttacttgcaatttctcctcgaaa gatattgacatgcgcatcaagatacacaagcaagtgaacagagctgaaattcgtctacaccctcacacacacacaca cacacacacatacacacacatcagtcggaaaactgtgtgttgcatacccgtctctagatgtacgctcttctgtagag ggggcatcttcttctagaggggggctggcaggacttctgctacgtacttcatctctctgactcaccaggccggacat ctctcttttttatgcacagggaatatcacttttctgttctgatgttctgatgtccgaccagtagttgagacctgttg cctcggccgtcttcttcataactccgcatgttgccttatcatgcagtatctccatcgtctcgtgatgaccccttctt cggtgcatctttggtccaataaatgatgaccaacacaatattctatctaccttactcaggactcatcaatcgtcgac ctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaactta atcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacag ttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcat atggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacg cgccctgacgggcttgtctgctcccggcatcc。

SEQ ID No:8 are：tgattacgaattcgagctcggtacccggggatcctctagagattgatgagtcctg agtaatcagatttgccccttttgaatccatgtacatatgtttgtaaatacaatacgagaattatgtgtgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgtgtaagcgtgtgcgtgcgtttccgttcctatctttttatcctttttcaatt agttcactttcttatttccatctttctctctctctctttctctctctatctccctccccctctctctctctctcaat ccctccctgtatctctctttcggttctcaactcttttcctatctcagttattccccacctttttctttctttcagtt cctatttatttttatttttcagtttcttcgcttccaaatttctctgtccttcattatctctccccctttatttctct ttctctctaactctcaacctctctatctatttgtctatcaatccttactcaggactcatcaatcgtcgacctgcagg catgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcct tgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgca gcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgc actctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctg acgggcttgtctgctcccgggatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggtttt caccgtc。

SEQ ID No:9 are：gattcgaattcgagctcggtacccggggatcctctagagattgatgagtcctgag taacataaaattatttttgactgttcagtatctctcctcaagcctgttctgcaaacatttacacaaattcattgggt atataattcagcactgtcctgagaaagacacacacacacacacacacacacacacacacacacacacacacacacac acacacacacacacacacacacacacacacacactcacacacacacacacacacacacacacacacacacacacaca cacacacacacacacacacacacacacacacacatgcacacacacacacacacacacacacacacacacacaccttc ccacacacacacactctcacacacacgcacataccaatgggagttcccgacactttccccaacactcacagatccgg cgctgcatgcgtgattttttgcttgtggtatctttttaataccactcgactgggaaagcactggcgttacagtctgt tatggtgctgcataatttccccatttccgaggtggcgtaatagcgaagaggcgcgcactgatcgtgtttctcctcac gtgatcagcttgtagggggaatgtgaccagatgcggtattttctcgtttcacatgtgtgcggcgttttcaacgacac atggtgcactatcagaacactctgtactgatgtcttgaaggataaatgtcaccgacacacgggaatcctcgatgtca cgcggtgttttgcgtgtgagcgcgcgcggcccccatatagtgcatattgtgaccgacacgtggagatgtgtgtgcga tagtgatacaacgacctctcacgaaaagctcgagagtatagggcgacgtgagagtatctatatttttaggtttct。

SEQ ID No:10 are：attacgaattcgagctcggtacccggggatcctctagagattgatgagtcctga gtaacacccacgagtcaggatatggcacatggccggtttatatgttgggaaattcaggcaaaaataatccgtatccc cgcatacacggagtcggacttgtctaccaccccagtcgagactctacccttcggcacatcctcgactggattcctcc ctgcccgaatgcaaggctgtcgcaagctatgcagcgaactgcctgacttgacaacagcctaagatctacgaatcaaa caagtagttactaaagccacgacttgtaatgctaacgatcggaaattttatagttggggtggctgtcgtctgttata atgtgtgtttgttgattttgggcgtatatattcataatctgtttcttttgttacgtagatggcgtctattccgtttg actgtattcccttccttccaaaagcagtaggaatacataaaatacgtacgaacacacacgcacacacacacgcacac acacacacacatactcagttttatggctatagatactttgaaaaaaaacataggtcatgatcttccactgactataa gtaatattcaggtttgagaaaaatccatcgcaaacggtaggaatgcataaaatatatactaacacacacacacgcac acacacatacacacacacacacatactcagtttactcaggactcatcaatcgtcgacctgcaggcatgcaagcttgg cactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccc cctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcga atggcgcctgatgcggtattttctccttacgcatctgtgcgg。

SEQ ID No:11 are：acgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagta acaaaatgtggaaacagtattacaatgagtataatgttcttcaagtctggtacttagatacatgtgtgtgtgtgtgt gtgtgtgtgtgtgtgtgtgtgtgtaacccgatatgtctatcagtctagtgcggcattctctcgttctggtcagctcc gcgtccgtggctgtctttgttgtaccgactatcctttactcaggactcatcaatcgtcgacctgcaggcatgcaagc ttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagt acaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttg tctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcat caccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttct tagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatat gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatt tccgtgtcgcccttattcccttttttgcggcattttgccttcctg。

SEQ ID No:12 are：tacgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagt aatatcagaaagaggcaactgaagtctctgggacatattatgagaaaggagggtctggaaaaccttatactcacagg acggatagagggaggtagagacaggggaaaacaccgcacgacatacttgaagggcctgagcgaatggatgacgggaa ggggcatgggagatacggggagtcaagccctgttgagagcttcgatagatagggggaagtggaaagccatgatagcc tacgtcctgaagggatacggcacatagaagaagaatgtttcgatgatgaaggtctctttaggcggaattggcaaaca ttatcccaacactttatttatacgcttgacaaaaggaaataaaaataaaacaaaaaacaaatcaaaataaagacgaa aacaataacaacaacaacaaccactaacatggcactaaaggcgggggtctagaatttatggtgtggttactcaggac tcatcaatcgtcgacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctg gcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgat cgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcgg tatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgc caacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggg agctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggtcc。

