WO2021097878A1 - 暗纹东方鲀snp分子标记及其在遗传育种的应用 - Google Patents

暗纹东方鲀snp分子标记及其在遗传育种的应用 Download PDF

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WO2021097878A1
WO2021097878A1 PCT/CN2019/121147 CN2019121147W WO2021097878A1 WO 2021097878 A1 WO2021097878 A1 WO 2021097878A1 CN 2019121147 W CN2019121147 W CN 2019121147W WO 2021097878 A1 WO2021097878 A1 WO 2021097878A1
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snp
type
base
tgf120
genetic
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French (fr)
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尹绍武
张鑫宇
王涛
裴雪莹
张红燕
郑翔
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南京师范大学
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Priority to KR1020207030876A priority Critical patent/KR102495383B1/ko
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    • G16B20/40—Population genetics; Linkage disequilibrium
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    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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Definitions

  • the invention belongs to aquatic breeding technology, and particularly relates to SNP molecular markers of obscurus puffer and its application in genetic breeding.
  • Takifugufasciatus (Takifugufasciatus), commonly known as pufferfish, is a valuable economic fish of the order Bonefish, Radial fish, and Takifugu. It is widely distributed in coastal areas and inland rivers of China, Japan and the Korean Peninsula. With the development of industrial technology, fishing technology and efficiency have been rapidly improved. Uncontrolled fishing and emission pollution have caused the population of obscured puffer fish to decline rapidly. The fishing flood of wild obscured puffer has basically disappeared, seriously threatening the diversity and ecological diversity. Stability has also caused a blow to the trade economy of the obscured puffer fish.
  • Fugu obscurus is a river-sea migratory fish with poor resistance to the environment and obvious stress response. Germplasm degradation such as increased disease and decreased meat quality often occurs. In addition, the ever-expanding market demand puts the existing obscured fish on the surface. The growth requirements of the Takifugu species are getting higher and higher, and the improvement of germplasm and the breeding of excellent strains have become an important method to break through the constraints of the Takifugu obscurus industry.
  • the low-temperature resistant Fugu obscurus can reduce the investment in temperature control equipment for artificial breeding of Fugu obscurus, and can also greatly increase the survival rate of the obscured during the winter. Therefore, to breed excellent obscured puffer fish strains on demand, we must not only start from one aspect, but also need to take into account a variety of required qualities, combined with growth, cold resistance and disease resistance, and other qualities for continuous breeding.
  • the standard head length (the ratio of head length to body length) is calculated as: individual head length/body length, which represents the ratio of head to torso. The smaller the standard head length, the smaller the head. For expensive fish like the obscured puffer, which hardly eats the head, the smaller the head ratio, the longer the torso, which means more edible parts. The economic value is greater.
  • SNPs Single nucleotide polymorphisms
  • SNPs Single Nucleotide Polymorphisms
  • a SNP site is mostly of three base types, the first is pure base type one, the second is pure base type two, and the third is base one and base two type (such as AA, CC, AC).
  • TGF- ⁇ Transforming growth factor ⁇
  • CIRP/CIRBP Cold-induced binding protein
  • the purpose of the present invention is to provide a good-growing, low-temperature-tolerant SNP molecular marker for Fugu obscurus and corresponding PCR primers, and to provide an informative means for accurately selecting broodstock with good genetic quality by using the molecular marker.
  • the present invention takes the obscured puffer fish population randomly selected from the breeding farm as the test material to develop the development of molecular markers for the obscured puffer growth traits and the establishment of a marker-assisted breeding system.
  • the SNP molecular markers of obscurus puffer provided by the present invention include the SNP molecular marker TGF120 related to body weight and standard head length and the SNP molecular marker CIRP41 related to cold resistance. Their base sequences are as SEQ ID NO:1 and SEQ, respectively. ID NO: 2.
  • TGF120 The partial genomic sequence of the SNP molecular marker source gene (TGF- ⁇ ) named TGF120 is shown in SEQ ID NO: 1. From the 5'end, there is a SNP at position 120 of the sequence: g.120C>ASNP
  • the base is C or A or AC.
  • the body weight of individuals with CC (CC means the site base is pure C, the same below) is significantly higher than that of the AA (AA means the site base is pure A, the same below), and the standard head length (head length)
  • the ratio to body length) is significantly lower than that of AA individuals;
  • the primer pair used to detect the SNP molecular marker named TGF120 should contain the primer pair.
  • CIRP41 The partial genomic sequence of the SNP molecular marker source gene (CIRP) named CIRP41 is shown in SEQ ID NO: 2. From the 5'end, there is SNP: g.41A>TSNP at position 41 of the sequence. , The base is A or T or AT. The low temperature tolerance of individuals with AA type at this site is significantly higher than that of TT (TT stands for pure T at the base site, the same below);
  • the primer pair used to detect the SNP molecular marker named CIRP41 should contain the primer pair.
  • the present invention also provides a system for judging the genetic quality of Takifugu obscurus, including: a SNP information selection module for providing the function of viewing and selecting SNP information and primers necessary for SNP amplification, the SNP information including the TGF120 marker as described above And CIRP41-labeled SNP name and SNP function, and the necessary primers for SNP amplification include the forward primer sequence and the reverse primer sequence as described above;
  • the SNP judgment module is used for judging the genetic quality level of the individual at the SNP locus according to the SNP marker base type obtained after amplification and sequencing of the individual to be tested: the base at the SNPg.120C>A locus is of the CC type For individuals, judge genetic quality as good GOOD, AA type judge genetic quality as poor BAD, AC type judge genetic quality as normal; For individuals with SNP locus SNPg.41A>T base showing AA type, judge genetic quality For better GOOD, TT type judges genetic quality as poor BAD, AT type judges genetic quality as normal NORMAL.
