A genetic marker for individual identification of otters and its application

Abstract

The present invention relates to a genetic marker for individual otter identification and its application, and relates to the field of biotechnology. The genetic marker includes at least 60 of the SNP sites 1-92. The genetic marker has rich polymorphic information content, can effectively identify individual otters, and is applied to kinship analysis and genetic diversity analysis based on this.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a genetic marker for individual otter identification and application thereof. Background Art

[0002] Otters belong to the order Carnivora, family Mustelidae, subfamily Lunae, with a total of 7 genera and 13 species, which are widely distributed in all lands except Antarctica and Oceania. Otters are top predators in many freshwater ecosystems and have very important ecological functions. They can be used as health indicator species and flagship species for freshwater ecosystems. There are three species of otters distributed in my country, namely the Eurasian otter (Lutralutra), the Asian small-clawed otter (Aonyx cinerea) and the smooth-coated otter (Lutrogale perspicillata). Historically, the Eurasian otter was widely distributed in my country, found in most provinces and regions in the southeast and central regions, and also in the Northeast and the Altai region of Xinjiang; the Asian small-clawed otter is distributed in southern provinces in my country, including Fujian, Guangdong, Guangxi, Guizhou, Yunnan, and southeastern Tibet; the smooth-coated otter is only distributed in southern Yunnan and the western border in my country, and may also be distributed in small quantities in parts of Guizhou and the Pearl River Estuary in Guangdong. In the IUCN Red List, the Eurasian otter is listed as Near Threatened (NT), while the Asian small-clawed otter and smooth-coated otter are Vulnerable (VU). In my country, the three otter species are listed as Class II national key protected wild animals.

[0003] Modern zoos are important institutions for the ex-situ conservation of wild animals, scientific research related to wild animals, and public education on the protection of wild animals. Their mission is no longer limited to traditional animal exhibitions, but they are important research bases for the preservation and breeding of animals, and are important places for biodiversity conservation. In recent years, domestic zoos have increasingly attached importance to the scientific management of captive populations of endangered species, and have carried out a series of studies on the pedigree, genetic diversity and breeding of endangered species. In particular, the identification and determination of individual identification and kinship have provided an important scientific basis for the management of captive animal populations on the one hand, and have also played a great role in promoting the protection of wild populations on the other.

[0004] my country has not yet established a sound pedigree registration system for captive Asian small-clawed otters, which cannot effectively avoid inbreeding and scientific research, thereby optimizing the population structure and promoting the sustainable development of captive Asian small-clawed otters. Therefore, it is urgent to establish a method for analyzing the kinship of individual Asian small-clawed otters. Summary of the invention

[0005] In view of the above problems, the present invention provides a genetic marker for identifying individual otters. The genetic marker has rich polymorphic information content, can effectively identify individual otters, and can be used as a basis for kinship analysis and genetic diversity analysis.

[0006] In order to achieve the above object, the present invention provides a genetic marker for individual otter identification, the genetic marker comprising at least 60 of the following SNP sites:

[0007] The SNP site 1 is located at otter chromosome NC_062278.1:29282801, and its base is C or T;

[0008] The SNP site 2 is located at otter chromosome NC_062278.1:53653392, and its base is A or C;

[0009] The SNP site 3 is located at otter chromosome NC_062278.1:77420684, and its base is A or C;

[0010] The SNP site 4 is located on the otter chromosome NC_062278.1:103766766, and its base is C or A;

[0011] The SNP site 5 is located at otter chromosome NC_062278.1:126700894, and its base is T or C;

[0012] The SNP site 6 is located on the otter chromosome NC_062278.1:151297453, and its base is G or A;

[0013] The SNP site 7 is located on the otter chromosome NC_062278.1:175880997, and its base is A or G;

[0014] The SNP site 8 is located on the otter chromosome NC_062278.1:200937948, and its base is T or C;

[0015] The SNP site 9 is located on the otter chromosome NC_062278.1:220353075, and its base is T or G;

[0016] The SNP site 10 is located at otter chromosome NC_062279.1:14048920, and its base is T or C;

[0017] The SNP site 11 is located at otter chromosome NC_062279.1:37515158, and its base is A or G;

[0018] The SNP site 12 is located at otter chromosome NC_062279.1:60498995, and its base is C or T;

[0019] The SNP site 13 is located at otter chromosome NC_062279.1:84719735, and its base is T or C;

[0020] The SNP site 14 is located at otter chromosome NC_062279.1:110340947, and its base is C or T;

[0021] The SNP site 15 is located on the otter chromosome NC_062279.1:132047633, and its base is G or A;

[0022] The SNP site 16 is located at otter chromosome NC_062279.1:154490011, and its base is T or C;

[0023] The SNP site 17 is located on the otter chromosome NC_062279.1:205590804, and its base is G or A;

[0024] The SNP site 18 is located on the otter chromosome NC_062280.1:12550419, and its base is G or A;

[0025] The SNP site 19 is located on the otter chromosome NC_062280.1:39563061, and its base is A or G;

[0026] The SNP site 20 is located at otter chromosome NC_062280.1:62585158, and its base is A or G;

[0027] The SNP site 21 is located on the otter chromosome NC_062280.1: 87980335, and its base is T or C; the SNP site 22 is located on the otter chromosome NC_062280.1: 112727804, and its base is C or T; the SNP site 23 is located on the otter chromosome NC_062280.1: 134132923, and its base is A or G; the SNP site 24 is located on the otter chromosome NC_062280.1: 161530021, and its base is A or G; the SNP site 25 is located on the otter chromosome NC_062280.1: 192311942, and its base is C or T; the SNP site 26 is located on the otter chromosome NC_062281.1: 160245, and its base is G or T;