SEQ ID No:13 are：tacgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagt aatatatctatcattcagttgtgtattttgactatctgttcttacaagtcttttcctgtttattcatctctcttcct ttctctattactccaaccactctcttgctcacctcttttctttctctgcttactcactcactcactcattcacgcac tcactcactcactcactcactcactcactcactcactcacgcactccgtttgtttctctctactttctccttctatt tgtcttcacatttgatccaatatttccatttttttctagatttatatctctttatatgcttttgtcttacctccttt tctctctttctctttctctccctatctctcttctttttctcctctctccttcactctctttttccctatcactttcc aattttccttatttccatcaacctctatttctctcccttctctctttctttcgtctttctctcttctcacttctttt ctcctcactacaatttctttttctttccctaattttttttctcttctcatctctctctttatctctccatctttcga tttcttctcttttctacacgctctttttctctacttcattcatattttatttctcttctccacggtctctttctctg cctacctcgctcattcttccctacttctttctttctcgcgctttattcttcactcactttttatcttttcctgtttt tcttccttactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcg tgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcg aagaggcccgcaccgatcgcccttcccaacagttgcgcag。

SEQ ID No:14 are：acgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagta aggttctattcatagttgttccgttgaaaacgatcttcatttttcaagcgtgctgggcaaatggtgcgtctgctgtg ttcactttttgcaaaagtgggatcacagctaacaccagaatatctttctttccttgccattcggttttttcttttgc catttttactacaattttttgtgattttccatttcctcattattatctcttctagagttgtgccacacacacacaca cacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacagatgagagagtga ggttttctcattactcaggactcatcacacacacacacacacacacacacacacacacacacacacacacacacaca tacacacatacaagaaagagacatattcacaattactcaggactcatcaatcgtcgacctgcaggcatgcaagcttg gcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcc ccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcg aatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtaca atctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtct gctcccggcatccgcttacagacaagctgtgaccgtctccggggagctgcatgtgtcagaggttttcaccgtcatca ccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtc。

SEQ ID No:15 are：cgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaa gttaacgaagtgaaaacaagtgttgcgagccgatgtatgcattttccccaacttgttgcaggcgttagaaacgtcga agtttgagttgaaaaataacacgctgtctcctgtaatgcccacagaacacatggatgtcttgctcgtgtgtccgcca tgatgaaatacaaagaaataacaagcaccaataaaaacctacaaacaaataaatatgtattgacaataaagatatac gagatataagtaacaaatgaatgtgaaaatcatttagggtttcaaagtatgtggactgtggagtgagtgaatgtgtg tacgtacgtacgggtatgtatgtgtgtgtgagtgtgtgtttactcaggactcatcaatcgtcgacctgcaggcatgc aagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcag cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctg aatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactct cagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacggg cttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccg tcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggt ttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttg。

SEQ ID No:16 are：cgaattcgagctcggtacccggggatcctctagagattgatgagtcctgagtaa atgcagaatccaacaatagttagctcctcattatttagaaaatgtccttttggggtgctaacggcccctataaataa gtggtcactcatgacccaacttctttttacctgtctttctacacaacatccaagatgtaagatcaggctcacagaaa gcatgcttgcatgcaccacatgctcacaaagacacctttatcctctctcaagacacacacccaaaacatgaacttgt gcaaaagaaacgtgcgctcgtgtgcactcactagcactcattcatgcatgcacacacacacacacacacacacacac ttagcatccatttcagatccacggtaatattttaggggtcctaatagctaattctttataactgagaattgcaattc ctacagtaatgtacgccatattactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactggccgtc gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctga tgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgat gccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatc cgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatgg。

SEQ ID No:17 are：tacgaattcgagctcggtacccggggatcctctagagattatgacttggataaa aaatatcaaagtattgtaaataataatggtaaaactaaaatgaataacgataattgttattggagtccgattagaaa aagttcacttggtcggaacggatgaattgatatagatgaaagcagaaaaacaggagaggaaatgatgagattaggag gtgtaaaaataaaagatgataagtccgaaagcaactaagcgaattttatgcataataaaacagaaagggagcattac tcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaa aaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatc tgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccga cacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgt ctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcc tatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcgga acccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttca ataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccctat。

SEQ ID No:18 are：attacgaattcgagctcggtacccggggatcctctagagattgatgagtcctga gtaaaagccccccacacacacacacacacgcacacactgcgaaactccataacgttttcatttcgtacacattcaca caattacaatttatttttctttcactcgcttgactctctctctctctctttctttccctctttctctctctctctct ctatttctctctctctcacacacacacatgtacacaggcatgcattctctctctctttttctctctttctctctttc acactttctcactcactcactcactcactcactcactcactcactcactcaccctctttctcttcctcagcatattg ctgctgccaccaattctacagaacgagttagtagcattagtagtcttgctgcttgttgttagtgttgagattggagt ccgtaatccacaaaacggctccagttcgcaaattccttttttcttcccttccgttagaagttacttttattagcata ttttagaaacttttataataacccatcgcttactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggca ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccc tttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaat ggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatc tgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgct cccggcatccgcttacagacaagctgtgaccgtctccc。

SEQ ID No:19 are：atgattacgaattcgagctcggtacccggggatcctctagagattgatgagtcc tgagtaacgtggggaatccaagctcatacactcggactgaggacaaaaattcctcgagccctcgcaaccgtcggttt cactgtcctctgggcgaagtcagagacgcaaataataaaaccttacccggctgatgacaggttattatacatacata catatatgtgtgtgtgtgtgtatgtgtgtgtgtgtgtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaaacgtatgcaatcgcttattatgaaagcga atattggctgcgtctattcgagtaatcacatttgtctattcaccatgacgccgttctaatcgtggggagtgaacttg cattacgtaaattgtttattactttccctcagatatgctttctgtcgtgcaaatataaggcatcactcgtctgcaga ttttactcaggactcatcaatcgtcgacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgac tgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaaga ggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctcctta cgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccag ccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgt gaccgtc。

The present invention is directed to the problem of being mentioned in background technology, and the technical scheme taken is：For Sepiella maindroni family The microsatellite marker of identification, the nucleotides sequence of the microsatellite marker are classified as SEQ ID No:1-19.The mark has highly polymorphic Property, and stability is high, is provided for the Population Genetics of Sepiella maindroni, Parentage determination and molecular mark technology Effective instrument.

Preferably, the screening technique for the microsatellite marker of Sepiella maindroni Parentage determination includes genomic DNA Extraction, the preparation of connection product, the amplification of connection product, strepavidin magnetic beads catch, the amplification of target DNA fragment, microsatellite The acquisition of mark.Method is simple, easily operated, and paramagnetic particle method substantially increases the bioaccumulation efficiency of repetitive sequence, is microsatellite mark The screening of note provides good condition.

