Molecular marker, primer and kit for identifying golden-screen geese and Taihu geese and application of molecular marker, primer and kit

Abstract

The invention relates to the technical field of germplasm resource identification, in particular to a molecular marker, a primer and a kit for identifying golden-screen geese and Taihu Lake geese and application of the molecular marker, the primer and the kit. According to the present invention, the difference between the pantoyl mercaptoethylamine enzyme (VNN1) gene sequences of the local Guizhou golden geese and the Jiangsu Taihu goose is utilized to find that the five polymorphic sites capable of accurately identifying the local Guizhou golden geese and the Jiangsu Taihu goose can be accurately identified, and the primers are designed based on the polymorphic sites so as to identify the local Guizhou golden geese and the Jiangsu Taihu goose; two different varieties of meat geese, namely local white geese in Guizhou and white geese in Taihu Lake, can be quickly and accurately identified, so that a molecular identification means is provided for purifying resources of the local variety of meat geese and accurately screening specific functional genes in germplasm resources of the local meat geese in a pure breeding and breeding process of the local variety of meat geese; moreover, a scientific basis is provided for retention of dominant genes of the local meat goose variety, and considerable economic benefits and use values are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of germplasm resource identification, in particular to a molecular marker, a primer and a kit for identifying Jinping goose and Taihu goose and an application thereof. Background Art

[0002] With the development of the meat goose industry, the demand for purebred breeding of high-quality meat goose breed resources is increasing. Guizhou Jinping geese and Taihu geese are both high-quality meat goose germplasm resources in my country, and are both local meat goose breed resources in the national livestock and poultry breed resources. In view of the characteristics of the goose eggs of Guizhou local white geese and Taihu white geese, which have similar body shape and appearance, there is a problem that it is not easy to distinguish local meat goose germplasm resources from foreign breed resources in the production of breeding geese. However, it will take time and increase the cost of raising geese to distinguish them after the meat geese grow up. Therefore, purebred breeding of germplasm resources is required in the breeding process of new meat goose varieties. In the breeding process, it is necessary to screen and identify some genes with special functions of local meat goose germplasm resources, especially to accurately select and breed the gene sequences with molecular characteristics unique to the genes of local resources. The breeding of a new variety includes two technologies: purebred breeding and hybridization. Whether it is the purebred breeding of the breed or the hybrid advantage, it is necessary to first accurately judge the breed of germplasm resources. Only with good breeding geese can high-quality new meat goose varieties be bred. However, the current breeding and selection process mostly adopts the traditional breeding method of judging the breed resources based on body shape and appearance. The breeding goose breed needs to have typical body shape and appearance characteristics after it grows up. This traditional breeding and selection method requires a long time period and is not conducive to the retention of superior genes. It is difficult to accurately select breeding geese and offspring with excellent traits.

[0003] Taihu geese are native to the Taihu Lake area of ​​the Yangtze River Delta. They are found in the areas around Taihu Lake in Jiangsu and Zhejiang provinces, the suburbs of Shanghai, most of Jiangsu Province, and Changwu, Liyang. Taihu geese are small to medium sized, with delicate and compact physiques and close-fitting feathers all over their bodies. There are no tumors or wrinkles. The neck is slender and arched, with no throat pouches or bags. From the appearance, there is not much difference between male and female geese. Male geese are taller and more majestic, often walking with their heads held high and their chests stretched out and wings spread out, with loud calls and love to chase and peck people; female geese are docile, with lower calls, smaller tumors than male geese, and shorter beaks. The feathers of the whole body are white, with occasional gray-brown spots on the corners of the eyes, the top of the head, and the waist and back; the beak, shanks, and webbed feet are all orange-red, with lighter color at the tip of the beak and white claws; the eyelids are light yellow, and the iris is gray-blue. The whole body of the gosling is milky yellow, with orange-yellow beaks, shanks, and webbed feet.