[0028] The SNP site 27 is located on the otter chromosome NC_062281.1: 21673124, and its base is G or A; the SNP site 28 is located on the otter chromosome NC_062281.1: 68759014, and its base is G or A; the SNP site 29 is located on the otter chromosome NC_062281.1: 90290431, and its base is A or G; the SNP site 30 is located on the otter chromosome NC_062281.1: 111500221, and its base is T or A; the SNP site 31 is located on the otter chromosome NC_062281.1: 132448525, and its base is T or C; the SNP site 32 is located on the otter chromosome NC _062281.1:153524347, its base is G or A; the SNP site 33 is located on the otter chromosome NC_062281.1:176560613, its base is G or A; the SNP site 34 is located on the otter chromosome NC_062281.1:197534750, its base is T or C; the SNP site 35 is located on the otter chromosome NC_062282.1:11145513, its base is C or T; the SNP site 36 is located on the otter chromosome NC_062282.1:61192832, its base is C or T; the SNP site 37 is located on the otter chromosome NC_062282.1:8419473 2, its base is T or C; the SNP site 38 is located on the otter chromosome NC_062282.1: 110340995, its base is A or G; the SNP site 39 is located on the otter chromosome NC_062282.1: 135662808, its base is C or T; the SNP site 40 is located on the otter chromosome NC_062283.1: 14060791, its base is C or G; the SNP site 41 is located on the otter chromosome NC_062283.1: 38100696, its base is C or T; the SNP site 42 is located on the otter chromosome NC_062283.1: 63833421, its base is C or T; the SNP site 4 3 is located on the otter chromosome NC_062283.1: 85276146, and its base is A or G; the SNP site 44 is located on the otter chromosome NC_062283.1: 107922003, and its base is T or C; the SNP site 45 is located on the otter chromosome NC_062283.1: 130579430, and its base is A or C; the SNP site 46 is located on the otter chromosome NC_062283.1: 152222422, and its base is T or C; the SNP site 47 is located on the otter chromosome NC_062284.1: 16690938, and its base is C or T; the SNP site 48 is located on the otter chromosome NC_062284.1: 16690938, and its base is C or T; the SNP site 49 is located on the otter chromosome NC_062284.1:39210100, its base is A or G; the SNP site 49 is located on the otter chromosome NC_062284.1:60640637, its base is C or T; the SNP site 50 is located on the otter chromosome NC_062284.1:81413981, its base is A or G; the SNP site 51 is located on the otter chromosome NC_062284.1:105593937, its base is G or A; the SNP site 52 is located on the otter chromosome NC_062284.1:129846973, its base is G or C; the SNP site 53 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 54 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 55 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 56 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 57 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 58 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 59 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 51 is located on the otter chromosome NC_062285.1:1054989, its Chromosome NC_062285.1: 24679449, the base is T or A; the SNP site 55 is located on the otter chromosome NC_062285.1: 50269007, the base is T or C; the SNP site 56 is located on the otter chromosome NC_062285.1: 76743726, the base is A or C; the SNP site 57 is located on the otter chromosome NC_062285.1: 102629337, the base is G or A; the SNP site 58 is located on the otter chromosome NC_062285.1: 126781695, the base is C or T; the SNP site 59 is located on the otter chromosome NC_062292.1: 642481, the base is C or T;.

[0029] The SNP site 60 is located on the otter chromosome NC_062292.1: 49172871, and its base is T or A; the SNP site 61 is located on the otter chromosome NC_062292.1: 73453848, and its base is C or T; the SNP site 62 is located on the otter chromosome NC_062292.1: 99375940, and its base is G or A; the SNP site 63 is located on the otter chromosome NC_062292.1: 125797454, and its base is A or G; the SNP site 64 is located on the otter chromosome NC_062287.1: 432333, and its base is G or A;

[0030] The SNP site 65 is located on the otter chromosome NC_062287.1: 25122190, and its base is C or T; the SNP site 66 is located on the otter chromosome NC_062287.1: 52214063, and its base is C or T; the SNP site 67 is located on the otter chromosome NC_062287.1: 79882440, and its base is A or T;

[0031] The SNP site 68 is located at otter chromosome NC_062287.1:106762161, and its base is C or T;

[0032] The SNP site 69 is located at otter chromosome NC_062288.1:14894220, and its base is C or T;

[0033] The SNP site 70 is located at otter chromosome NC_062288.1:45078342, and its base is A or T;

[0034] The SNP site 71 is located on the otter chromosome NC_062288.1:79929504, and its base is G or A;

[0035] The SNP site 72 is located at otter chromosome NC_062288.1:103690730, and its base is C or T;

[0036] The SNP site 73 is located at otter chromosome NC_062289.1:15755696, and its base is C or T;

[0037] The SNP site 74 is located at otter chromosome NC_062289.1:41188592, and its base is C or T;

[0038] The SNP site 75 is located on the otter chromosome NC_062289.1:64790204, and its base is G or A;

[0039] The SNP site 76 is located on the otter chromosome NC_062289.1:87777063, and its base is A or G;

[0040] The SNP site 77 is located at otter chromosome NC_062290.1:11396992, and its base is G or A;

[0041] The SNP site 78 is located at otter chromosome NC_062290.1:33757642, and its base is T or C;

[0042] The SNP site 79 is located at otter chromosome NC_062290.1:58042763, and its base is A or C;

[0043] The SNP site 80 is located on the otter chromosome NC_062290.1:82046565, and its base is G or A;

[0044] The SNP site 81 is located at otter chromosome NC_062291.1:35320951, and its base is C or T;

[0045] The SNP site 82 is located on the otter chromosome NC_062291.1:58881100, and its base is G or A;

