CN106591462B - Primer, kit and identification method for identifying genetic relationship between Yangtze river finless porpoise individuals - Google Patents

Abstract

The invention provides a primer, a kit and an identification method for identifying half sibling, full sibling and parent-parent genetic relationship among Yangtze river finless porpoise individuals, wherein the primer comprises 20 pairs of microsatellite primers for amplification. The identification method of the invention can quickly and accurately identify the half-sibling, full-sibling and parental genetic relationship among the Yangtze river finless porpoise individuals, and has wide application in germplasm resource protection of the Yangtze river finless porpoise. The previous microsatellite research is mostly used for identifying the genetic structure and the paternal rights of the Changjiang river finless porpoise population, and does not relate to the identification of half siblings, full siblings and parent-parent relativity among individuals.

Description

Primer, kit and identification method for identifying genetic relationship between Yangtze river finless porpoise individuals

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a kit and an identification method for identifying half-sib, full-sib and parental genetic relationship among Yangtze river finless porpoise individuals.

Background

The Changjiang river finless porpoise (Neophocaena phocaenoides asiaaeeae orientalis) is a small tooth whale, is listed as a national secondary protective animal, is mainly distributed in the downstream main stream in the Yangtze river, Dongting lake and Poyang lake and the main branch stream thereof, and is a relatively independent fresh water subspecies. The results of international union research on the long finless porpoise class conducted in the end of 2006 showed that the number was about 1800 at that time and continued to decline at a rate of at least 5% per year. Since the ecological environment of Yangtze river is unlikely to be improved obviously in a short period, the development of ex-situ protection and the enhancement of artificial breeding are considered as the most promising two protection measures for avoiding the extinction of Yangtze river porpoise, and natural protection areas have been established in the first Swan continent of Hubei stone, the holy of Anhui, and the like, so that a better protection effect is shown.

In the region of migratory protection, inbreeding depression is likely to be caused due to the undersized population. In order to prevent inbreeding, genetic relationship identification needs to be carried out on individuals in a population, so as to establish a reliable genetic pedigree for guiding scientific breeding management and exchange of Changjiang river finless porpoise individuals in different protection intervals. Since the finless porpoise in the Yangtze river lives in water, the relativity between the groups is difficult to determine by observation and external characteristics, so that a new technology with high feasibility is urgently needed to solve the paternity test problem of finless porpoise breeding groups. In recent years, microsatellites, as a novel molecular marker, are widely applied to population dynamic structural analysis and genetic relationship identification among individuals, and are genetic markers based on genetic materials among individuals. Compared with other molecular markers, the microsatellite molecular marker has the characteristics of large quantity, wide and uniform distribution and high polymorphic information content, is widely applied to forensic identification, and is easily suitable for identifying half-sibling and full-sibling individual relationship of Changjiang river finless porpoise and parent-parent relationship.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims to provide a primer, a kit and an identification method for identifying half siblings, full siblings and parent-parent genetic relationships among Yangtze river finless porpoise individuals, which are beneficial to quickly and accurately identifying the half siblings, the full siblings and parent-parent genetic relationships among the Yangtze river finless porpoise individuals and have wide application in germplasm resource protection of the Yangtze river finless porpoise.

The technical scheme is as follows: a primer for identifying the genetic relationship between individual Changjiang river finless porpoise comprises 20 pairs of microsatellite primers for amplification, and the sequences of the microsatellite primers are as follows: JTSSR1 SEQ ID NO. 1-2, JTSSR2 SEQ ID NO. 3-4, JTSSR3SEQ ID NO. 5-6, JTSSR4 SEQ ID NO. 7-8, JTSSR5 SEQ ID NO. 9-10, JTSSR6 SEQ ID NO. 11-12, JTSSR7 SEQ ID NO. 13-14, JTSSR8 SEQ ID NO. 15-16, JTSSR9 SEQ ID NO. 17-18, JTSSR10 SEQ ID NO. 19-20, JTSSR11 SEQ ID NO. 21-22, JT 12 SEQ ID NO. 23-24, JTSSR13 SEQ ID NO. 25-26, SSR14 SEQ ID NO. 27-28, JT 15 SEQ ID NO. 29-30, JTSSR16 SEQ ID NO. 31-32, JTSSR 638 SEQ ID NO. 33-34, SSR 6335 SEQ ID NO. 36, SEQ ID NO. 92-40, and SEQ ID NO. 59640.

The 5' end of at least one primer in each pair of primer pairs is marked by fluorescent dye.

Each pair of primers was labeled by staining with either FAM or HEX.

According to another aspect of the invention, a detection kit comprising the primer set is also provided.

The kit comprises the following components: DNA template, 10 × reaction buffer, Mg²⁺dNTP, upstream and downstream primers, Taq enzyme and sterile double distilled water.