Preferably, in the extraction process of genomic DNA, Sepiella maindroni genomic DNA is extracted, with restriction enzyme Mbol By genomic DNA at 37 DEG C digestion 3h, reclaim 400-1000bp DNA fragmentations.Under the conditions of being somebody's turn to do, 400- in digestion products The DNA fragmentation concentration of 1000bp sizes is high, improves the utilization rate of Sepiella maindroni.

Preferably, in the preparation process of connection product, by joint A (5'-GATCGTCGACGGTACCGAATTCT-3', SEQ ID No:And joint B (5'-GTCAAGAATTCGGTACCGTCGAC-3', SEQ ID No 20):21) equal proportion mixes, Continue 10min at 95 DEG C, be then connected with digestion recovery DNA fragmentation, obtain connection product.Joint is connected to Sepiella maindroni DNA fragmentation, for the DNA fragmentation provide one section known to sequence, be very easy to the amplification of connection product.

Preferably, in the amplification procedure of connection product, using connection product as template, performing PCR is entered as primer using joint B and expanded Increase, PCR amplification system is：5 10 × buffer of μ L, 4 μ L dNTP, 2U Taq polymerases, 2 μ L joints B, 2 μ L template DNAs, add Aqua sterilisa is to the μ L of final volume 50；PCR amplification conditions are：94 DEG C of pre-degeneration 2min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C Extend 30s, expand 30 circulations, last 72 DEG C of extensions 7min.Under the conditions of being somebody's turn to do, Taq polymerase activity is high, and target sequence denaturation is thorough Bottom, effective amplification amount can be reached.

Preferably, in strepavidin magnetic beads capture-process, the amplified production and biotinylated probes (CA) 12 of connection product are taken （20mmol/L）Mixing, 5min first is denatured at 95 DEG C, then hybridizes 60min at 65 DEG C, add the magnetic bead for being coated with streptomysin, mixed 30min is closed, after magnetic bead adsorbs, magnetic bead is fixed with magnetic frame, removes hybrid mixed liquid.DNA magnetic bead has been adsorbed in magnetic field Under the conditions of can assemble or disperse, avoid the flows hand-manipulated such as centrifugation, improve the automation journeys of DNA purge processes Degree.

Preferably, in the amplification procedure of elution fragment, magnetic bead is mixed with TE buffer solutions, in 95 DEG C of water-bath 5min, treated After magnetic frame fixes magnetic bead, magnet stand is separated, the supernatant containing microsatellite DNA fragment is obtained, using the supernatant as template, to connect Head B is primer, enters performing PCR amplification, and PCR amplification system is：5 10 × buffer of μ L, 4 μ L dNTP, 2U Taq polymerases, 2 μ L Joint B, 2 μ L template DNAs, add aqua sterilisa to the μ L of final volume 50；PCR amplification conditions are：94 DEG C of pre-degeneration 2min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, expands 30 circulations, last 72 DEG C of extensions 7min.Pcr amplification product is purified, is connect And be connected with pMD18-T carriers, convert DH5 α competent cells, be coated on the LB containing IPTG, X-Gal and ampicillin and put down Plate, 37 DEG C of overnight incubations, enter performing PCR amplification, detection by template of bacterium solution, picking part clone serves the survey of Hai Ying fine horses biotech firm Sequence, obtain microsatellite sequence.Sequence to be measured is cloned on carrier and is sequenced, the template amount obtained is big, and purity is high, improves sequencing Efficiency and the degree of accuracy.

Preferably, in the acquisition process of microsatellite marker, designed according to the flanking sequence at sequencing gained microsatellite both ends Primer, design of primers condition are：G/C content is 40%-60%, and annealing temperature is 48-60 DEG C, primer length 18bp-24bp, right The 5' ends of primer are modified with FAM, and Bing carries out pcr amplification reaction by template of Sepiella maindroni genomic DNA, by 5 μ L 10 × Buffer, 4 μ L dNTP, 2U Taq polymerases, 2 μ L sense primers, 2 μ L anti-sense primers, the mixing of 2 μ L template DNAs, add aqua sterilisa To the μ L of final volume 50, by mixture in 95 DEG C of pre-degeneration 5min, followed by 35 circulations, each circulation include 94 DEG C of denaturation 45s, 57 DEG C of annealing 45s, 72 DEG C of extension 1min, are placed in 72 DEG C of extension 10min again after the completion of circulation.Each PCR primer is poly- in 6% denaturation Each primer polymorphism is detected on acrylamide gel, filter out the amplification of non-specific band less, result it is stable, rich polymorphism Microsatellite marker 20, its nucleotide sequence is as shown in SEQ ID No.1 to SEQ ID No.20.Pass through the special of fluorescence labeling Property probe PCR primer is marked tracking, greatly simplifies the process of quantitative detection, and automation process is reproducible, High sensitivity, high specificity, as a result clearly.

Compared with prior art, the advantage of the invention is that：What the present invention screened is used for Sepiella maindroni Parentage determination Microsatellite marker allele number it is more, polymorphism is high, can be used for the population genetic variations of Sepiella maindroni, heredity Thremmatology assessment and paternity test etc.；Nucleotides sequence is classified as SEQ ID No:1-19 microsatellite marker be used for identify Man without Sepia andreana family, the limitation that genetic background is fuzzy, molecular labeling quantity is few in current Sepiella maindroni germplasm monitoring is compensate for, And this method efficiency high, qualification cycle are short.