[0046] The SNP site 83 is located at otter chromosome NC_062291.1:88842652, and its base is C or T;

[0047] The SNP site 84 is located at otter chromosome NC_062292.1:12616147, and its base is T or C;

[0048] The SNP site 85 is located on the otter chromosome NC_062292.1:65508548, and its base is G or A;

[0049] The SNP site 86 is located at otter chromosome NC_062293.1:17189832, and its base is A or C;

[0050] The SNP site 87 is located on the otter chromosome NC_062294.1:312132, and its base is G or A;

[0051] The SNP site 88 is located at otter chromosome NC_062294.1:35873144, and its base is G or A;

[0052] The SNP site 89 is located on the otter chromosome NC_062294.1:60056374, and its base is G or C;

[0053] The SNP site 90 is located at otter chromosome NC_062296.1:1621281, and its base is C or T;

[0054] The SNP site 91 is located at otter chromosome NC_062296.1:38485632, and its base is T or C;

[0055] Or the SNP site 92 is located at otter chromosome NC_062296.1:82234974, and its base is T or A;

[0056] The reference genome of the SNP site is the Asian small-clawed otter reference genome Aonyx_cinereus_HiC.fasta.gz.

[0057] Single nucleotide polymorphism (SNP) genetic markers are the third generation of genetic markers, which have attracted the attention of researchers for their advantages such as low mutation rate, stable inheritance, short amplicon and high universality, and are particularly suitable for severely degraded, highly corrupted and old samples. The inventors proposed the above 92 SNPs sites to form genetic markers, which have rich polymorphic information content and can effectively identify individual otters, and are used on this basis for kinship analysis and genetic diversity analysis.

[0058] In one embodiment, the genetic markers include 92 of the following SNP sites:

[0059] The SNP site 1 is located at otter chromosome NC_062278.1:29282801, and its base is C or T;

[0060] The SNP site 2 is located at otter chromosome NC_062278.1:53653392, and its base is A or C;

[0061] The SNP site 3 is located at otter chromosome NC_062278.1:77420684, and its base is A or C;

[0062] The SNP site 4 is located on the otter chromosome NC_062278.1:103766766, and its base is C or A;

[0063] The SNP site 5 is located at otter chromosome NC_062278.1:126700894, and its base is T or C;

[0064] The SNP site 6 is located on the otter chromosome NC_062278.1:151297453, and its base is G or A;

[0065] The SNP site 7 is located on the otter chromosome NC_062278.1:175880997, and its base is A or G;

[0066] The SNP site 8 is located on the otter chromosome NC_062278.1:200937948, and its base is T or C;

[0067] The SNP site 9 is located on the otter chromosome NC_062278.1:220353075, and its base is T or G;

[0068] The SNP site 10 is located at otter chromosome NC_062279.1:14048920, and its base is T or C;

[0069] The SNP site 11 is located at otter chromosome NC_062279.1:37515158, and its base is A or G;

[0070] The SNP site 12 is located at otter chromosome NC_062279.1:60498995, and its base is C or T;

[0071] The SNP site 13 is located at otter chromosome NC_062279.1:84719735, and its base is T or C;

[0072] The SNP site 14 is located at otter chromosome NC_062279.1:110340947, and its base is C or T;

[0073] The SNP site 15 is located on the otter chromosome NC_062279.1:132047633, and its base is G or A;

[0074] The SNP site 16 is located on the otter chromosome NC_062279.1: 154490011, and its base is T or C; the SNP site 17 is located on the otter chromosome NC_062279.1: 205590804, and its base is G or A; the SNP site 18 is located on the otter chromosome NC_062280.1: 12550419, and its base is G or A; the SNP site 19 is located on the otter chromosome NC_062280.1: 39563061, and its base is A or G; the SNP site 20 is located on the otter chromosome NC_062280.1: 62585158, and its base is A or G; the SNP site 21 is located on the otter chromosome NC_062 280.1:87980335, the base is T or C; the SNP site 22 is located on the otter chromosome NC_062280.1:112727804, the base is C or T; the SNP site 23 is located on the otter chromosome NC_062280.1:134132923, the base is A or G; the SNP site 24 is located on the otter chromosome NC_062280.1:161530021, the base is A or G; the SNP site 25 is located on the otter chromosome NC_062280.1:192311942, the base is C or T; the SNP site 26 is located on the otter chromosome NC_062281.1:160245, the base is G or T;