According to another aspect of the invention, the above kit is also provided for identifying the genetic relationship between individual Changjiang river finless porpoise, comprising the following steps:

step 1, collecting blood of Yangtze river finless porpoise to be identified, and extracting genome DNA by adopting an alkaline lysis method;

step 2, taking the genome DNA obtained in the step 1 as a template to carry out PCR amplification;

step 3, carrying out capillary electrophoresis detection on the PCR amplification product obtained in the step 2 by using an ABI3730 sequencer full-automatic nucleic acid protein analyzer;

step 4, converting the fragment size of the sequencing peak diagram obtained in the step 3 into a microsatellite marker to obtain the genotype of each individual;

and 5, analyzing by using genetic analysis software, and identifying the relationship among half-siblings, full-siblings and parents of the Changjiang river finless porpoise individuals.

Has the advantages that: the method can quickly and accurately identify the half-sibling, full-sibling and parental genetic relationship among the Yangtze river finless porpoise individuals, and has wide application in germ plasm resource protection of the Yangtze river finless porpoise.

Drawings

Fig. 1 is a graph showing the genetic relationship analysis of seven individuals of the Changjiang river finless porpoise in example 1, with 1 as an unknown male parent, #1 as an unknown female parent, 3 as a female parent, and 4 as a male parent. 1, 2, 6 and 7 are filial generations;

FIG. 2 is a graph showing the relationship analysis of seven individuals of Jiangjiang finless porpoise in example 2.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

1. Developing the microsatellite markers of the Changjiang river finless porpoise by a magnetic bead enrichment method, and screening out primers with high polymorphism and good amplification efficiency from a plurality of microsatellite markers by PCR amplification. Selecting 20 pairs of microsatellite primers, labeling different primers by using FAM and HEX fluorescence respectively, and simultaneously detecting two sites according to fluorescence colors, wherein the sequence of the primers is shown in a table below.

2. Blood of seven Yangtze river finless porpoise is collected, and the individual positions are numbered as 1, 2, 3, 4, 5, 6 and 7. Wherein No.3 is mature female finless porpoise (hypothetical female parent), No. 4 and No.5 are mature male finless porpoise (hypothetical male parent), and No.1, No.2, No. 6 and No.7 are offspring bred by hypothetical male parent. The blood of the seven-head Changjiang river finless porpoise is taken and put into a 1.5mL centrifuge tube, 150 μ L of alkaline lysis solution (0.25mM NaOH,0.25mM EDTA) is added, the sample is heated to 95 ℃ for 10 minutes, the sample is rapidly cooled to 4 ℃, and 150 μ L of neutral solution (45mM Tris-HCl) is added to obtain the DNA of the sample. Storing at 4 deg.C for use.

3. And amplifying the DNA of the sample by using a PCR instrument. The total volume of the PCR reaction was 15. mu.L, 2. mu.L of DNA template (about 25 ng/. mu.L), 1.5. mu.L of 10 × reaction buffer, Mg²⁺2mmol/L, dNTP 200. mu. mol/L, upstream and downstream primers 0.15. mu. mol/L, Taq enzyme 0.25U, sterile double distilled water to make up the total volume to 15. mu.L. The PCR reaction conditions are as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 20s, 58-60 ℃ for 30s and 72 ℃ for 30 s; 72 ℃ extension for 5min, wherein the annealing temperature varies for different primers.

4. And (3) carrying out capillary electrophoresis detection on the PCR amplification product by using an ABI3730 sequencer full-automatic nucleic acid protein analyzer, and converting the fragment size of a sequencing peak image into a microsatellite marker to obtain the genotype of each individual.

5. The gene types of seven individuals of the Yangtze river finless porpoise are analyzed by using Colony genetic analysis software, and the half sibling, the full sibling and the parent-parent genetic relationship among the seven individuals are identified. The analytical results are shown in FIG. 1. It can be seen from figure 1 that the male parents of individual No.1, individual No.2 and individual No.7 are individual No. 4; the female parents of individuals No.1 and No. 6 are individuals No. 3.

Example 2

1. Developing the microsatellite markers of the Changjiang river finless porpoise by a magnetic bead enrichment method, and screening out primers with high polymorphism and good amplification efficiency from a plurality of microsatellite markers by PCR amplification. Selecting 20 pairs of microsatellite primers, labeling different primers by using FAM and HEX fluorescence respectively, and detecting two sites simultaneously according to the fluorescence color, wherein the sequence of the primers is the same as that in example 1.