[0004] Jinping geese are a new breed of meat geese that has been cultivated in Jinping County, Guizhou Province in recent years. Jinping County has developed the ecological goose industry as the leading industry of "one county, one industry", and strived to create an industrial development system integrating goose breeding, goose farming, goose sales and goose down product processing to drive farmers to increase their income and become rich. Jinping geese include the blood source of meat goose breed resources such as Guizhou Zhijin White Goose, Sichuan White Goose, and Yuzhou White Goose. The feathers on the whole body are white, with a small amount of gray-brown spots on the corners of the eyes, the top of the head, and the waist and back; the beak, shanks, and webs are all orange-red, the tip of the beak is lighter, and the claws are white; the eyelids are light yellow, and the iris is gray-blue. The whole body of the gosling is milky yellow, and the beak, shanks, and webs are orange-yellow.

[0005] With the development of molecular technology, molecular technology has been applied to different fields. This technology uses molecular biology technology to conduct early molecular identification of Guizhou's local Jinping geese and Taihu geese from the breeding egg or gosling stage to solve the above-mentioned problems. It can achieve the purpose of early accurate identification of Guizhou's local Jinping geese and Taihu geese and shorten the breeding cycle of new varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular identification technology and application for early identification of Guizhou native Jinping geese and Taihu white geese, so as to solve the problems of difficult distinction in the production management of early breeding goose eggs and goslings and long breeding cycle in the above-mentioned breeding goose breeding process. Through the designed primer pairs, accurate distinction and identification of the breeding eggs and goslings of Guizhou native white geese and Taihu white geese can be achieved, which is of great significance to the purebred breeding, selection and cultivation of new varieties of Guizhou local meat geese.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The primers (SEQ ID No.2-3) designed by the present invention are used to clone the CDs region sequence of the pantethioethylamine (VNN1) gene of Guizhou Jinping goose. The sequence is shown in SEQ ID No.1. The sequence is compared with the CDs region sequence of the VNN1 gene of Taihu goose (NCBI Accession No: KY399733.1). It is found that the differences in nucleotide sequences between the two meat goose breeds lead to the following mutations in amino acids: the 1225th position (A / G) leads to the difference in amino acid sequence at position 410 (glycine G / arginine R); the mutations at three sites, 1426th position (C / G), 1429th position (T / G) and 1431st position (A / C), lead to the nucleotide sequence (CTCTTA / GTCGTC) leading to the difference in amino acid sequence at positions 476-477, which is that two leucines are replaced by two valines (leucine L, leucine L / Valine V, Valine V); mutations and deletions at positions 1411 (T / A) and others result in the nucleotide sequence 1407-1418 (A-ATAGCACATCA / ACAAAAC-CAACA) leading to the amino acid sequence at positions 470-473 (NSTS / TKPT).

[0009] The VNN1 gene of Guizhou Jinping goose and Taihu goose was analyzed and it was found that the nucleotide sequence of the CDs region of the VNN1 gene had 5 SNP sites, specifically: 1225th position A / G, 1411th position T / A, 1426th position C / G, 1429th position T / G, and 1431st position A / C. When the nucleotide sequence of the CDS region of the VNN1 gene was 1225th position A, 1411th position T, 1426th position C, 1429th position T and 1431st position A, the tested breed was Jinping white goose; when the nucleotide sequence of the CDs region of the VNN1 gene was 1225th position G, 1411th position A, 1426th position G, 1429th position G and 1431st position C, the tested breed was Taihu white goose.

[0010] Another object of the present invention is to provide a kit for identifying the varieties of Jinping White Goose and Taihu White Goose, wherein the kit contains primers as shown in SEQ ID No. 2-3.