[0075] The SNP site 27 is located on the otter chromosome NC_062281.1: 21673124, and its base is G or A; the SNP site 28 is located on the otter chromosome NC_062281.1: 68759014, and its base is G or A; the SNP site 29 is located on the otter chromosome NC_062281.1: 90290431, and its base is A or G; the SNP site 30 is located on the otter chromosome NC_062281.1: 111500221, and its base is T or A; the SNP site 31 is located on the otter chromosome NC_062281.1: 132448525, and its base is T or C; the SNP site 32 is located on the otter chromosome NC _062281.1:153524347, its base is G or A; the SNP site 33 is located on the otter chromosome NC_062281.1:176560613, its base is G or A; the SNP site 34 is located on the otter chromosome NC_062281.1:197534750, its base is T or C; the SNP site 35 is located on the otter chromosome NC_062282.1:11145513, its base is C or T; the SNP site 36 is located on the otter chromosome NC_062282.1:61192832, its base is C or T; the SNP site 37 is located on the otter chromosome NC_062282.1:8419473 2, its base is T or C; the SNP site 38 is located on the otter chromosome NC_062282.1: 110340995, its base is A or G; the SNP site 39 is located on the otter chromosome NC_062282.1: 135662808, its base is C or T; the SNP site 40 is located on the otter chromosome NC_062283.1: 14060791, its base is C or G; the SNP site 41 is located on the otter chromosome NC_062283.1: 38100696, its base is C or T; the SNP site 42 is located on the otter chromosome NC_062283.1: 63833421, its base is C or T; the SNP site 4 3 is located on the otter chromosome NC_062283.1: 85276146, and its base is A or G; the SNP site 44 is located on the otter chromosome NC_062283.1: 107922003, and its base is T or C; the SNP site 45 is located on the otter chromosome NC_062283.1: 130579430, and its base is A or C; the SNP site 46 is located on the otter chromosome NC_062283.1: 152222422, and its base is T or C; the SNP site 47 is located on the otter chromosome NC_062284.1: 16690938, and its base is C or T; the SNP site 48 is located on the otter chromosome NC_062284.1: 16690938, and its base is C or T; the SNP site 49 is located on the otter chromosome NC_062284.1:39210100, its base is A or G; the SNP site 49 is located on the otter chromosome NC_062284.1:60640637, its base is C or T; the SNP site 50 is located on the otter chromosome NC_062284.1:81413981, its base is A or G; the SNP site 51 is located on the otter chromosome NC_062284.1:105593937, its base is G or A; the SNP site 52 is located on the otter chromosome NC_062284.1:129846973, its base is G or C; the SNP site 53 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 54 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 55 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 56 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 57 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 58 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 59 is located on the otter chromosome NC_062285.1:1054989, its base is C or T; the SNP site 51 is located on the otter chromosome NC_062285.1:1054989, its Chromosome NC_062285.1: 24679449, the base is T or A; the SNP site 55 is located on the otter chromosome NC_062285.1: 50269007, the base is T or C; the SNP site 56 is located on the otter chromosome NC_062285.1: 76743726, the base is A or C; the SNP site 57 is located on the otter chromosome NC_062285.1: 102629337, the base is G or A; the SNP site 58 is located on the otter chromosome NC_062285.1: 126781695, the base is C or T; the SNP site 59 is located on the otter chromosome NC_062292.1: 642481, the base is C or T;.

[0076] The SNP site 60 is located on the otter chromosome NC_062292.1: 49172871, and its base is T or A; the SNP site 61 is located on the otter chromosome NC_062292.1: 73453848, and its base is C or T; the SNP site 62 is located on the otter chromosome NC_062292.1: 99375940, and its base is G or A;

[0077] The SNP site 63 is located on the otter chromosome NC_062292.1:125797454, and its base is A or G;

[0078] The SNP site 64 is located on the otter chromosome NC_062287.1:432333, and its base is G or A;

[0079] The SNP site 65 is located at otter chromosome NC_062287.1:25122190, and its base is C or T;

[0080] The SNP site 66 is located at otter chromosome NC_062287.1:52214063, and its base is C or T;

[0081] The SNP site 67 is located at otter chromosome NC_062287.1:79882440, and its base is A or T;

[0082] The SNP site 68 is located at otter chromosome NC_062287.1:106762161, and its base is C or T;

[0083] The SNP site 69 is located at otter chromosome NC_062288.1:14894220, and its base is C or T;

[0084] The SNP site 70 is located at otter chromosome NC_062288.1:45078342, and its base is A or T;

[0085] The SNP site 71 is located on the otter chromosome NC_062288.1:79929504, and its base is G or A;

[0086] The SNP site 72 is located at otter chromosome NC_062288.1:103690730, and its base is C or T;

[0087] The SNP site 73 is located at otter chromosome NC_062289.1:15755696, and its base is C or T;

[0088] The SNP site 74 is located at otter chromosome NC_062289.1:41188592, and its base is C or T;

[0089] The SNP site 75 is located on the otter chromosome NC_062289.1:64790204, and its base is G or A;

[0090] The SNP site 76 is located on the otter chromosome NC_062289.1:87777063, and its base is A or G;

[0091] The SNP site 77 is located at otter chromosome NC_062290.1:11396992, and its base is G or A;

[0092] The SNP site 78 is located at otter chromosome NC_062290.1:33757642, and its base is T or C;

[0093] The SNP site 79 is located at otter chromosome NC_062290.1:58042763, and its base is A or C;

[0094] The SNP site 80 is located on the otter chromosome NC_062290.1:82046565, and its base is G or A;

[0095] The SNP site 81 is located at otter chromosome NC_062291.1:35320951, and its base is C or T;

[0096] The SNP site 82 is located on the otter chromosome NC_062291.1:58881100, and its base is G or A;

[0097] The SNP site 83 is located at otter chromosome NC_062291.1:88842652, and its base is C or T;

[0098] The SNP site 84 is located at otter chromosome NC_062292.1:12616147, and its base is T or C;

[0099] The SNP site 85 is located on the otter chromosome NC_062292.1:65508548, and its base is G or A;

[0100] The SNP site 86 is located at otter chromosome NC_062293.1:17189832, and its base is A or C;

[0101] The SNP site 87 is located on the otter chromosome NC_062294.1:312132, and its base is G or A;

[0102] The SNP site 88 is located at otter chromosome NC_062294.1:35873144, and its base is G or A;

[0103] The SNP site 89 is located on the otter chromosome NC_062294.1:60056374, and its base is G or C;

[0104] The SNP site 90 is located at otter chromosome NC_062296.1:1621281, and its base is C or T;

[0105] The SNP site 91 is located at otter chromosome NC_062296.1:38485632, and its base is T or C;

[0106] The SNP site 92 is located in otter chromosome NC_062296.1:82234974, and its base is T or A.