  • the output module is used to output the judgment result of the SNP judgment module.
  • the use of the SNP molecular markers, primer pairs and the kit in the breeding of obscured puffer is mainly as a method for detecting the growth of obscured puffer and the advantages and disadvantages of low temperature tolerance, through the application of the kit and the positive
  • the reverse primer is used to detect the SNP molecular markers of the obscured puffer to be screened to determine the pros and cons of the various traits of the obscured puffer, and the system is to store and integrate the information of these SNPs.
  • Fast and convenient SNP collection method is convenient for the retention, transmission and use of information.
  • the method for judging the genetic quality of obscured pufferfish specifically includes the following steps:
  • the weight of the SNPg.120C>A site CC base type individual with the label name TGF120 was significantly higher than that of the AA type individual .
  • the standard head length is significantly lower than that of the AA type individuals; the AA base type individuals at the SNPg.41A>T locus labeled CIRP41 have significantly higher cold resistance performance than the TT type individuals.
  • the present invention has the following advantages:
  • the SNP locus disclosed in the present invention can be used for molecular marker-assisted breeding, and is not restricted by the sex of Fugu obscurus. It can be used for parent selection of Fugu obscurus and significantly enhances the genetic quality of Fugu obscurus.
  • the present invention uses the two pairs of primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 to detect the base sequence shown in SEQ ID NO: 1 from the 5'end
  • the base sequence shown at position 120 and SEQ ID NO: 2 is based on the base type at position 41 at the 5'end, and the result is accurate and stable.
  • the system uses Matlab GUI to design the interface and enter the SNP information.
  • the conditions and results are clear and intuitive, the information is comprehensive, it is expandable, durable and open source, the data capacity is huge, and the results are displayed concisely, which is convenient for all people to use.
  • Fig. 1 is an operation flowchart according to an embodiment of the present invention
  • Fig. 2 is a system interface when the base type of the TGF120 label is CC according to an embodiment of the present invention.
  • the present invention is based on the two pairs of primers described in SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 on the extraction of genomic DNA, PCR amplification, amplification product sequencing and Analysis of sequencing results.
  • the SNP sites (SNPg.120C>A) that are significantly related to the growth of the obscured puffer and the SNP sites (SNPg.41A>T) that are significantly related to the cold tolerance are obtained.
  • the genetic quality judgment program related to the growth and cold resistance of the obscured puffer can be obtained, which can be applied to the current and future obscured orientals. Takifugu molecular markers assist the breeding process.
  • the main steps of the process include:
  • the fish used in the experiment of the present invention were taken from Jiangyin Shengang Sanxian Breeding Co., Ltd. and Jiangsu Zhongyang Group Co., Ltd., with 600 random pufferfish about five months old.
  • Use a vernier caliper and an electronic balance to measure the head length, body length, and body weight of 300 of the three growth indicators, and take the corresponding tail fins and store them in 95% ethanol at -20°C for genomic DNA extraction; place the remaining 300 tails in a storage area.
  • the amplified length of the PCR product labeled with TGF120 is about 350 bp, and the PCR primers are:
  • Reverse primer (SEQ ID NO: 4): 5’-CGGTAGATTAAGTTAAGTTCC-3’;
  • the amplified length of the PCR product labeled with SNP41 is about 420bp, and the PCR primers are:
  • Reverse primer (SEQ ID NO: 6): 5'-CCCGTCTGTATTTGTTATG-3'.
  • the PCR reaction system is 20 ⁇ L: Master Mix 10 ⁇ L, each of the forward and reverse primers is 0.8 ⁇ L, the DNA template is 1 ⁇ L, and the sterilized water is 7.4 ⁇ L.
  • a total of 35 cycles of PCR reaction including 95°C pre-denaturation for 5 minutes.
  • Each cycle includes denaturation at 95°C for 30s, tempering at 60°C for 30s, and extension at 72°C for 30s; after the end of the cycle, it is extended at 72°C for 5 minutes.
  • the PCR amplification products of the 600-tailed Fugu obscurus were sequenced and spliced in both directions. Based on the sequencing results, the SNP locus of Fugu obscurus was typed.
  • the base patterns and growth characteristics of the SNP sites of 300 individuals of Fugu obscurus are shown in Table 1 (see Table 1 at the end), and the base patterns and cold resistance performance of the SNP sites of the remaining 300 individuals are shown in Table 2 ( See table 2 at the end).
  • the SPSS (25.0) GLM program was used to analyze the association between gene polymorphisms and traits, and statistical data was expressed as mean ⁇ standard deviation.
  • Table 4 shows the correlation between different base types of SNP and cold resistance.
  • SNPg.120C>A site has a significant difference in body weight between type CC and type AA (P ⁇ 0.05), and the standard head length (the ratio of head length to body length) has a significant difference (P ⁇ 0.05). It proves that SNPg.120C>A is a SNP site that is significantly related to growth and can distinguish two different growths of obscured puffer.
  • SNPg.41A>T site AA type and TT type imbalance temperature has a significant difference (P ⁇ 0.05). It proves that SNPg.41A>T is a SNP site that is significantly related to cold resistance and can distinguish two kinds of obscured Takifugu obscurus with different cold resistance.
  • the cold resistance performance of pufferfish is significantly related, which is reflected in that the number of AA individuals who survive under low temperature is significantly more than that of TT individuals (P ⁇ 0.05).
  • individuals with base type CC at SNPg.120C>A can be preferentially selected as good breeding parents, and individuals with base type AA at SNPg.41A>T are preferentially selected as breeding parents for cold-resistant strains.
  • the individuals whose base type is CC at the SNPg.120C>A locus in TGF120 can be selected preferentially, and SNPg.41A in CIRP41 can be selected first.