[0011] Another aspect of the present invention is to provide a method for identifying Jinping White Goose and Taihu White Goose varieties by using the VNN1 gene as a molecular marker as shown in SEQ ID No. 1. The total mRNA is extracted by the Trizol method, reverse transcribed into cDNA, and then PCR amplified using the primers described in claim 4. The PCR product is detected by 1.5% agarose gel electrophoresis. The PCR product after detection is directly sequenced by Sanger using the upstream primers. The sequenced sequences are compared and it is found that at the 1225th A / G, the 1411th T / A, the 1426th C / G, the 1429th T / G, the 1431st A / C The nucleotide sequence at 5 positions; when the 1225th, 1411th, 1426th, 1429th, and 1431st positions are A, T, C, T, and A respectively, it is a Jinping White Goose; when the 1225th, 1411th, 1426th, 1429th, and 1431st positions are G, A, G, G, and C respectively, it is a Taihu White Goose.

[0012] Furthermore, the PCR amplification reaction system is: 24 μL of PCR mixture, 2 μL of upstream and downstream primers, 4 μL of cDNA template and 18 μL of double distilled water;

[0013] The reaction procedure was as follows: pre-denaturation at 95°C for 5 min; 25 cycles of 94°C for 45 s, 50°C for 40 s, and 72°C for 40 s; and extension at 72°C for 5 min.

[0014] The beneficial technical effect of the present invention is as follows: the present invention utilizes the difference in the pantetheinase (VNN1) gene sequence between the local Jinping geese in Guizhou and the Taihu geese in Jiangsu to discover 5 polymorphic sites that can accurately identify the local Jinping geese in Guizhou and the Taihu geese in Jiangsu, and designs primers based on the polymorphic sites to achieve rapid and accurate identification of two different breeds of meat geese, the local white geese in Guizhou and the Taihu white geese, which provides a molecular identification means for purifying the resources of the local breed of meat geese and accurately screening out the unique functional genes in the germplasm resources of the local meat goose during the pure breeding and selection of the local breed of meat geese, and provides a scientific basis for the retention of the superior genes of the local meat goose breed, and has considerable economic benefits and use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1Nucleotide sequence comparison of VNN1 gene of Jingping goose and Taihu goose at NCBI; the nucleotide sequence represented by Sbjct in the figure is the nucleotide sequence of Jingping goose; the nucleotide sequence represented by Query is the nucleotide sequence of Taihu goose.

[0017] Figure 2 Comparison of amino acid sequences corresponding to the VNN1 gene of Jingping goose and Taihu goose; the amino acid sequence represented by Sbjct in the figure is the nucleotide sequence of Jingping goose; the amino acid sequence represented by Query is the nucleotide sequence of Taihu goose. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1

[0020] A method for identifying the varieties of Guizhou Jinping goose and Taihu goose

[0021] 1. Primer design

[0022] The primers designed to clone the VNN1 gene sequence of Jinping goose were as follows:

[0023] Upstream primer (SEQ ID No. 2): 5′-CCTGGGGAATTTGAGGTGCT-3′;

[0024] Downstream primer (SEQ ID No. 3): 5'-TTATGGGGAGTTTCGCAGGG-3'.

[0025] 2. PCR amplification

[0026] Select breeding eggs or goslings of Guizhou Jinping geese and Taihu white geese, which are similar in size and appearance, and use tweezers to cut a small piece of tissue. Then, use the Trizol method to extract total mRNA, reverse transcribe it into cDNA, and then amplify it using the designed primers.

[0027] The PCR reaction system is as follows: 50 μL of PCR amplification reaction system includes: 24 μL of PCR mixture, 2 μL of upstream and downstream primers, 4 μL of cDNA template and 18 μL of double distilled water.

[0028] The PCR amplification reaction program was as follows: pre-change at 95°C for 5 min; 94°C for 45 s, 50°C for 40 s, and 72°C for 40 s, for 25 cycles; and extension at 72°C for 5 min.

[0029] 3. Electrophoresis detection

[0030] The PCR product was detected by 1.5% agarose gel electrophoresis, and a specific band appeared at 715 bp, and its fragment size was consistent with the expected result. The detected PCR product was sent to Hunan Qingke Biological Co., Ltd. for Sanger direct sequencing using upstream primers.