[0107] The present invention also provides a primer for detecting the genetic marker, comprising at least 60 of the following primers 1-92:

[0108] Primer 1 includes SEQ NO.1 and SEQ NO.2, primer 2 includes SEQ NO.3 and SEQ NO.4, primer 3 includes SEQ NO.5 and SEQ NO.6, primer 4 includes SEQ NO.7 and SEQ NO.8, primer 5 includes SEQ NO.9 and SEQ NO.10, primer 6 includes SEQ NO.11 and SEQ NO.12, primer 7 includes SEQ NO.13 and SEQ NO.14, primer 8 includes SEQ NO.15 and SEQ NO.16, primer 9 includes SEQ NO.17 and SEQ NO.18, primer 10 includes SEQ NO.19 and SEQ NO.20, primer 11 includes SEQ NO.21 and SEQ NO.22, primer 12 includes SEQ NO.23 and SEQ NO.24, primer 13 includes SEQ NO.25 and SEQ NO.26, primer 14 includes SEQ NO.27 and SEQ NO.28, primer 15 includes SEQ NO.29 and SEQ NO.30, primer 16 includes SEQ NO.31 and SEQ NO.32, primer 17 includes SEQ NO.33 and SEQ NO.34, primer 18 includes SEQ NO.35 and SEQ NO.36, primer 19 includes SEQ NO.37 and SEQ NO.38, primer 20 includes SEQ NO.39 and SEQ NO.40, primer 21 includes SEQ NO.41 and SEQ NO.42, primer 22 includes SEQ NO.43 and SEQ NO.44, primer 23 includes SEQ NO.45 and SEQ NO.46, primer 24 includes SEQ NO.47 and SEQ NO.48, primer 25 includes SEQ NO.49 and SEQ NO.50, primer 26 includes SEQ NO.51 and SEQ NO.52, primer 27 includes SEQ NO.53 and SEQ NO.54, primer 28 includes SEQ NO.55 and SEQ NO.56, primer 29 includes SEQ NO.57 and SEQ NO.58, primer 30 includes SEQ NO. NO.59 and SEQ NO.60, primer 31 includes SEQ NO.61 and SEQ NO.62, primer 32 includes SEQ NO.63 and SEQ NO.64, primer 33 includes SEQ NO.65 and SEQ NO.66, primer 34 includes SEQ NO.67 and SEQ NO.68, primer 35 includes SEQ NO.69 and SEQ NO.70, primer 36 includes SEQ NO.71 and SEQ NO.72, primer 37 includes SEQ NO.73 and SEQ NO.74, primer 38 includes SEQ NO.75 and SEQ NO.76, primer 39 includes SEQ NO.77 and SEQ NO.78, primer 40 includes SEQ NO.79 and SEQ NO.80, primer 41 includes SEQ NO.81 and SEQ NO.82, primer 42 includes SEQ NO.83 and SEQ NO.84, primer 43 includes SEQ NO.85 and SEQ NO.86, primer 44 includes SEQ NO.87 and SEQ NO.88, primer 45 includes SEQ NO.89 and SEQ NO.90, primer 46 includes SEQ NO.91 and SEQ NO.92, primer 47 includes SEQ NO.93 and SEQ NO.94, primer 48 includes SEQ NO.95 and SEQ NO.96, primer 49 includes SEQ NO.97 and SEQ NO.98, primer 50 includes SEQ NO.99 and SEQ NO.100, primer 51 includes SEQ NO.101 and SEQ NO.102, primer 52 includes SEQ NO.103 and SEQ NO.104, primer 53 includes SEQ NO.105 and SEQ NO.106, primer 54 includes SEQ No. 107 and SEQ No. 108, primer 55 includes SEQ No. 109 and SEQ No. 110, primer 56 includes SEQ No. 111 and SEQ No. 112, primer 57 includes SEQ No. 113 and SEQ No. 114, primer 58 includes SEQ No. 115 and SEQ No. 116, primer 59 includes SEQ No. 117 and SEQ No. 118, primer 60 includes SEQ No. 119 and SEQ No. 120, primer 61 includes SEQ No. 121 and SEQ No. 122, primer 62 includes SEQ No. 123 and SEQ No. 124, primer 63 includes SEQ No. 125 and SEQ No. 126, primer 64 includes SEQ No. 127 and SEQ No. 128, primer 65 includes SEQ No. 129 and SEQ No. 130, primer 66 includes SEQ NO.131 and SEQ NO.132, primer 67 includes SEQ NO.133 and SEQ NO.134, primer 68 includes SEQ NO.135 and SEQ NO.136, primer 69 includes SEQ NO.137 and SEQ NO.138, primer 70 includes SEQ NO.139 and SEQ NO.140, primer 71 includes SEQ NO.141 and SEQ NO.142, primer 72 includes SEQ NO.143 and SEQ NO.144, primer 73 includes SEQ NO.145 and SEQ NO.146, primer 74 includes SEQ NO.147 and SEQ NO.148, primer 75 includes SEQ NO.149 and SEQ NO.150, primer 76 includes SEQ NO.151 and SEQ NO.152, primer 77 includes SEQ NO.153 and SEQ NO.154, primer 78 includes SEQ NO.155 and SEQ NO.156, primer 79 includes SEQ NO.157 and SEQ NO.158, primer 80 includes SEQ NO.159 and SEQ NO.160, primer 81 includes SEQ NO.161 and SEQ NO.162, primer 82 includes SEQ NO.163 and SEQ NO.164, primer 83 includes SEQ NO.165 and SEQ NO.166, primer 84 includes SEQ NO.167 and SEQ NO.168, primer 85 includes SEQ NO.169 and SEQ NO.170, primer 86 includes SEQ NO.171 and SEQ NO.172, primer 87 includes SEQ NO.173 and SEQ NO.174, primer 88 includes SEQ NO.175 and SEQ NO.176, primer 89 includes SEQ NO.177 and SEQ NO.178, primer 90 includes SEQ No. 179 and SEQ No. 180, primer 91 includes SEQ No. 181 and SEQ No. 182, and primer 92 includes SEQ No. 183 and SEQ No. 184.