  • the individuals whose base type is AA at the C site are breeding parents, which has important guiding significance for the selection and breeding of excellent new lines of Fugu obscurus.
  • the present invention writes a genetic quality judgment system based on Matlab, including:
  • the SNP entry module is used to add biological information of the raw SNP, including the SNP name, SNP function, primer information, and traits associated with the base type.
  • the SNP name and function describe the relationship between a SNP locus and a trait.
  • the primer information is The primer sequence designed by using genomic DNA according to the SNP site, that is, the primer base sequence of the SNP, includes a forward primer sequence and a reverse primer sequence.
  • the system provides GUI pages for users to enter.
  • step (e) After the SNP site correlation analysis is obtained in step (e), the code shown in CODE ID NO:1 (see the end of the specification) is entered through the GUI provided by the system, and the purpose is to convert the SNPg.120 (TGF120 mark) and SNPg.41 (CIRP41 mark) biological information entry system, forming a complete judgment system. details as follows:
  • str ['For[SNP name],[SNP function],F-Primer:[forward primer sequence] and R-Primer:[reverse primer sequence]']; where "SNP name” is the SNP site "SNP function” refers to which traits the site is related to, "forward primer sequence” is the sequence of the forward primer for detecting the site, and "reverse primer sequence” is the sequence of the reverse primer for detecting the site .
  • SNP name is the SNP site
  • SNP function refers to which traits the site is related to
  • forward primer sequence is the sequence of the forward primer for detecting the site
  • reverse primer sequence is the sequence of the reverse primer for detecting the site .
  • the information of TGF120 and CIRP41 should be added in this format:
  • str ['For TGF120, body weight-related SNP, F-Primer: GCATAGCTCCTCAGGGTA and R-Primer: CGGTAGATTAAGTTAAGTTCC'] and ['For CIRP41, cold resistance-related SNP, F-Primer: AAAGTAGGTCACTGGTGC and CCGCT-Primer: CATGTGT ].
  • SNP SNP There are three meanings of SNP represented by each base type of SNP, good GOOD, bad BAD, and normal NORMAL.
  • the adding method is to add text display, and the command used should conform to this rule:
  • the SNP information selection module is used to provide the function of viewing and selecting SNP information and the necessary primers for SNP amplification.
  • the SNP information is the name and function of the SNP, that is, the relationship between this SNP site and a certain trait, and the necessary primers for SNP amplification are SNP primer base sequence; the system provides a GUI page for users to choose.
  • the SNP base type that can be verified can be selected from TGF120 and CIRP41. After selecting the SNP molecular marker, it will automatically correlate and display the corresponding The primer sequence information.
  • the SNP judgment module obtains the trait primers to be screened from the SNP information selection module and performs amplification and sequencing on the unknown individual.
  • the judgment module determines that the individual is at the SNP position based on the SNP marker base type obtained after amplification and sequencing. The genetic quality level of the point.
  • the body weight of the CC type individuals is significantly higher than the AA type individuals, and the standard head length is smaller than the AA type individuals (P ⁇ 0.05), and the CC type individuals have a more trunk. Longer, heavier growth.
  • the SNPg.120 is obtained as CC (that is, the sequencing peak graph is represented as pure C)
  • the feedback result is GOOD
  • AA that is, the sequencing peak graph is represented as pure A
  • the AC type is NORMAL (That is, the sequencing peak map is reflected as AC)
  • AA individuals with SNP locus SNPg.41A>T have a higher probability of being able to withstand low temperature environmental stimuli than TT individuals.
  • SNPg.41 can be obtained by measurement.
  • the feedback result is GOOD, for TT, it is BAD, and for AT type is NORMAL.
  • the output module is used to display and output the judgment result of the SNP judgment module.
  • step (c) Perform PCR amplification and sequencing according to step (c) to obtain the base type of the corresponding site of the parent to be tested;
  • FIG. 1 shows the interface when the base type of the TGF120 label is CC in an embodiment.
  • the present invention selects 600 samples of Fugu obscurus and performs genomic DNA extraction, amplification and SNP analysis for each individual, and finds that SNPg.120C>A is related to the body weight and standard head of Fugu obscurus. There is a significant correlation between the length, and it can effectively distinguish two different growths of obscured puffer, and SNPg.41A>T has a significant correlation with the cold tolerance of obscured puffer, and it can effectively distinguish two different types. Obscured puffer with cold-resistant ability. Entering two kinds of SNP marker biological information into the quality judgment system can save users other than non-entrants the time and resource consumption of verifying the markers.
  • New users only need to open the system to understand the information and meaning of the corresponding mark, and can independently add newly verified mark information to the system, enriching the open source functions of the system, greatly improving the efficiency of breeding promotion, and accelerating the breeding process. Therefore, in the selection process of high genetic quality Fugu obscurus parents, parents with excellent genetic quality can be selected according to the third-generation molecular markers suggested by the system. For example, individuals with SNPg.120C>A site as CC are preferred.
  • Growing breeding parents individuals whose SNPg.41A>T site is AA are used as low-temperature-tolerant breeding parents to achieve precise breeding, convenient promotion, and high-tech breeding goals.
  • the content of the system can be supplemented by scientific research and production personnel, and the content of its marking can be expanded.
  • the system can easily obtain each marker information for broodstock screening, so as to realize the purpose of popularizing science and technology to workers at all levels.