[0031] 4. Results Analysis

[0032] 1. Nucleotide sequence mutation, analysis results such as Figure 1 shown.

[0033] ①There are 16 mutations at one nucleotide site: at position 54 (G / T), position 217 (C / T), position 508 (G / T), position 700 (G / A), position 1213 (C / T), position 1225 (A / G), position 1452 (G / A), position 1260 (G / A), position 1392 (A / G), position 1396 (C / T), position 1411 (T / A), position 1426 (C / G), position 1429 (T / G), position 1431 (A / C), position 1452 (A / T), and position 1452 (A / G).

[0034] ②There are 4 mutations at 2 nucleotide sites: 1380-1381 (GC / AT), 1399-1400 (GA / AT), 1436-1437 (CA / TG), and 1449-1450 (AA / TC).

[0035] ③One of the three nucleotide sites mutated: 1383-1385 (ACC / GAA).

[0036] 2. Amino acid sequence mutations, analysis results are as follows Figure 2 shown.

[0037] ① There are 5 mutations at one amino acid site: 410 (G / R), 462 (P / N), 467 (E / I), 479 (A / V), and 485 (C / W).

[0038] ②There is one mutation at two amino acid sites: at position 476-477 (LL / VV).

[0039] ③One of the four amino acid sites mutated: at position 470-473 (NSTS / TKPT).

[0040] It was finally determined that when the 1225th, 1411th, 1426th, 1429th, and 1431st are A, T, C, T, A respectively, it is the Guizhou Jinping Goose; when the 1225th, 1411th, 1426th, 1429th, and 1431st are G, A, G, G, C respectively, it is the Taihu Goose.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of VNN1 gene as a molecular marker in the identification of Jinping goose and Taihu goose varieties, characterized in that: The nucleotide sequence of the CDs region of the VNN1 gene has 5 SNP sites, specifically: A / G at position 1225, T / A at position 1411, C / G at position 1426, T / G at position 1429, and A / C at position 1431.

2. The application according to claim 1, characterized in that: When the nucleotide sequence of the CDs region of the VNN1 gene is A at position 1225, T at position 1411, C at position 1426, T at position 1429 and A at position 1431, the breed to be tested is Jinping goose; when the nucleotide sequence of the CDS region of the VNN1 gene is G at position 1225, A at position 1411, G at position 1426, G at position 1429 and C at position 1431, the breed to be tested is Taihu goose.

3. Primers for amplifying the VNN1 gene according to claim 1, characterized in that: The primer sequences are shown in SEQ ID No. 2-3.

4. A kit for identifying the species of Jinping goose and Taihu goose, characterized in that: The kit contains the primers according to claim 3.

5. A method for identifying Jinping goose and Taihu goose breeds using VNN1 gene as a molecular marker, characterized in that: Total mRNA was extracted by the Trizol method, reverse transcribed into cDNA, and then PCR amplified using the primers described in claim 3. The PCR products were detected by 1.5% agarose gel electrophoresis. The PCR products after detection were directly sequenced by Sanger using upstream primers, and the sequencing sequences were compared to find nucleotide sequences at five positions of the VNN1 gene: A / G at position 1225, T / A at position 1411, C / G at position 1426, T / G at position 1429, and A / C at position 1431; when positions 1225, 1411, 1426, 1429, and 1431 are A, T, C, T, and A, respectively, it is a Jinping goose; when positions 1225, 1411, 1426, 1429, and 1431 are G, A, G, G, and C, respectively, it is a Taihu goose.

6. The method according to claim 5, characterized in that: The PCR amplification: The reaction system was: 24 μL of PCR mixture, 2 μL of upstream and downstream primers, 4 μL of cDNA template, and 18 μL of double distilled water; The reaction procedure was as follows: pre-denaturation at 95°C for 5 min; 25 cycles of 94°C for 45 s, 50°C for 40 s, and 72°C for 40 s; and extension at 72°C for 5 min.

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