Embodiment

Below by embodiment, the present invention will be further described：

Embodiment 1：

For the microsatellite marker of Sepiella maindroni Parentage determination, the nucleotides sequence of the microsatellite marker is classified as SEQ ID No: 1-19, its screening technique comprise the following steps：

1) extraction of genomic DNA：Sepiella maindroni genomic DNA is extracted, with restriction enzyme Mbol by genomic DNA at 37 DEG C Lower digestion 3h, reclaim 400-1000bp DNA fragmentations.Under the conditions of being somebody's turn to do, the DNA fragmentation of 400-1000bp sizes is dense in digestion products Degree is high, improves the utilization rate of Sepiella maindroni；

2) preparation of connection product：By joint A (5'-GATCGTCGACGGTACCGAATTCT-3') and joint B (5'- GTCAAGAATTCGGTACCGTCGAC-3') equal proportion is mixed, and makes final concentration of 25 μm of ol/L, and 10min, cooling are incubated at 95 DEG C To room temperature, then it is connected with digestion recovery DNA fragmentation at 15 DEG C, obtains connection product.Joint is connected to Sepiella maindroni DNA Fragment, for the DNA fragmentation provide one section known to sequence, be very easy to the amplification of connection product；

3) amplification of connection product：Using connection product as template, enter performing PCR amplification by primer of joint B, PCR amplification system is： 5 10 × buffer of μ L, 4 μ L dNTP, 2U Taq polymerases, 2 μ L joints B, 2 μ L template DNAs, add aqua sterilisa to the μ of final volume 50 L；PCR amplification conditions are：94 DEG C of pre-degeneration 2min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, expand 30 and follow Ring, last 72 DEG C of extensions 7min.Under the conditions of being somebody's turn to do, Taq polymerase activity is high, and target sequence denaturation is thorough, can reach effective expansion Increment；

4) strepavidin magnetic beads are caught：Take 25 μ L pcr amplification products and 5 μ L biotinylated probes (CA) 12（20mmol/L）Hybridization, Hybridization solution is 6 × SSC+0.1%SDS, and cumulative volume is 100 μ L, is first denatured 5min at 95 DEG C, then hybridizes 60min at 65 DEG C, The magnetic bead for being coated with streptomysin is added, 30min is mixed, after magnetic bead adsorbs, fixes magnetic bead with magnetic frame, remove hybrid mixed Liquid.Having adsorbed DNA magnetic bead can assemble or disperse under magnetic field condition, avoid the flows hand-manipulated such as centrifugation, carry The high automaticity of DNA purge processes；

5) amplification of fragment is eluted：Magnetic bead is mixed with TE buffer solutions, in 95 DEG C of water-bath 5min, after magnetic frame fixes magnetic bead, Magnet stand is separated, the supernatant containing microsatellite DNA fragment is obtained, using the supernatant as template, using joint B as primer, enters performing PCR Amplification, PCR amplification system are：5 10 × buffer of μ L, 4 μ L dNTP, 2U Taq polymerases, 2 μ L joints B, 2 μ L template DNAs, Add aqua sterilisa to the μ L of final volume 50；PCR amplification conditions are：94 DEG C of pre-degenerations 2min, 94 DEG C of denaturation 30s, 55 DEG C of 30s that anneal, 72 DEG C extension 30s, expand 30 circulation, it is last 72 DEG C extension 7min.Pcr amplification product is purified, is then connected with pMD18-T carriers Connect, convert DH5 α competent cells, be coated on the LB flat boards containing IPTG, X-Gal and ampicillin, 37 DEG C of overnight incubations, Enter performing PCR amplification, detection by template of bacterium solution, picking part clone serves the sequencing of Hai Ying fine horses biotech firm, obtains microsatellite sequence. Sequence to be measured is cloned on carrier and is sequenced, the template amount obtained is big, and purity is high, improves efficiency and the degree of accuracy of sequencing；

6) acquisition of microsatellite marker：Primer, design of primers condition are designed according to the flanking sequence at sequencing gained microsatellite both ends For：G/C content is 40%-60%, and annealing temperature is 48-60 DEG C, primer length 18bp-24bp, and the 5' ends of primer are repaiied with FAM Decorations, Bing using Sepiella maindroni genomic DNA be template progress pcr amplification reaction, by 5 10 × buffer of μ L, 4 μ L dNTP, 2U Taq polymerases, 2 μ L sense primers, 2 μ L anti-sense primers, the mixing of 2 μ L template DNAs, add aqua sterilisa to the μ L of final volume 50, will be mixed Compound is in 95 DEG C of pre-degeneration 5min, followed by 35 circulations, and each circulation includes 94 DEG C of denaturation 45s, 57 DEG C of 45s that anneal, 72 DEG C Extend 1min, be placed in 72 DEG C of extension 10min after the completion of circulation again.Each PCR primer is examined on 6% denaturing polyacrylamide gel Survey each primer polymorphism, filter out the amplification of non-specific band less, result is stable, the microsatellite marker 20 of rich polymorphism, Its nucleotide sequence is as shown in SEQ ID No.1 to SEQ ID No.20.By the specific probe of fluorescence labeling to PCR primer Tracking is marked, greatly simplifies the process of quantitative detection, and automation process is reproducible, high sensitivity, specificity By force, as a result clearly.

Embodiment 2：

Nucleotides sequence is classified as SEQ ID No:Application of the 1-19 microsatellite marker in Sepiella maindroni Parentage determination, including Following steps：

1) FAM marks are carried out to the 5' ends of primer designed by the step 6) of embodiment 1；

2) extract the genomic DNA of Sepiella maindroni, extracted DNA expanded with the primer in step 1), by 5 μ L 10 × Buffer, 4 μ L dNTP, 2U Taq polymerases, 2 μ L sense primers, 2 μ L anti-sense primers, the mixing of 2 μ L template DNAs, add aqua sterilisa To the μ L of final volume 50,0.1 μ L diphenyl-bromomethanes and 0.1 μ L3- methyl benzophenones are added, by mixture in 95 DEG C of pre- changes Property 5min, followed by 35 circulations, each circulation include 94 DEG C of denaturation 45s, 57 DEG C of annealing 45s, and 72 DEG C extend 1min, have circulated Cheng Houzai is placed in 72 DEG C of extension 10min.Under the conditions of being somebody's turn to do, Taq polymerase activity is high, and target sequence denaturation is thorough, can reach effective Amplification amount；Added diphenyl-bromomethane, 3- methyl benzophenones and buffer have synergy, make Taq polymerase active The site in region is mutated, and realizes the modification to enzyme gene, improves specificity, expression quantity and the enzyme of Taq polymerase Vigor, the efficiency of amplification is greatly improved, shorten the cycle of Parentage determination；

3) pcr amplification product is gone into electropherotyping in polyacrylamide denaturant gel, taken pictures after cma staining, and The genotype data of every cuttlefish is obtained using software Gel-Pro Analyzer`4.5, it is each using PopGen32 software statistics The gene frequency (P) of microsatellite marker locus, number of alleles (Na), effective number of allele (Ne), observation heterozygosis (Ho) is spent, heterozygosity (He) it is expected, breathes out moral Weinberg equilibrium P_HW, calculate polymorphism information content PIC.