2. Blood of seven Yangtze river finless porpoise is collected, and the individual positions are numbered as 1, 2, 3, 4, 5, 6 and 7. Wherein No.3 is mature female finless porpoise (hypothetical female parent), No. 4 and No.5 are mature male finless porpoise (hypothetical male parent), and No.1, No.2, No. 6 and No.7 are offspring bred by hypothetical male parent. The blood of the seven-head Changjiang river finless porpoise is taken and put into a 1.5mL centrifuge tube, 150 μ L of alkaline lysis solution (0.25mM NaOH,0.25mM EDTA) is added, the sample is heated to 95 ℃ for 10 minutes, the sample is rapidly cooled to 4 ℃, and 150 μ L of neutral solution (45mM Tris-HCl) is added to obtain the DNA of the sample. Storing at 4 deg.C for use.

3. Using a PCR instrumentAmplifying the DNA of the sample. The total volume of the PCR reaction was 15. mu.L, 2. mu.L of DNA template (about 25 ng/. mu.L), 1.5. mu.L of 10 × reaction buffer, Mg²⁺2mmol/L, dNTP 200. mu. mol/L, upstream and downstream primers 0.15. mu. mol/L, Taq enzyme 0.25U, sterile double distilled water to make up the total volume to 15. mu.L. The PCR reaction conditions are as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 20s, 58-60 ℃ for 30s and 72 ℃ for 30 s; 72 ℃ extension for 5min, wherein the annealing temperature varies for different primers.

4. And (3) carrying out capillary electrophoresis detection on the PCR amplification product by using an ABI3730 sequencer full-automatic nucleic acid protein analyzer, and converting the fragment size of a sequencing peak image into a microsatellite marker to obtain the genotype of each individual.

5. The gene types of seven individuals of the Yangtze river finless porpoise are analyzed by using Colony genetic analysis software, and the half sibling, the full sibling and the parent-parent genetic relationship among the seven individuals are identified. The analytical results are shown in FIG. 2. From figure 2 it can be seen that individuals No.2 and No.7 are homozygotes; individuals No.1 and No. 6, individuals No.1 and No.2, and individuals No.1 and No.7 are half-siblings.

SEQUENCE LISTING

<110> research center of freshwater fishery of Chinese aquatic science research institute

<120> a primer for identifying half-sib, full-sib and parental relationship between Yangtze river finless porpoise individuals,

Kit and identification method

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Claims (6)

1. A primer combination for identifying genetic relationship among Yangtze river finless porpoise individuals is characterized in that: comprises 20 pairs of microsatellite primers for amplification, and the sequences of the microsatellite primers are as follows: JTSSR1 SEQ ID NO. 1-2, JTSSR2 SEQ ID NO. 3-4, JTSSR3SEQ ID NO. 5-6, JTSSR4 SEQ ID NO. 7-8, JTSSR5 SEQ ID NO. 9-10, JTSSR6 SEQ ID NO. 11-12, JTSSR7 SEQ ID NO. 13-14, JTSSR8 SEQ ID NO. 15-16, JTSSR9 SEQ ID NO. 17-18, JTSSR10 SEQ ID NO. 19-20, JTSSR11 SEQ ID NO. 21-22, JT 12 SEQ ID NO. 23-24, JT 13 SEQ ID NO. 25-26, JTSSR14 SEQ ID NO. 27-28, JT6392 SEQ ID NO. 29-30, JTSSR16 SEQ ID NO. 31-32, JTSSR 636 SEQ ID NO. 33-34, SSR18 SEQ ID NO. 35-19, JTSSR15 SEQ ID NO. 38 SEQ ID NO. 29-36 and JTSSR 3639 SEQ ID NO. 73740.

2. The primer combination for identifying relativity between individual Changjiang river finless porpoise as claimed in claim 1, wherein: the 5' end of at least one primer in each pair of primer pairs is marked by fluorescent dye.

3. The primer combination for identifying relativity between individual Changjiang river finless porpoise as claimed in claim 2, wherein: each pair of primers was labeled by staining with either FAM or HEX.

4. Kit comprising a combination of primers according to any one of claims 1 to 3 for identifying the genetic relationship between individual Changjiang river finless porpoise.

5. The kit of claim 4, wherein: consists of the following components: DNA template, 10 × reaction buffer, Mg²⁺dNTP, upstream and downstream primers, Taq enzyme and sterile double distilled water.

6. The method for identifying the genetic relationship between individual Changjiang river finless porpoise with the kit of claim 4, which comprises: the method comprises the following steps:

step 1, collecting blood of Yangtze river finless porpoise to be identified, and extracting genome DNA by adopting an alkaline lysis method;

step 2, taking the genome DNA obtained in the step 1 as a template to carry out PCR amplification;

step 3, carrying out capillary electrophoresis detection on the PCR amplification product obtained in the step 2 by using an ABI3730 sequencer full-automatic nucleic acid protein analyzer;

step 4, converting the fragment size of the sequencing peak diagram obtained in the step 3 into a microsatellite marker to obtain the genotype of each individual;

and 5, analyzing by using genetic analysis software, and identifying the relationship among half-siblings, full-siblings and parents of the Changjiang river finless porpoise individuals.

Images