  • Table 1 Relationship between base type of SNP locus and body length, head length, body weight and standard head length of 300 individuals of Fugu obscurus

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Abstract

本发明公开了一种暗纹东方鲀SNP分子标记及其在遗传育种的应用。公开的暗纹东方鲀SNP分子标记包括体重、标准头长相关的SNP分子标记TGF120和与抗寒能力相关的SNP分子标记CIRP41，它们所在碱基序列分别如SEQ ID NO:1和SEQ ID NO:2所示，所述TGF120位于SEQ ID NO:1所示序列的第120位m，碱基为C或A，所述CIRP41位于SEQ ID NO:2所示序列的第41位y，碱基为A或T。本发明还提供了一种暗纹东方鲀遗传质量判断系统。

Description

暗纹东方鲀SNP分子标记及其在遗传育种的应用 技术领域
本发明属于水产育种技术，尤其涉及暗纹东方鲀的SNP分子标记以及在遗传育种中的应用。
背景技术
暗纹东方鲀(Takifugufasciatus)俗名河豚，为硬骨鱼纲，辐鳍鱼亚纲，鲀形目的名贵经济鱼类，广泛分布于中国、日本和朝鲜半岛国家的沿海地区和内陆河流。随着工业技术的发展，捕捞技术与效率迅速提升，无节制的捕捞与排放污染使得暗纹东方鲀种群数量迅速下跌，野生暗纹东方鲀的渔汛已经基本消失，严重威胁了生态的多样性与稳定性，也致使暗纹东方鲀贸易经济遭受打击。所以，对暗纹东方鲀一类的名贵鱼物种进行人工选育来缓解市场供求矛盾，不仅是一种高效促进河豚产业发展的措施，也是减缓野生暗纹东方鲀种质资源压力的一种方法。
暗纹东方鲀属江海洄游性鱼类，对环境抵抗力较差，应激反应明显，时常出现病害增加、肉质下降等种质退化现象，再加上不断扩大的市场需求对现有的暗纹东方鲀品种长势要求越来越高，种质改良和优良品系繁育已成为突破暗纹东方鲀产业制约的重要方法。随着暗纹东方鲀的人工育种进程的开展，其良种的繁育已经逐渐进入正途，但仍面临多种问题：尽管已经保证了养殖密度的适宜与饵料充足，仍然有不少个体会出现与同期其他个体相差较大的长势，而这多体现在体重上；有的个体虽然与其它个体体重差异不大，却躯干短小，这意味着可食用部分不多；自然条件下，暗纹东方鲀生存的自然水体多为海水，进入河流和人工养殖环境后有可能受低温环境刺激而导致批量死亡，同时人工养殖的暗纹东方鲀需要在人工水环境中进行高密度越冬，一旦控温出现失温就会造成巨大损失。耐低温的暗纹东方鲀可以减少人工繁育暗纹东方鲀时的控温设备投入，也可大大增加暗纹东方鲀的越冬存活率。所以，按需繁育优良的暗纹东方鲀品系，不能仅从一方面入手，还需要将多种需要的品质考虑在内，结合生长、抗寒抗病等多种品质进行不断地选育。标准头长(头长与体长的比值)计算方式为：个体头长/个体体长，表现的是头部与躯干的比例。标准头长越小，说明头部越小，对于暗纹东方鲀这样几乎不食用头部的名贵商品鱼来说，头部比例越小说明躯干则越长，也意味着可食用部分更多，经济价值更大。
分子育种也称为分子标记辅助育种，利用了DNA分子标记对育种目标进行标记选择，从而达到选育、改良特定遗传性状的目的，是一种传统遗传技术与现代分子生物学技术有机结合的新育种策略。单核苷酸多态性(Single nucleotide polymorphisms,SNPs)标记是第三代分子标记，具有多态性高、遗传稳定和检测方便等优点，已被广泛应用于各种分子育种的研究领域。一个SNP位点大多为三种碱基型，第一为纯碱基一型，第二为纯碱基二型，第三为碱基一与碱基二兼有型(如AA，CC，AC)。虽然不是所有的SNP位点都会给个体带来表型差异，但通常情况下带来表型差异的个体中纯碱基一型与纯碱基二型都有显著的性状差异，而兼有型则介于二者之间。转化生长因子β(TGF-β)是一组新近发现的调节细胞生长和分化的TGF-β超家族，具有促进细胞生长、成骨、组织修复的作用，对生物生长与分化都有重要调节作用；冷诱导结合蛋白(CIRP/CIRBP)是一种应激应答因子，特别是在寒冷应激时的上调最为明显。对暗纹东方鲀而言，这两种基因的相关研究与SNP报道都未见发布。
发明内容
发明目的：本发明的目的是提供长势好、耐低温的暗纹东方鲀SNP分子标记以及 相应PCR引物，并提供利用该分子标记实现准确选择遗传质量良好的亲鱼的信息化手段。
技术方案：本发明以养殖场随机选取暗纹东方鲀群体为试材，开展暗纹东方鲀生长性状分子标记的开发及标记辅助育种体系的建立。
本发明提供的暗纹东方鲀SNP分子标记，包括体重、标准头长相关的SNP分子标记TGF120和与抗寒能力相关的SNP分子标记CIRP41，它们所在碱基序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
所述名为TGF120的SNP分子标记来源基因(TGF-β)的部分基因组序列如SEQ ID NO：1所示，自5’端起，该序列的第120位m存在SNP：g.120C>ASNP位点，碱基为C或A或AC。该位点呈现CC(CC表示位点碱基为纯C，下同)型的个体体重显著高于AA(AA表示位点碱基为纯A，下同)型个体，标准头长(头长与体长的比值)显著低于AA型个体；
用于检测所述名为TGF120的SNP分子标记的引物对，其正向引物序列为5’-GCATAGCTCCTCAGGGTA-3’(SEQ ID NO：3)；反向引物序列为5-’CGGTAGATTAAGTTAAGTTCC-3’(SEQ ID NO：4)。用于检测所述名为TGF120的SNP分子标记的试剂盒，应当包含所述的引物对。