Embodiment of above is merely to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this area Member, without departing from the spirit and scope of the present invention, it can also make a variety of changes and modification.Therefore, it is all equivalent Technical scheme fall within scope of the invention, scope of patent protection of the invention should be defined by the claims.

Sequence table

<110>Zhejiang Ocean university

<120>Microsatellite marker and application for Sepiella maindroni Parentage determination

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 891

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 1

attacgaatt cgagctcggt acccggggat cctctagaga ttgatgagtc ctgagtaaat 60

ctgtctgctg caaagagctt gtcaaataaa tttgttcatc tttataagtg acgtttcaat 120

tgaactggct tcgatcatca tcatcatcat catcatcatc atcatcatca tcatcatcat 180

catcatcatc atcaggattt gttcttttat tttgcgattt tgaatacaag tggttctgtt 240

ggaaactttg cagattagga tctcctgaca ggctccagaa ctgacccaat catgtgaaaa 300

ggctgatgga ttgttcggtg aagatcgtca tggatgagtt cacaaatatt ttctaaattt 360

tccggctctc tgctgatagc ttttcgactt caaccaatac atgactggcc ttgaaatgag 420

gatctctctc tcactcttcc acacacacac acacacacac acacaaatat ccagtttgtg 480

acaaagcttt atcctatgcc ttagaacttg tcaagatatt tggtaatgta acaaaagtga 540

caatgaccct ttgtacatgt agatttcata tttccttatt caaaaattgt ggatgagaaa 600

atatatatca cctgaaagca gttcccagca tacaatctgg tacaactgtg cttcatcttg 660

gaacgtccta attgcaatag aaaatgtgta ataaaaactg aaacaattac tcaggactca 720

tcaatcgtcg acctgcaggc atgcaagctt ggcactggcc gtcgttttac aacgtcgtga 780

ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 840

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc g 891

<210> 2

<211> 961

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 2

cgaattcgag ctcggtaccc ggggatcctc tagagattga tgagtcctga gtaactgcca 60

gccgggtcac agttaggcat tcaaacacca actcgacatt caacgccagt ctgaagactt 120

ggcattcaaa cgcgagtcgg gtcggcacat caaagaaagt ttggcatcgc agaagacaat 180

cagacccgga catattttcg cgaacaagtc ggcacaaaag gattctcaag tcaaagatag 240

ccacacccgt acacaccagg atctgaccaa gtcgagaacc aagtacagaa actcaaaacc 300

ggatcttcgc acacagagaa caaccgccgt tctccgggtt acacacgcct ggcatatctc 360

gtttcacaca cacacacaca cacacacaca cacacacaca cgcacacaca cacacatatg 420

tatacacaca catatctcta cttatataaa gttgctgtct ctgtcattct gtttttctgt 480

ctgtttccat tactcaggac tcatcaatcg tcgacctgca ggcatgcaag cttggcactg 540

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 600

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 660

tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 720

catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 780

gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 840

ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 900

aggttttcac cgtcatcacc gaaacgcgcg agaacgaaag ggcctcgtga tacgcctatt 960

t 961

<210> 3

<211> 950

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 3

acgaattcga gctcggtacc cggggatcct ctagagattg atgagtcctg agtaagtatg 60

cacattacgg gaatagcttg caattcttca cacacacaca cacacacaca cacacacaca 120

cacacacaca cacacactct ctctctctct ctccctctct ctctctctct ctgtgaaaca 180

cacatacagg aatacacata cgtgtatcgg cgttggttga taatgatgtt ttatttgacc 240

tgcgatatac ttttatattc gacgaatttt gcgtagcagt aaatatatac tatttacatc 300

attcgaactg ttcttcccat ctttgtcgtc atattcagaa caacgtttcg gttgttgtac 360

gctccagcct tcttcggttg gttggaatgt cgaactgaat cctttatata tctcattgaa 420

gtagtctgtt ctagttccac gtgccatgcc ttttctgggt gtcaattacc agttatctcc 480

tgttactcag gactcatcaa tcgtcgacct gcaggcatgc aagcttggca ctggccgtcg 540

ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 600

atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 660

agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 720

gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 780

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 840

cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 900

caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 950

<210> 4

<211> 909

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 4

tacgaattcg agctcggtac ccggggatcc tctagagatt gatgagtcct gagtaactca 60

caccgtcttt tgcaagtgaa cgttctcgca ttcgcgcccg caatccattc tcgaacacgc 120

agcgcaccac gtgttgtttt tttttgatgg acgcggacgc acatcatcat caacggtagt 180

cgacagttcg ttctgctttt catttccgct acaataaggc atgtttgtgc gaataacatg 240

cacgctctct ctctctctct cactcataca cactctctct ctctcactca ctcacacaca 300

cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 360

cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 420

cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 480

cacacacaca cacacacaca cacacacaat cgcagtaccc caggtcaagt gagtcagcca 540

cttactcagg actcatcact cgtcgacgtg cacgcatgcg agcgtgacac tgtgcgtcgt 600

ttacaacgtc gtgactggaa aaccgtgcgt accaacttat cgcgtgcagc acatcccttc 660

gcagctggcg tatagcgaga gccgcacgat cgccttccac agtgcgcagc gtgatgcgaa 720

tgcgctgatg cggatttctc tcacgcatct gtgcggtatt cacaccgcat atggtgcact 780

ctcagtacaa tctgctctga tgcgcataga taagccagac cagaatcagc aacaccgctg 840

acgcgccctg acgctgtctg tctgcatcgc atcgcggctg tacgttctga ctgcatgttc 900

gagttcacg 909

<210> 5

<211> 949

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 5

cgaattcgag ctcggtaccc ggggatcctc tagagattga tgagtcctga gtaaagcgga 60

taaaattata tttatgtctg tatacccctc attaggggta ttggttgatc tggtctgtgt 120

ttgtttttgt gtttttttta ttgtttgtgt gttttctgta agatacagcc acttgctcac 180

cgccgggaag tcgcctctct gtgcctcttt tataagtact gtcacggctt ctgttcggat 240

gagctccgca ctatggtccc tagcgtgtac agattcaagc gcaatactcg tctagcagcg 300

cgatctcatc aatttacagt caagctggct agatgtaacc gcagtttcta tgctaacagt 360

ttcttttccc gcacctccag attgtggaac tcctcccggc cttttgcttt cctgacggct 420

tcaacctgca aaggttcaag tgcaacgtca atcgttacct gctctcttcg taacccagta 480

tacccttcct tctgtaaccc ctagcatgca agtggcgctc ctgccttgct tggggtgact 540

gattactcag gactcatcaa tcgtcgacct gcaggcatgc aagcttggca ctggccgtcg 600

ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 660

atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 720

agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 780

gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 840