[0109] In one embodiment, the primer set includes primers 1-92.

[0110] The present invention also provides a kit for identifying individual otters, comprising the primers.

[0111] In one embodiment, the kit includes a PCR system, and the PCR system includes the primers.

[0112] In one embodiment, the PCR system includes a first round of PCR and a second round of PCR, and the first round of PCR and the second round of PCR are multiplex PCR.

[0113] In one embodiment, the reaction conditions of the first round of PCR are: 93-98°C, 12-18 min; 93-95°C, 28-32 s, 60-63°C, 10-14 min, 70-74°C, 28-32 s, 3-7 cycles; 92-96°C, 28-32 s, 60-63°C, 0.8-1.2 min, 70-74°C, 28-32 s, 23-27 cycles.

[0114] In one embodiment, the reaction conditions of the second round of PCR are: 93-98°C, 12-18 min; 93-95°C, 28-32 s, 58-62°C, 2-6 min, 70-74°C, 28-32 s, 3-7 cycles; 92-96°C, 28-32 s, 60-63°C, 0.8-1.2 min, 70-74°C, 28-32 s, 18-22 cycles.

[0115] The present invention also provides application of the genetic marker, the primer set or the kit in individual otter identification.

[0116] In one embodiment, the individual identification includes: genotyping of otter species, identification of otter species, analysis of otter relatedness, paternity testing of otters, analysis of genetic diversity of otter populations, and / or analysis of genetic structure of otter populations.

[0117] The present invention also provides a method for identifying individual otters, comprising the following steps: extracting DNA from a sample to be tested, performing PCR amplification using the kit to obtain a PCR amplification product, performing high-throughput sequencing to obtain genotype data of the genetic marker, and performing individual identification based on the genotype data.

[0118] This method uses the genetic markers and primer sets composed of the above-mentioned SNPs sites to establish a method for individual identification and kinship of Asian small-clawed otters, and establishes an accurate, reliable and safe molecular biological method.

[0119] In one embodiment, the sample to be tested includes muscle and / or hair.

[0120] Compared with the prior art, the present invention has the following beneficial effects:

[0121] The present invention discloses a genetic marker for individual otter identification and its application. The genetic marker includes 92 SNPs sites, which are rich in genetic polymorphism information, and the primer set established on this basis has high stability. The Asian small-clawed otter individuals can be accurately identified, and even hair samples can be accurately identified, so sampling is convenient, stress-free, non-traumatic, and more practical. The method constructed using the above genetic markers and primer sets has an individual identification rate of 100% for Asian small-clawed otters, and can also be used to analyze the kinship of Asian small-clawed otters and the genetic diversity of populations. The present invention can clarify and improve the pedigree of Asian small-clawed otters and the kinship between individuals, promote the breeding and management level of Asian small-clawed otter populations, and improve economic benefits. DETAILED DESCRIPTION

[0122] For ease of understanding of the present invention, the present invention will be described more fully below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0124] source:

[0125] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0126] Example

[0127] 1. Screening of SNP sites.

[0128] 1. Use Genomic DNA from 10 Asian small-clawed otters was extracted using the Blood Tissue DNA Kit.

[0129] 2. Using the Illumina HiSEQ_X-ten platform, reads of each individual were obtained by whole genome resequencing, with a data volume of about 45Gb for each sample. The raw data was filtered, and poor quality and short sequences were filtered out using fastp. The filtered sequences were compared with the Asian small-clawed otter reference genome Aonyx_cinereus_HiC.fasta.gz (for details of the reference genome, see: https: / / www.dnazoo.org / assemblies / aonyx_cinereus) using BWA software, and SNPs sites were detected and screened on the Haplotype Caller tool of GATK 4 software.

[0130] By screening the SNP data set of the 10 samples, we obtained SNPs sites with stable inheritance in the population. The filtering criteria were as follows: (1) the screened SNPs should be evenly distributed on each chromosome; (2) the minimum allele frequency should be greater than 0.39; (3) the minimum distance between two SNPs on the genome should be 9.9 Mb; (4) the 500 bp sequence containing the SNP site had only one copy when compared with the genome; and there were no insertions or deletions on both sides of the 500 bp. Through the above analysis, 92 SNP sites were selected, as shown in the following table.

[0131] Table 1 Allele types of 92 SNP loci

[0132]

[0133]

[0134]

[0135] 2. SNP locus typing.

[0136] 1. Extract sample DNA from the validation population.

[0137] Fifteen samples of Asian small-clawed otters were randomly collected, including 10 muscle samples and 5 hair samples from 12 individuals, and 3 samples contained tissue and feather samples. Magen Magen Hipure Tissue DNA Kit (D3131-02) and Hipure Forensic DNA Kit (D3130-02) were used to extract genomic DNA from Asian small-clawed otters' tissues and hair, respectively.

[0138] 2. Amplification of SNP sites.

[0139] The multiplex PCR method was used to perform PCR amplification on the 92 SNP sites screened above. According to the sequences on both sides of the target site, specific primers for PCR amplification of the 92 SNP sites screened above were designed. The primer information is shown in the following table.

[0140] The multiplex PCR amplification system was as follows: 5 μL DNA, 1 μL Buffer (10×), 2 μL premixed primer combination (50 nM), 1 μL dNTP (2.5 mM), 3.0 μL ddH2O, 1 μL Mg 2+ (100 mM), 0.1 μL enzyme (5 U / μL), and 10 μL paraffin oil.