所述名为CIRP41的SNP分子标记来源基因(CIRP)的部分基因组序列如SEQ ID NO：2所示，自5’端起，该序列的第41位y存在SNP：g.41A>TSNP位点，碱基为A或T或AT。该位点呈现AA型的个体耐低温表现显著高于TT(TT表示碱基位点为纯T，下同)型个体；
用于检测所述名为CIRP41的SNP分子标记的引物对，其正向引物序列为5’-AAAGTAGGTCACTGGTGC-3’(SEQ ID NO：5)；反向引物序列为5-’CCCGTCTGTATTTGTTATG-3’(SEQ ID NO：6)。用于检测所述名为CIRP41的SNP分子标记的试剂盒，应当包含所述的引物对。
本发明还提供一种暗纹东方鲀遗传质量判断系统，包括：SNP信息选择模块，用于提供查看和选择SNP信息及SNP扩增必要引物的功能，所述SNP信息包括如上所述的TGF120标记和CIRP41标记的SNP名称、SNP功能，所述SNP扩增必要引物包括如上所述的正向引物序列和反向引物序列；
SNP判断模块，用于根据对待测个体进行扩增测序后所得的SNP标记碱基型，判断所述个体在该SNP位点的遗传质量等级：对于SNPg.120C>A位点碱基为CC型个体，判断遗传质量为较好GOOD，为AA型判断遗传质量为较差BAD，为AC型判断遗传质量为一般NORMAL；对于SNP位点SNPg.41A＞T碱基呈现AA型个体，判断遗传质量为较好GOOD，TT型判断遗传质量为较差BAD，AT型判断遗传质量为一般NORMAL。
输出模块，用于输出SNP判断模块的判断结果。
所述的SNP分子标记、引物对和所述的试剂盒在暗纹东方鲀选育中的用途主要是作为一种检测暗纹东方鲀长势、低温耐性优劣的方法，通过应用试剂盒与正反向引物对待筛选暗纹东方鲀进行所述的SNP分子标记的检测，确定所述待测暗纹东方鲀各性状的优劣，而所述系统是对这些SNP信息的储存与整合，是更快速更便捷的SNP汇集方法，便于信息的保留、传递与使用。
所述判断暗纹东方鲀遗传质量的方法，具体包括以下步骤：
a)获得暗纹东方鲀群体；
b)提取暗纹东方鲀尾鳍DNA；
c)基于前述的引物对或试剂盒，对暗纹东方鲀的基因组DNA进行PCR扩增，以获得PCR扩增产物；
d)对PCR扩增产物进行测序，基于测序结果，确定SNP分子标记的碱基型；
e)TGF120标记SNPg.120C>A位点碱基型与暗纹东方鲀生长性状的相关性分析；
f)CIRP41标记SNPg.41A>T位点碱基型与暗纹东方鲀抗寒表现的相关性分析；
g)在前述系统中录入对应标记的信息以及各碱基型意义，用于后续直接进行比对分析。
通过对暗纹东方鲀基因组DNA提取、PCR扩增、扩增产物测序以及测序结果分析，所述标记名为TGF120的SNPg.120C>A位点CC碱基型个体的体重显著高于AA型个体，标准头长显著低于AA型个体；标记名为CIRP41的SNPg.41A>T位点的AA碱基型个体抗寒表现显著高于TT型个体。录入系统后通过Matlab GUI界面可直观显示出判断必须条件与遗传质量判断结果。
技术效果：相对于现有技术，本发明具有以下优势：
1)本发明公开的SNP位点可以进行分子标记辅助育种，不受暗纹东方鲀的性别限制，可用于暗纹东方鲀的亲本选择，显著体高暗纹东方鲀的遗传质量。
2)本发明通过SEQ ID NO：3与SEQ ID NO：4，SEQ ID NO：5与SEQ ID NO：6所示的两对引物，检测SEQ ID NO：1所示碱基序列自5’端第120位与SEQ ID NO：2所示碱基序列自5’端第41位碱基型的方法，结果精确稳定。
3)根据本发明所提供的两种与暗纹东方鲀优良性状相关的SNP分子标记，还建立一种可不断补充数据的暗纹东方鲀遗传质量判断系统，可经由不断补充数据以覆盖暗纹东方鲀更全面的性状，实现利用暗纹东方鲀多种基因上与优良遗传性状显著相关的多个SNP位点系统地、全面地进行分子标记辅助育种。
4)该系统通过Matlab GUI设计界面和录入SNP信息，条件与结果清晰直观，信息收录全面，具有可扩展性，持久性与开源性，数据容量巨大，结果显示简洁，便于各人群使用。
附图说明
图1是根据本发明实施例的操作流程图；
图2是根据本发明实施例的TGF120标记碱基型为CC时的系统界面。
具体实施方式
本发明基于SEQ ID NO：3和SEQ ID NO：4以及SEQ ID NO：5和SEQ ID NO：6所述的两对引物对暗纹东方鲀基因组DNA提取、PCR扩增、扩增产物测序以及测序结果分析。得到了与暗纹东方鲀生长显著相关的SNP位点(SNPg.120C>A)以及与抗寒能力显著相关的SNP位点(SNPg.41A>T)。将两个位点的相关生物信息录入本发明所提的遗传质量判断系统，即可得到与暗纹东方鲀生长和抗寒能力相关的遗传质量判断程序，可应用于目前与将来的暗纹东方鲀分子标记辅助育种进程。
所述过程主要步骤包括：
(a)暗纹东方鲀群体的获得；
(b)暗纹东方鲀基因组DNA提取；
(c)基于所述SNP引物，对暗纹东方鲀的基因组DNA进行PCR扩增；
(d)对PCR扩增产物进行测序，基于测序结果，确定SNP的碱基型；
(e)SNP位点碱基型与暗纹东方鲀体重、抗寒能力的相关性分析；
(f)SNP位点生物信息的录入与判断系统建立。
下面结合具体实施例进行详细说明。
(a)暗纹东方鲀群体的获得。