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 900

cggcatccgc ttacagacag ctgtgaccgt ctccgggagc tgcatgtgt 949

<210> 6

<211> 943

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 6

gattacgaat tcgagctcgg tacccgggga tcctctagag attgatgagt cctgagtaaa 60

cataaaaata cataaacaca caattcatct tttcgcctcc gatctttatc agatcagtga 120

gcggaatgtt ccgttgttct ttcgaaacat tatattttta tcaactcttt tccaattgtc 180

aataatttat aagtttgtcg ggatcatata cagaaattta tcacacacac acacacacac 240

acacacacac acacacacac acacacacac acacacacac acacacacac acacacacac 300

acacacacac acacacatat atataaatat atatgcaagg ctcaaatata agtaaacact 360

gtaataacat ttacggtaat gccagattac tcaggactca tcaatcgtcg acctgcaggc 420

atgcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 480

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 540

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc 600

ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta 660

caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 720

cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 780

ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 840

tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 900

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tat 943

<210> 7

<211> 934

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 7

attacgaatt cgagctcggt acccggggat cctctagaga ttgatgagtc ctgagtaaaa 60

aagtaagtca ttagaatgac actacgcagg tgacttttga atctgtgatt tacttgcaat 120

ttctcctcga aagatattga catgcgcatc aagatacaca agcaagtgaa cagagctgaa 180

attcgtctac accctcacac acacacacac acacacacat acacacacat cagtcggaaa 240

actgtgtgtt gcatacccgt ctctagatgt acgctcttct gtagaggggg catcttcttc 300

tagagggggg ctggcaggac ttctgctacg tacttcatct ctctgactca ccaggccgga 360

catctctctt ttttatgcac agggaatatc acttttctgt tctgatgttc tgatgtccga 420

ccagtagttg agacctgttg cctcggccgt cttcttcata actccgcatg ttgccttatc 480

atgcagtatc tccatcgtct cgtgatgacc ccttcttcgg tgcatctttg gtccaataaa 540

tgatgaccaa cacaatattc tatctacctt actcaggact catcaatcgt cgacctgcag 600

gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 660

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 720

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat 780

gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 840

tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 900

cgcgccctga cgggcttgtc tgctcccggc atcc 934

<210> 8

<211> 909

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 8

tgattacgaa ttcgagctcg gtacccgggg atcctctaga gattgatgag tcctgagtaa 60

tcagatttgc cccttttgaa tccatgtaca tatgtttgta aatacaatac gagaattatg 120

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg taagcgtgtg cgtgcgtttc 180

cgttcctatc tttttatcct ttttcaatta gttcactttc ttatttccat ctttctctct 240

ctctctttct ctctctatct ccctccccct ctctctctct ctcaatccct ccctgtatct 300

ctctttcggt tctcaactct tttcctatct cagttattcc ccaccttttt ctttctttca 360

gttcctattt atttttattt ttcagtttct tcgcttccaa atttctctgt ccttcattat 420

ctctccccct ttatttctct ttctctctaa ctctcaacct ctctatctat ttgtctatca 480

atccttactc aggactcatc aatcgtcgac ctgcaggcat gcaagcttgg cactggccgt 540

cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 600

acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 660

acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct 720

gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata 780

gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 840

cccgggatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 900

ttcaccgtc 909

<210> 9

<211> 900

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 9

gattcgaatt cgagctcggt acccggggat cctctagaga ttgatgagtc ctgagtaaca 60

taaaattatt tttgactgtt cagtatctct cctcaagcct gttctgcaaa catttacaca 120

aattcattgg gtatataatt cagcactgtc ctgagaaaga cacacacaca cacacacaca 180

cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 240

cactcacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 300

cacacacaca cacacacaca tgcacacaca cacacacaca cacacacaca cacacacacc 360

ttcccacaca cacacactct cacacacacg cacataccaa tgggagttcc cgacactttc 420

cccaacactc acagatccgg cgctgcatgc gtgatttttt gcttgtggta tctttttaat 480

accactcgac tgggaaagca ctggcgttac agtctgttat ggtgctgcat aatttcccca 540

tttccgaggt ggcgtaatag cgaagaggcg cgcactgatc gtgtttctcc tcacgtgatc 600

agcttgtagg gggaatgtga ccagatgcgg tattttctcg tttcacatgt gtgcggcgtt 660

ttcaacgaca catggtgcac tatcagaaca ctctgtactg atgtcttgaa ggataaatgt 720

caccgacaca cgggaatcct cgatgtcacg cggtgttttg cgtgtgagcg cgcgcggccc 780

ccatatagtg catattgtga ccgacacgtg gagatgtgtg tgcgatagtg atacaacgac 840

ctctcacgaa aagctcgaga gtatagggcg acgtgagagt atctatattt ttaggtttct 900

<210> 10

<211> 943

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 10

attacgaatt cgagctcggt acccggggat cctctagaga ttgatgagtc ctgagtaaca 60

cccacgagtc aggatatggc acatggccgg tttatatgtt gggaaattca ggcaaaaata 120

atccgtatcc ccgcatacac ggagtcggac ttgtctacca ccccagtcga gactctaccc 180

ttcggcacat cctcgactgg attcctccct gcccgaatgc aaggctgtcg caagctatgc 240

agcgaactgc ctgacttgac aacagcctaa gatctacgaa tcaaacaagt agttactaaa 300

gccacgactt gtaatgctaa cgatcggaaa ttttatagtt ggggtggctg tcgtctgtta 360

taatgtgtgt ttgttgattt tgggcgtata tattcataat ctgtttcttt tgttacgtag 420

atggcgtcta ttccgtttga ctgtattccc ttccttccaa aagcagtagg aatacataaa 480

atacgtacga acacacacgc acacacacac gcacacacac acacacatac tcagttttat 540

ggctatagat actttgaaaa aaaacatagg tcatgatctt ccactgacta taagtaatat 600

tcaggtttga gaaaaatcca tcgcaaacgg taggaatgca taaaatatat actaacacac 660

acacacgcac acacacatac acacacacac acatactcag tttactcagg actcatcaat 720

cgtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg 780

aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 840

gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 900

aatggcgcct gatgcggtat tttctcctta cgcatctgtg cgg 943

<210> 11

<211> 946

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 11

acgaattcga gctcggtacc cggggatcct ctagagattg atgagtcctg agtaacaaaa 60

tgtggaaaca gtattacaat gagtataatg ttcttcaagt ctggtactta gatacatgtg 120

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtaaccc gatatgtcta tcagtctagt 180