[0141] The PCR reaction conditions were: 95°C, 15 min; 94°C, 30 s, 61.5°C, 12 min, 72°C, 30 s, 5 cycles; 94°C, 30 s, 61.5°C, 1 min, 72°C, 30 s, 25 cycles.

[0142] The second round of PCR was performed using the first round PCR product as a template, and adapters were added before and after each sample to facilitate subsequent high-throughput sequencing.

[0143] Add 50 μL ddH2O to the PCR product of each sample in the first round, centrifuge briefly, let stand at room temperature for 15 min, and then use the diluted PCR product as a template for the second round of amplification.

[0144] The reaction system for the second round of amplification was as follows: 10 μL template, 3.6 μL ddH2O, 3.6 μL dNTP (2 μM) Barcode, 2 μL Buffer (10×), 1 μL Mg 2+ (100 mM), 0.8 μL dNTP (2.5 mM), 0.1 μL enzyme (5 U / μL) and 20 μL paraffin oil.

[0145] The reaction conditions for the second round of PCR amplification were: 95°C, 15 min; 94°C, 30 s, 60°C, 4 min, 72°C, 30 s, 5 cycles; 94°C, 30 s, 61.5°C, 1 min, 72°C, 30 s, 20 cycles.

[0146] Table 2 PCR amplification primer information of 92 SNPs loci

[0147]

[0148]

[0149]

[0150]

[0151] 3. High-throughput sequencing and typing of SNP sites.

[0152] Take 10 μL of each of the two rounds of PCR products and mix them, shake and let stand for 24 hours, then cut the gel and purify the mixed PCR products, dilute the purified PCR products according to the requirements of the Illumina X-ten sequencing platform, and perform high-throughput sequencing on the amplicons. Use bioinformatics methods to analyze the sequencing results of different samples, and finally obtain the genotypes of 92 SNP sites in each sample.

[0153] This time, 15 Asian small-clawed otter verification samples were tested. The verification samples were divided into tissues and hair with follicles. The results showed that both samples can accurately identify individuals. Among them, hair with follicles is non-stressful to animals and easy to collect. This method is more practical for Asian small-clawed otters where blood collection is difficult. Finally, information on 92 SNP sites was obtained for each sample.

[0154] This method can sequence millions of DNA molecules at a time, with an average sequencing depth of 10215× for each SNP site and a SNP detection rate of up to 99.88%, making the SNP typing of the present invention highly accurate and sensitive.

[0155] The typing results of 92 SNP loci are shown in the table below, of which 83 loci are heterozygous, and 9 homozygous loci appear, which may be due to the fact that the samples are siblings. The average observed heterozygosity (HO) of the 92 SNP loci is 0.404, the expected heterozygosity (HE) is 0.505, and the average polymorphic information content (PIC) is 0.365. These results show that the screened SNP group is also suitable for samples with close kinship, and can accurately perform individual identification and paternity testing, and can also analyze the genetic diversity and kinship of the Asian lower-clawed otter population.

[0156] Table 3 Genotyping statistics of 92 SNP loci

[0157]

[0158]

[0159]

[0160] 4. Individual identification.

[0161] According to the 92 SNP genotypes detected for each individual, individual identification was performed using the identity analysis software Cervus3.0.7.

[0162] The 92 screened SNPs were used for individual identification, and the PID values ​​were all 0. All the results were judged as "Excluded", indicating that individual identification can be performed accurately.

[0163] Among the 15 samples, the genotypes of the 92 SNP sites of 3 hair samples and 3 muscle samples are all the same, which is displayed as "Exact match", that is, they are considered to be the same individual. The other samples are marked as "Excluded" in pairs. According to the 92 SNP site group provided by the present invention, each SNP site is a double allele, so each SNP can be considered as 3 genotypes, forming a 92-bit digital label, which can theoretically give 3 92 The 92 SNPs in this group can provide individual molecular tags for Asian small-clawed otters, meeting the detection needs of the Asian small-clawed otter population. Therefore, through this group of 92 SNP loci, individual identification and genetic diversity assessment of Asian small-clawed otters can be performed.

[0164] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0165] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a primer for detecting genetic markers for individual identification of otters in preparing a kit, It is characterized in that The genetic markers include 92 of the following SNP loci: The reference genome of the SNP site is the Asian small-clawed otter reference genome Aonyx_cinereus_HiC.fasta.gz.