本发明实验所用鱼取自江阴市申港三鲜养殖有限公司和江苏中洋集团股份有限公司五月龄左右的随机东方鲀600尾。用游标卡尺和电子天平测量其中300尾的头长、体长、体重三项生长指标，并取对应尾鳍于-20℃的95％乙醇中保存，以备基因组DNA提取；将剩余300尾置于可控温的循环水体中，在保持其它环境条件不变的情况下以每小时-1℃的温度降温，取出因水温关系失衡的个体并记录失衡温度，取对应尾鳍于-20℃的95％乙醇中保存，以备基因组DNA提取。
(b)暗纹东方鲀DNA的提取。
(1)取20～50mg的尾鳍，去离子水清洗尽量去除酒精，加入200μl组织裂解液TL后剪碎。完成后加入20μl(20mg/ml)的Proteinase K，震荡混匀1分钟，置于55℃裂解2h，至裂解液澄清。
(2)依次加入200μl结合液CB和100μl异丙醇，摇匀，13,000rpm离心5分钟。
(3)将上清液仔细吸出到GenClean Column中(放入收集管)，避免吸到沉淀与杂质。
(4)10,000rpm离心0.5分钟，取下GenClean Column，倒掉收集管中废液。
(5)加入500μl抑制物去除液IR，12,000rpm离心30秒，倒掉废液。
(6)加入700μl漂洗液WB，12,000rpm离心30秒，倒掉废液。
(7)加入500μl漂洗液WB，12,000rpm离心30秒，倒掉废液。
(8)将GenClean Column放回空收集管中，13,000rpm离心2分钟。
(9)取出GenClean Column，放入一个干净的离心管中，在吸附膜的中间部位加100μl洗脱缓冲液EB(洗脱缓冲液事先在70℃水浴中预热)，室温放置3分钟后12,000rpm离心1分钟。
(10)-20℃长期保存，4℃短期保存防止反复冻融。
(c)基于所述SNP引物，对暗纹东方鲀的基因组DNA进行PCR扩增：
TGF120标记的PCR产物扩增长度约为350bp，PCR引物为：
正向引物(SEQ ID NO：3)：5’-GCATAGCTCCTCAGGGTA-3’；
反向引物(SEQ ID NO：4)：5’-CGGTAGATTAAGTTAAGTTCC-3’；
SNP41标记的PCR产物扩增长度约为420bp，PCR引物为：
正向引物(SEQ ID NO：5)：5’-AAAGTAGGTCACTGGTGC-3’；
反向引物(SEQ ID NO：6)：5’-CCCGTCTGTATTTGTTATG-3’。
PCR反应体系为20μL:Master Mix 10μL，正反向引物各0.8μL，DNA模板1μL，灭菌水7.4μL。
PCR反应共计35个循环，包括95℃预变性5min。每个循环包括95℃变性30s，60℃褪火30s，72℃延伸30s；循环结束后于72℃延伸5min。
(d)对PCR扩增产物进行测序，基于测序结果，确定SNP型。
基于Hiseq2000高通量测序平台和ABI3730测序仪对上述的暗纹东方鲀600尾个体的PCR扩增产物进行双向测序与拼接。基于测序结果，将暗纹东方鲀SNP位点分型。
(e)SNP位点碱基型与暗纹东方鲀生长性状的相关性分析。
暗纹东方鲀300尾个体的SNP位点碱基型与生长性状如表1所示(见末尾表1)，其余300尾个体的SNP位点碱基型与抗寒表现如表2所示(见末尾表2)。
根据表1的数据，利用SPSS(25.0)GLM程序进行基因多态性与性状间的关联分析，统计数据用平均值±标准偏差表示。SNP不同碱基型与生长性状相关性如表3所示。
根据表2的数据，利用SPSS(25.0)GLM程序进行基因多态性与性状间的关联分析，统计数据用平均值±标准偏差表示。SNP不同碱基型与抗寒能力相关性如表4所示。
表3.暗纹东方鲀TGF-β基因SNP位点与生长的相关性：
Figure PCTCN2019121147-appb-000001
*表中表达方式为均值±标准偏差；平均值差值的显著性水平为0.05。
结果显示：SNPg.120C>A位点CC型与AA型的体重具有显著差异(P<0.05)，标准头长(头长与体长的比值)具有显著差异(P<0.05)。证明SNPg.120C>A是一个与生长显著相关，且可以区分两种不同长势的暗纹东方鲀的SNP位点。
表4.暗纹东方鲀CIRP基因SNP位点与生长的相关性：
Figure PCTCN2019121147-appb-000002
*表中表达方式为均值±标准偏差；平均值差值的显著性水平为0.05。
结果显示：SNPg.41A>T位点AA型与TT型的失衡温度具有显著差异(P<0.05)。证明SNPg.41A>T是一个与抗寒能力显著相关，且可以区分两种不同抗寒能力的暗纹东方鲀的SNP位点。
分析可知，位于TGF-β基因上的SNP位点SNPg.120C>A与暗纹东方鲀生长显著相关，具体体现在CC型个体的体重显著高于AA型个体，而CC型个体的标准头长小于AA型个体(P<0.05)，CC型个体呈现出躯干更长，体重更大，因此可食用部分相对更多的长势；位于CIRP基因上的SNP位点SNPg.41A＞T与暗纹东方鲀抗寒表现显著相关，具体体现在低温下存活的AA型个体显著多于TT型个体(P＜0.05)，AA型的个体 更能承受低温的环境刺激，越冬能力更强。因此可以优先选择SNPg.120C>A位点碱基型为CC的个体作为长势优良的育种亲本，优先选择SNPg.41A>T位点碱基型为AA的个体作为抗寒品系的育种亲本。在以快长、抗寒为选育目标的暗纹东方鲀遗传育种研究过程中，可以优先选择标记TGF120中SNPg.120C>A位点碱基型为CC的个体，且标记CIRP41中SNPg.41A＞C位点碱基型为AA的个体为育种亲本，这对于暗纹东方鲀优良新品系的选育具有重要的指导意义。
(f)SNP位点生物信息的录入以及判断系统建立与使用。
本发明根据Matlab编写了遗传质量判断系统，包括：