gcggcattct ctcgttctgg tcagctccgc gtccgtggct gtctttgttg taccgactat 240

cctttactca ggactcatca atcgtcgacc tgcaggcatg caagcttggc actggccgtc 300

gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 360

catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 420

cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 480

tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 540

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 600

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 660

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 720

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 780

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 840

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 900

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctg 946

<210> 12

<211> 960

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 12

tacgaattcg agctcggtac ccggggatcc tctagagatt gatgagtcct gagtaatatc 60

agaaagaggc aactgaagtc tctgggacat attatgagaa aggagggtct ggaaaacctt 120

atactcacag gacggataga gggaggtaga gacaggggaa aacaccgcac gacatacttg 180

aagggcctga gcgaatggat gacgggaagg ggcatgggag atacggggag tcaagccctg 240

ttgagagctt cgatagatag ggggaagtgg aaagccatga tagcctacgt cctgaaggga 300

tacggcacat agaagaagaa tgtttcgatg atgaaggtct ctttaggcgg aattggcaaa 360

cattatccca acactttatt tatacgcttg acaaaaggaa ataaaaataa aacaaaaaac 420

aaatcaaaat aaagacgaaa acaataacaa caacaacaac cactaacatg gcactaaagg 480

cgggggtcta gaatttatgg tgtggttact caggactcat caatcgtcga cctgcaggca 540

tgcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 600

ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 660

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 720

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 780

aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 840

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 900

gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggtcc 960

<210> 13

<211> 960

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 13

tacgaattcg agctcggtac ccggggatcc tctagagatt gatgagtcct gagtaatatc 60

agaaagaggc aactgaagtc tctgggacat attatgagaa aggagggtct ggaaaacctt 120

atactcacag gacggataga gggaggtaga gacaggggaa aacaccgcac gacatacttg 180

aagggcctga gcgaatggat gacgggaagg ggcatgggag atacggggag tcaagccctg 240

ttgagagctt cgatagatag ggggaagtgg aaagccatga tagcctacgt cctgaaggga 300

tacggcacat agaagaagaa tgtttcgatg atgaaggtct ctttaggcgg aattggcaaa 360

cattatccca acactttatt tatacgcttg acaaaaggaa ataaaaataa aacaaaaaac 420

aaatcaaaat aaagacgaaa acaataacaa caacaacaac cactaacatg gcactaaagg 480

cgggggtcta gaatttatgg tgtggttact caggactcat caatcgtcga cctgcaggca 540

tgcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 600

ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 660

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 720

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 780

aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 840

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 900

gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggtcc 960

<210> 14

<211> 958

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 14

acgaattcga gctcggtacc cggggatcct ctagagattg atgagtcctg agtaaggttc 60

tattcatagt tgttccgttg aaaacgatct tcatttttca agcgtgctgg gcaaatggtg 120

cgtctgctgt gttcactttt tgcaaaagtg ggatcacagc taacaccaga atatctttct 180

ttccttgcca ttcggttttt tcttttgcca tttttactac aattttttgt gattttccat 240

ttcctcatta ttatctcttc tagagttgtg ccacacacac acacacacac acacacacac 300

acacacacac acacacacac acacacacac acacacacac acacacacag atgagagagt 360

gaggttttct cattactcag gactcatcac acacacacac acacacacac acacacacac 420

acacacacac acacacacat acacacatac aagaaagaga catattcaca attactcagg 480

actcatcaat cgtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt 540

cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 600

gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 660

ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 720

caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 780

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 840

tacagacaag ctgtgaccgt ctccggggag ctgcatgtgt cagaggtttt caccgtcatc 900

accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtc 958

<210> 15

<211> 955

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 15

cgaattcgag ctcggtaccc ggggatcctc tagagattga tgagtcctga gtaagttaac 60

gaagtgaaaa caagtgttgc gagccgatgt atgcattttc cccaacttgt tgcaggcgtt 120

agaaacgtcg aagtttgagt tgaaaaataa cacgctgtct cctgtaatgc ccacagaaca 180

catggatgtc ttgctcgtgt gtccgccatg atgaaataca aagaaataac aagcaccaat 240

aaaaacctac aaacaaataa atatgtattg acaataaaga tatacgagat ataagtaaca 300

aatgaatgtg aaaatcattt agggtttcaa agtatgtgga ctgtggagtg agtgaatgtg 360

tgtacgtacg tacgggtatg tatgtgtgtg tgagtgtgtg tttactcagg actcatcaat 420

cgtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg 480

aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 540

gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 600

aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 660

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 720

caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 780

ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 840

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 900

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttg 955

<210> 16

<211> 958

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 16

cgaattcgag ctcggtaccc ggggatcctc tagagattga tgagtcctga gtaaatgcag 60

aatccaacaa tagttagctc ctcattattt agaaaatgtc cttttggggt gctaacggcc 120

cctataaata agtggtcact catgacccaa cttcttttta cctgtctttc tacacaacat 180

ccaagatgta agatcaggct cacagaaagc atgcttgcat gcaccacatg ctcacaaaga 240

cacctttatc ctctctcaag acacacaccc aaaacatgaa cttgtgcaaa agaaacgtgc 300

gctcgtgtgc actcactagc actcattcat gcatgcacac acacacacac acacacacac 360

acttagcatc catttcagat ccacggtaat attttagggg tcctaatagc taattcttta 420

taactgagaa ttgcaattcc tacagtaatg tacgccatat tactcaggac tcatcaatcg 480

tcgacctgca ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa 540

aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 600

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 660

tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 720

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 780

acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 840

gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 900

agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatgg 958

<210> 17

<211> 954

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 17

tacgaattcg agctcggtac ccggggatcc tctagagatt atgacttgga taaaaaatat 60

caaagtattg taaataataa tggtaaaact aaaatgaata acgataattg ttattggagt 120

ccgattagaa aaagttcact tggtcggaac ggatgaattg atatagatga aagcagaaaa 180

acaggagagg aaatgatgag attaggaggt gtaaaaataa aagatgataa gtccgaaagc 240

aactaagcga attttatgca taataaaaca gaaagggagc attactcagg actcatcaat 300

cgtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg 360

aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 420

gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 480

aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 540

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 600

caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 660

ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 720

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 780

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 840

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 900

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc ctat 954

<210> 18

<211> 939

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 18

attacgaatt cgagctcggt acccggggat cctctagaga ttgatgagtc ctgagtaaaa 60

gccccccaca cacacacaca cacgcacaca ctgcgaaact ccataacgtt ttcatttcgt 120

acacattcac acaattacaa tttatttttc tttcactcgc ttgactctct ctctctctct 180

ttctttccct ctttctctct ctctctctct atttctctct ctctcacaca cacacatgta 240

cacaggcatg cattctctct ctctttttct ctctttctct ctttcacact ttctcactca 300

ctcactcact cactcactca ctcactcact cactcaccct ctttctcttc ctcagcatat 360

tgctgctgcc accaattcta cagaacgagt tagtagcatt agtagtcttg ctgcttgttg 420

ttagtgttga gattggagtc cgtaatccac aaaacggctc cagttcgcaa attccttttt 480

tcttcccttc cgttagaagt tacttttatt agcatatttt agaaactttt ataataaccc 540

atcgcttact caggactcat caatcgtcga cctgcaggca tgcaagcttg gcactggccg 600

tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 660

cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 720

aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 780

tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 840

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 900

tcccggcatc cgcttacaga caagctgtga ccgtctccc 939

<210> 19

<211> 908

<212> DNA

<213>Sepiella maindroni (Sepiella maindroni de Rochebrune)

<400> 19

atgattacga attcgagctc ggtacccggg gatcctctag agattgatga gtcctgagta 60

acgtggggaa tccaagctca tacactcgga ctgaggacaa aaattcctcg agccctcgca 120

accgtcggtt tcactgtcct ctgggcgaag tcagagacgc aaataataaa accttacccg 180

gctgatgaca ggttattata catacataca tatatgtgtg tgtgtgtgta tgtgtgtgtg 240

tgtgtatgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 300

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg taaacgtatg caatcgctta ttatgaaagc 360

gaatattggc tgcgtctatt cgagtaatca catttgtcta ttcaccatga cgccgttcta 420

atcgtgggga gtgaacttgc attacgtaaa ttgtttatta ctttccctca gatatgcttt 480

ctgtcgtgca aatataaggc atcactcgtc tgcagatttt actcaggact catcaatcgt 540

cgacctgcag gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa 600

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 660

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 720

ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 780

gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 840

cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 900

tgaccgtc 908

<210> 20

<211> 23

<212> DNA

<213>Artificial synthesized (Saccharum)

<400> 20

gatcgtcgac ggtaccgaat tct 23

<210> 21

<211> 23

<212> DNA

<213>Artificial synthesized (Saccharum)

<400> 21

gtcaagaatt cggtaccgtc gac 23

Claims (7)

1. the microsatellite marker for Sepiella maindroni Parentage determination, it is characterised in that：The nucleotides of the microsatellite marker Sequence is SEQ ID No:1-19.

2. the microsatellite marker according to claim 1 for Sepiella maindroni Parentage determination, it is characterised in that：It is described The screening technique of microsatellite marker comprises the following steps：

1) extraction of genomic DNA：Sepiella maindroni genomic DNA is extracted, with restriction enzyme Mbol by genomic DNA digestion, Reclaim 400-1000bp DNA fragmentations；

2) preparation of connection product：By joint A (5'-GATCGTCGACGGTACCGAATTCT-3') and joint B (5'- GTCAAGAATTCGGTACCGTCGAC-3') equal proportion mixes, and is then connected with digestion recovery DNA fragmentation, obtains connection product；

3) amplification of connection product：Using connection product as template, enter performing PCR amplification by primer of joint B；

4) strepavidin magnetic beads are caught：Take the amplified production and biotinylated probes (CA) 12 of connection product（20mmol/L）Hybridization, adds Enter to be coated with the magnetic bead mixing of streptomysin, remove hybrid mixed liquid；

5) elution of DNA fragmentation：Magnetic bead is added to TE buffer solutions, 5min is incubated at 95 DEG C, separates magnetic bead, obtain defending containing micro- The supernatant of star DNA fragmentation；

6) amplification of fragment is eluted：Using joint B as primer, using the supernatant containing microsatellite DNA fragment as template, enter performing PCR Amplification, purify pcr amplification product, then be connected with pMD18-T carriers, conversion DH5 α competent cells, be coated on containing IPTG, X-Gal and ampicillin LB flat boards, 37 DEG C of overnight incubations, enter performing PCR amplification by template of bacterium solution, detect, sequencing；

7) acquisition of microsatellite marker：Primer is designed according to the flanking sequence at sequencing gained microsatellite both ends, to the 5' ends of primer Modified with FAM, Bing carries out pcr amplification reaction by template of Sepiella maindroni genomic DNA, by each PCR primer in 6% denaturation Each primer polymorphism is detected on polyacrylamide gel, filter out the amplification of non-specific band less, result is stable, rich polymorphism Microsatellite marker 20, its nucleotide sequence is as shown in SEQ ID No.1 to SEQ ID No.20.

3. the microsatellite marker according to claim 2 for Sepiella maindroni Parentage determination, it is characterised in that：It is described Digestion temperature is 37 DEG C in step 1), and the digestion time is 3h.

4. the microsatellite marker according to claim 2 for Sepiella maindroni Parentage determination, it is characterised in that：It is described Mixing temperature is 95 DEG C in step 2), incorporation time 10min.

5. the microsatellite marker according to claim 2 for Sepiella maindroni Parentage determination, it is characterised in that：It is described Hybridization conditions are in step 4)：5min first is denatured at 95 DEG C, then hybridizes 60min at 65 DEG C.

6. the microsatellite marker according to claim 2 for Sepiella maindroni Parentage determination, it is characterised in that：It is described Design of primers condition is in step 7)：G/C content is 40%-60%, and annealing temperature is 48-60 DEG C, primer length 18bp-24bp.

7. nucleotides sequence is classified as SEQ ID No:Application of the 1-19 microsatellite marker in Sepiella maindroni Parentage determination, It is characterized in that nucleotides sequence is classified as SEQ ID No:1-19 microsatellite marker is used to identify in Sepiella maindroni family Purposes.