2. A primer set for detecting the genetic marker according to claim 1, It is characterized in that The following primers 1-92 are included: Primer 1 includes SEQ NO.1 and SEQ NO.2, primer 2 includes SEQ NO.3 and SEQ NO.4, primer 3 includes SEQ NO.5 and SEQ NO.6, primer 4 includes SEQ NO.7 and SEQ NO.8, primer 5 includes SEQ NO.9 and SEQ NO.10, primer 6 includes SEQ NO.11 and SEQ NO.12, primer 7 includes SEQ NO.13 and SEQ NO.14, primer 8 includes SEQ NO.15 and SEQ NO.16, primer 9 includes SEQ NO.17 and SEQ NO.18, primer 10 includes SEQ NO.19 and SEQ NO.20, primer 11 includes SEQ NO.21 and SEQ NO.22, primer 12 includes SEQ NO.23 and SEQ NO.24, primer 13 includes SEQ NO.25 and SEQ NO.26, primer 14 includes SEQ NO.27 and SEQ NO.28, primer 15 includes SEQ NO.29 and SEQ NO.30, primer 16 includes SEQ NO.31 and SEQ NO.32, primer 17 includes SEQ NO.33 and SEQ NO.34, primer 18 includes SEQ NO.35 and SEQ NO.36, primer 19 includes SEQ NO.37 and SEQ NO.38, primer 20 includes SEQ NO.39 and SEQ NO.40, primer 21 includes SEQ NO.41 and SEQ NO.42, primer 22 includes SEQ NO.43 and SEQ NO.44, primer 23 includes SEQ NO.45 and SEQ NO.46, primer 24 includes SEQ NO.47 and SEQ NO.48, primer 25 includes SEQ NO.49 and SEQ NO.50, primer 26 includes SEQ NO.51 and SEQ NO.52, primer 27 includes SEQ NO.53 and SEQ NO.54, primer 28 includes SEQ NO.55 and SEQ NO.56, primer 29 includes SEQ NO.57 and SEQ NO.58, primer 30 includes SEQ NO. NO.59 and SEQ NO.60, primer 31 includes SEQ NO.61 and SEQ NO.62, primer 32 includes SEQ NO.63 and SEQ NO.64, primer 33 includes SEQ NO.65 and SEQ NO.66, primer 34 includes SEQ NO.67 and SEQ NO.68, primer 35 includes SEQ NO.69 and SEQ NO.70, primer 36 includes SEQ NO.71 and SEQ NO.72, primer 37 includes SEQ NO.73 and SEQ NO.74, primer 38 includes SEQ NO.75 and SEQ NO.76, primer 39 includes SEQ NO.77 and SEQ NO.78, primer 40 includes SEQ NO.79 and SEQ NO.80, primer 41 includes SEQ NO.81 and SEQ NO.82, primer 42 includes SEQ NO.83 and SEQ NO.84, primer 43 includes SEQ NO.85 and SEQ NO.86, primer 44 includes SEQ NO.87 and SEQ NO.88, primer 45 includes SEQ NO.89 and SEQ NO.90, primer 46 includes SEQ NO.91 and SEQ NO.92, primer 47 includes SEQ NO.93 and SEQ NO.94, primer 48 includes SEQ NO.95 and SEQ NO.96, primer 49 includes SEQ NO.97 and SEQ NO.98, primer 50 includes SEQ NO.99 and SEQ NO.100, primer 51 includes SEQ NO.101 and SEQ NO.102, primer 52 includes SEQ NO.103 and SEQ NO.104, primer 53 includes SEQ NO.105 and SEQ NO.106 No. 106, primer 54 includes SEQ No. 107 and SEQ No. 108, primer 55 includes SEQ No. 109 and SEQ No. 110, primer 56 includes SEQ No. 111 and SEQ No. 112, primer 57 includes SEQ No. 113 and SEQ No. 114, primer 58 includes SEQ No. 115 and SEQ No. 116, primer 59 includes SEQ No. 117 and SEQ No. 118, primer 60 includes SEQ No. 119 and SEQ No. 120, primer 61 includes SEQ No. 121 and SEQ No. 122, primer 62 includes SEQ No. 123 and SEQ No. 124, primer 63 includes SEQ No. 125 and SEQ No. 126, primer 64 includes SEQ No. 127 and SEQ No. 128, primer 65 includes SEQ No. 129 and SEQ No.

130. No. 130, primer 66 includes SEQ No. 131 and SEQ No. 132, primer 67 includes SEQ No. 133 and SEQ No. 134, primer 68 includes SEQ No. 135 and SEQ No. 136, primer 69 includes SEQ No. 137 and SEQ No. 138, primer 70 includes SEQ No. 139 and SEQ No. 140, primer 71 includes SEQ No. 141 and SEQ No. 142, primer 72 includes SEQ No. 143 and SEQ No. 144, primer 73 includes SEQ No. 145 and SEQ No. 146, primer 74 includes SEQ No. 147 and SEQ No. 148, primer 75 includes SEQ No. 149 and SEQ No. 150, primer 76 includes SEQ No.151 and SEQ NO.152, primer 77 includes SEQ NO.153 and SEQ NO.154, primer 78 includes SEQ NO.155 and SEQ NO.156, primer 79 includes SEQ NO.157 and SEQ NO.158, primer 80 includes SEQ NO.159 and SEQ NO.160, primer 81 includes SEQ NO.161 and SEQ NO.162, primer 82 includes SEQ NO.163 and SEQ NO.164, primer 83 includes SEQ NO.165 and SEQ NO.166, primer 84 includes SEQ NO.167 and SEQ NO.168, primer 85 includes SEQ NO.169 and SEQ NO.170, primer 86 includes SEQ NO.171 and SEQ NO.172, primer 87 includes SEQ NO.173 and SEQ NO.174, primer 88 includes SEQ NO.175 and SEQ NO.176, primer 89 includes SEQ No. 177 and SEQ No. 178, primer 90 includes SEQ No. 179 and SEQ No. 180, primer 91 includes SEQ No. 181 and SEQ No. 182, and primer 92 includes SEQ No. 183 and SEQ No.

184.

3. A kit for identifying individual otters, It is characterized in that Comprising the primer set described in claim 2.

4. The kit according to claim 3, It is characterized in that The kit comprises a PCR system, and the PCR system comprises the primer set according to claim 2.

5. Use of the genetic marker according to claim 1, the primer set according to claim 2 or the kit according to any one of claims 3 to 4 in individual otter identification.

6. The use according to claim 5, It is characterized in that The individual identification includes: genotyping of individual otters, analysis of otter kinship, paternity testing of otters, genetic diversity analysis of otter populations and / or analysis of genetic structure.

7. A method for identifying individual otters, It is characterized in that The following steps are involved: Extract DNA from the sample to be tested, perform PCR amplification using the kit described in any one of claims 3-4 to obtain PCR amplification products, perform high-throughput sequencing to obtain genotype data of the genetic marker described in claim 1, and perform individual identification based on the genotype data.

8. The method according to claim 7, It is characterized in that The sample to be tested includes muscle and / or hair.

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