SNP录入模块，用于添加生SNP生物信息，包括SNP名称、SNP功能、引物信息、碱基型所关联的性状，SNP名称和功能描述了一个SNP位点与某一性状的关系，引物信息是利用基因组DNA根据所述SNP位点所设计得到的引物序列，即SNP的引物碱基序列，包括正向引物序列和反向引物序列。系统提供GUI页面供用户录入。
在本实施例中，在步骤(e)得到SNP位点相关性分析后，通过系统提供的GUI录入CODE ID NO:1(见说明书末尾)所示的代码，目的是将所述的SNPg.120(TGF120标记)与SNPg.41(CIRP41标记)生物信息录入系统，形成完整的判断系统。具体如下：
添加所需要radiobutton按钮，若需要添加SNP信息及引物信息，按照Matlab GUI通用radiobutton规则添加。添加方式为添加文本显示，采用的命令应符合此规则：
str＝['For[SNP名称],[SNP功能],F-Primer:[正向引物序列]and R-Primer:[反向引物序列]']；其中，“SNP名称”为该SNP位点的命名，“SNP功能”为该位点与何种性状有关，“正向引物序列”为检测该位点的正向引物序列，“反向引物序列”为检测该位点的反向引物序列。本实施例中应按此格式添加TGF120与CIRP41的信息：
str＝['For TGF120,body weight-related SNP,F-Primer:GCATAGCTCCTCAGGGTA and R-Primer:CGGTAGATTAAGTTAAGTTCC']和['For CIRP41,cold resistance-related SNP,F-Primer:AAAGTAGGTCACTGGTGC and R-Primer:CCCGTCTGTATTTGTTATG']。
SNP每个碱基型所代表的SNP意义共有三种，较好GOOD,较差BAD,一般NORMAL。添加方式为添加文本显示，采用的命令应符合此规则：
①str＝['[SNP名称][三种碱基型中对性状有促进作用的碱基型GOOD]']；
②str＝['[SNP名称][三种碱基型中混合型碱基型NORMAL]']；
③str＝['[SNP名称][三种碱基型中对性状无促进作用的碱基型BAD]']
SNP信息选择模块，用于提供查看和选择SNP信息及SNP扩增必要引物的功能，所述SNP信息是SNP名称、功能，即此SNP位点与某一性状的关系，SNP扩增必要引物即SNP的引物碱基序列；系统提供GUI页面供用户选择，在当前实现中，可供验证的SNP碱基型可从TGF120位点与CIRP41位点中选择，在选择SNP分子标记后自动关联显示相应的引物序列信息。
SNP判断模块，从SNP信息选择模块中获得拟筛选性状引物并对未知个体进行扩增测序后，在判断模块中根据扩增测序后所得的SNP标记碱基型，判断所述个体在该SNP位点的遗传质量等级。
针对本模块，由于SNPg.120C>A位点中，碱基呈现CC型个体的体重显著高于AA型个体，且标准头长小于AA型个体(P<0.05)，CC型个体呈现出躯干更长，体重更大的长势。如有未知个体经过测序得到SNPg.120为CC(即测序峰图体现为纯C)，回馈结果即为GOOD，为AA(即测序峰图体现为纯A)则为BAD，AC型为NORMAL(即测序峰图体现为AC)；SNP位点SNPg.41A＞T的AA型个体相对于TT型个体而言有更 高几率能承受低温的环境刺激，则如有未知个体经过测量得到SNPg.41为AA，回馈结果即为GOOD，为TT则为BAD，AT型为NORMAL。
输出模块，用于显示和输出SNP判断模块的判断结果。
参照图1，该质量判断系统的应用场景如下：
开始选育前，标记待选亲本，选育时，剪取小部分对应尾鳍组织进行基因组DNA提取；
根据需求调取对应标记的引物信息进行合成：从本系统调取CIRP41的引物信息合成验证抗寒能力引物；调取TGF120的信息合成验证长势优良的引物。合成采用一般PCR引物合成方法，本发明不做赘述；
根据步骤(c)进行PCR扩增和测序，得到待测亲本对应位点的碱基型；
根据得到的碱基型点击系统中对应标记的对应碱基型按钮，从显示框得到判断结果。GOOD判断为有较好遗传质量，BAD判断为遗传质量较差，NORMAL判断为遗传质量一般。图2示出了一实施例中TGF120标记碱基型为CC时的界面。
本发明通过选取600尾暗纹东方鲀样本并对每个个体进行基因组DNA提取、扩增和单核苷酸多态位点分析，发现SNPg.120C>A与暗纹东方鲀的体重、标准头长存在显著的相关性，能够有效地分辨两种不同长势的暗纹东方鲀，而SNPg.41A>T与暗纹东方鲀的抗寒能力存在显著的相关性，并且能够有效地分辨两种不同抗寒能力的暗纹东方鲀。将两种SNP标记生物信息录入质量判断系统，可省去非录入者以外的使用者验证标记的时间与资源消耗。新使用者仅需打开系统即可了解对应标记的信息与意义，并且可以自主添加新验证过的标记信息到系统，丰富系统的开源功能，大大提高了育种推广效率，加速了育种进程。因此，在高遗传质量暗纹东方鲀亲本的选择过程中，可以根据系统所提示的第三代分子标记选择优良遗传质量的亲本，例如优先选择SNPg.120C>A位点为CC的个体作为好长势的育种亲本，SNPg.41A>T位点为AA的个体作为耐低温的育种亲本，以达到精确选育，便捷推广，投入高新技术的育种目标。
本系统经推广进入各阶层科研院所后，可经科研、生产人员补充系统内容，将其标记内容涵盖方面扩大。当某一育种设施需要选择优良遗传质量亲鱼时即可通过此系统便捷获取各个标记信息进行亲鱼筛选，实现将科学技术推广至各层工作者的目的。
表1.暗纹东方鲀300尾个体SNP位点碱基型与体长、头长、体重和标准头长的关系
Figure PCTCN2019121147-appb-000003
Figure PCTCN2019121147-appb-000004
Figure PCTCN2019121147-appb-000005
Figure PCTCN2019121147-appb-000006
Figure PCTCN2019121147-appb-000007
表2.暗纹东方鲀300尾个体SNP位点碱基型与耐受低温的关系
Figure PCTCN2019121147-appb-000008
Figure PCTCN2019121147-appb-000009
Figure PCTCN2019121147-appb-000010
CODE ID NO:1
Figure PCTCN2019121147-appb-000011
Figure PCTCN2019121147-appb-000012
Figure PCTCN2019121147-appb-000013
Figure PCTCN2019121147-appb-000014
Figure PCTCN2019121147-appb-000015
Figure PCTCN2019121147-appb-000016
Figure PCTCN2019121147-appb-000017
Figure PCTCN2019121147-appb-000018

Claims (10)

  1. 暗纹东方鲀SNP分子标记，其特征在于，包括体重、标准头长相关的SNP分子标记TGF120和与抗寒能力相关的SNP分子标记CIRP41，它们所在碱基序列分别如SEQ ID NO:1和SEQ ID NO:2所示，所述TGF120位于SEQ ID NO:1所示序列的第120位m，碱基为C或A，所述CIRP41位于SEQ ID NO:2所示序列的第41位y，碱基为A或T。
  2. 用于检测权利要求1所述的SNP分子标记的特异性PCR引物，其特征在于，包括正向引物、反向引物，其碱基序列分别为：
    适用于TGF120的正向引物：5′-GCATAGCTCCTCAGGGTA-3′；
    适用于TGF120的反向引物：5′-CGGTAGATTAAGTTAAGTTCC-3′；
    适用于CIRP41的正向引物：5′-AAAGTAGGTCACTGGTGC-3′；
    适用于CIRP41的反向引物：5′-CCCGTCTGTATTTGTTATG-3′。
  3. 用于检测权利要求1所述的SNP分子标记的试剂盒，其特征在于，包括权利要求2所述的特异性引物。
  4. 权利要求1所述的SNP分子标记在检测或辅助检测暗纹东方鲀的遗传育种质量中的应用，其特征在于，包括以下步骤：
    1)提取待测暗纹东方鲀的基因组DNA；
    2)利用权利要求2所述的特异性引物或权利要求3所述的试剂盒进行PCR扩增；
    3)检测PCR扩增产物SNP位点TGF120的碱基型是CC、AA还是CA，和/或检测SNP位点CIRP41的碱基型是TT、AA还是TA；
    4)选择SNP TGF120位点的CC型个体和/或SNP CIRP41位点AA型个体用作育种亲本。
  5. 根据权利要求4所述应用，其特征在于，所述PCR扩增的反应体系为20μL:Master Mix 10μL，正反向引物各0.8μL，DNA模板1μL，灭菌水7.4μL；PCR反应共计35个循环，包括95℃预变性5min，每个循环包括95℃变性30s，60℃褪火30s，72℃延伸30s；循环结束后于72℃延伸5min。
  6. 一种暗纹东方鲀遗传质量判断系统，其特征在于，包括：
    SNP信息选择模块，用于提供查看和选择SNP信息及SNP扩增必要引物的功能，所述SNP信息包括如权利要求1所述的TGF120标记和CIRP41标记的SNP名称、SNP功能，所述SNP扩增必要引物包括如权利要求2所述的正向引物序列和反向引物序列；
    SNP判断模块，用于根据对待测个体进行扩增测序后所得的SNP标记碱基型，判 断所述个体在该SNP位点的遗传质量等级；
    输出模块，用于输出SNP判断模块的判断结果。
  7. 根据权利要求6所述的暗纹东方鲀遗传质量判断系统，其特征在于，所述SNP判断模块对于SNPg.120C>A位点碱基为CC型个体，判断遗传质量为较好GOOD，为AA型判断遗传质量为较差BAD，为AC型判断遗传质量为一般NORMAL；对于SNP位点SNPg.41A＞T碱基呈现AA型个体，判断遗传质量为较好GOOD，呈TT型判断遗传质量为较差BAD，呈AT型判断遗传质量为一般NORMAL。
  8. 根据权利要求6所述的暗纹东方鲀遗传质量判断系统，其特征在于，还包括：SNP录入模块，用于添加SNP分子标记生物信息，包括SNP名称、SNP功能、正向引物序列和反向引物序列、碱基型所关联的性状。
  9. 根据权利要求6-8中任一项所述的暗纹东方鲀遗传质量判断系统，其特征在于，所述系统由Matlab软件实现。
  10. 权利要求6-8中任一项所述的暗纹东方鲀遗传质量判断系统的应用，其特征在于，包括以下步骤：
    1)提取待测暗纹东方鲀的基因组DNA；
    2)根据需求从系统调取对应标记的引物信息进行合成；
    3)进行PCR扩增和测序，得到待测亲本对应位点的碱基型；
    4)根据得到的碱基型点击系统中对应标记的对应碱基型按钮，从显示框得到判断结果。
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