A method for molecular marker site-assisted detection of buffalo growth traits and its application

Abstract

The present invention discloses a method and application of a molecular marker site-assisted detection method for buffalo growth traits, wherein the molecular marker is located at the 247th base of the buffalo reference sequence XM_044935926 in the NCBI database, the base is C or T, and the nucleotide sequence of the molecular marker is as SEQ ID NO.1; the growth traits include the cross height and body oblique length of the buffalo. The molecular marker site obtained by the present invention can screen out and retain buffalo individuals carrying the dominant genotype TT at the molecular level, improve the accuracy of individual genetic assessment, shorten the generation interval in the buffalo breeding process, and effectively improve the efficiency and accuracy of seed selection.

Description

Technical Field

[0001] The invention relates to the technical field of molecular genetic markers, and in particular to a method for molecular marker site-assisted detection of buffalo growth traits and an application thereof. Background Art

[0002] Buffalo is an important livestock in tropical and subtropical regions, playing a pivotal role in the animal husbandry and agricultural economy of many countries, providing meat and milk for people in at least 67 countries. Buffaloes are treasures all over. The first treasure is buffalo milk, which accounts for more than 15% of the world's total milk supply. Compared with other milks, buffalo milk contains more fat, lactose, protein and minerals, and is often used as a raw material for making butter, high-quality cheese and other high-quality dairy products; the second treasure is buffalo meat, which has less fat and cholesterol than other beef; the third is buffalo skin, which can be used to make high-quality leather products. my country is the third largest country in buffalo breeding, and the breeding areas are mainly distributed in southern my country. The buffalo has the characteristics of tolerance to roughage, moisture, heat and disease, and high nutritional value of dairy products. Due to the low production performance of its population, river buffalo for dairy use were introduced in the 1950s to carry out genetic improvement of local swamp buffalo. However, compared with other cattle species, there is still a large gap in scientific research on buffalo, and it is urgent to continue to obtain better growth traits of buffalo through variety improvement or breeding to improve its economic value.

[0003] The fundamental purpose of breeding is to select favorable genes or favorable genotypes that express advantageous traits. Since the growth traits of buffalo are mainly quantitative traits, they are controlled by the joint action of multiple micro-effect genes. They are greatly affected by the environment during the breeding process and the separation generations after hybridization cannot be clearly grouped. Traditional animal breeding is essentially to break the dynamic balance of gene frequencies produced by random mating groups through multiple selections, selections and other breeding techniques, so as to achieve the purpose of increasing the frequency of favorable genotypes (or favorable genes) in the group. Therefore, the breeding cycle required for breeding buffalo according to traditional animal breeding methods is long and the effect is poor. At present, the genetic characteristics of buffalo can be studied in depth by studying the correlation between the polymorphism of related genes and growth traits, which can better understand the growth and development laws and genetic characteristics of buffalo, thereby providing a scientific basis for the precise selection and optimization of buffalo breeding. Therefore, the application of molecular marker technology in buffalo genetic breeding is of great significance, and it also has a positive role in improving the production efficiency and economic benefits of buffalo.

[0004] However, buffalo breeding technology is hampered by the relatively few auxiliary studies on molecular marker loci of buffalo and the fact that relevant molecular marker loci have not yet been discovered.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a method for molecular marker site-assisted detection of buffalo growth traits and its application, aiming to overcome the problem that there are few molecular marker sites associated with buffalo growth traits available for use during buffalo breeding, resulting in long buffalo selection and / or breeding cycles and poor results.

[0007] To achieve the above object, the present invention provides a molecular marker associated with the growth traits of buffalo, wherein the molecular marker is located at the 247th base of the buffalo reference sequence XM_044935926 in the NCBI database, the base is C or T, and the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1; wherein the specific details of SEQ ID NO.1 are as follows:

[0008] .

[0009] Preferably, in the above technical solution, the growth traits associated with the molecular markers include the cross height and body oblique length of the buffalo.

[0010] Preferably, in the above technical scheme, the dominant genotype individual of the molecular marker is the TT genotype individual; the principle is: the cross height of the buffalo of the TT genotype individual is better than the cross height of the buffalo of the CT genotype individual and the difference is significant, and the body oblique length of the buffalo of the TT genotype individual is better than the body oblique length of the buffalo of the CC genotype individual and the difference is significant.

[0011] A PCR amplification primer for amplifying the nucleotide sequence of the molecular marker as described above, the PCR amplification primer comprises a forward primer and a reverse primer, the forward primer is specifically shown as SEQ ID NO.2, the reverse primer is specifically shown as SEQ ID NO.3, wherein,

[0012] SEQ ID NO. 2 is F5′-AGGCACCTCCTCTTCATCT-3′;

[0013] SEQ ID NO. 3 is R5'-TGGAAGGCGAAATCAGTGG-3'.

[0014] A PCR amplification primer as described above is used in a kit for detecting molecular markers associated with growth traits of buffalo.

[0015] An application of the PCR amplification primer as described above in buffalo breeding.

[0016] A method for molecular marker site-assisted detection of buffalo growth traits, sequencing the nucleotide sequence of buffalo to be bred, genotyping the molecular markers according to the sequencing results, and selecting and retaining buffalo individuals carrying dominant genotypes for seed selection and / or breeding.

[0017] Preferably, in the above technical solution, the method comprises the following steps:

[0018] (1) designing amplification primers according to the nucleotide sequence SEQ ID NO.1 of the molecular marker, wherein the amplification primers consist of a forward primer and a reverse primer;

[0019] (2) using the amplification primers to perform PCR amplification using the blood genomic DNA of the buffalo to be bred as a template to obtain an amplification product;

[0020] (3) sequencing the amplified products, genotyping the molecular markers according to the sequencing results, and selecting buffalo individuals carrying the dominant genotype for seed selection and / or breeding.

[0021] Preferably, in the above technical solution, in step S3, the amplification product is sequenced, the molecular marker is genotyped according to the sequencing result, and the buffalo individuals carrying the dominant genotype are selected to carry out seed selection and / or breeding work, specifically:

[0022] (1) sequencing the amplified product, and genotyping the molecular marker according to the sequencing result, into CC genotype, CT genotype and TT genotype;

[0023] (2) According to the correlation analysis between polymorphism and growth traits of buffalo, the height of the cross section of buffalo with TT genotype is better than that of buffalo with CT genotype, and the body oblique length of buffalo with TT genotype is better than that of buffalo with CC genotype, which indicates that TT genotype is the dominant genotype.

[0024] (3) Select and retain buffalo individuals carrying the TT genotype for seed selection and / or breeding.

[0025] A method for assisting detection of buffalo growth traits using molecular marker sites as described above is used in buffalo selection and / or breeding.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention provides for the first time molecular marker loci associated with growth traits such as body height, cross height and body length in Chinese buffalo populations, and uses the molecular marker loci for buffalo selection and / or breeding to assist in detecting the genotype of buffalo, thereby predicting the growth traits of buffalo in advance based on the genotype, laying a theoretical foundation for genetic breeding and optimized breeding of buffalo.

[0028] (2) Screening out and retaining buffalo individuals carrying the dominant genotype TT at the molecular level not only improves the accuracy of individual genetic assessment, but also shortens the generation interval in the buffalo breeding process, effectively improving the efficiency and accuracy of seed selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is an electrophoretic diagram of the PCR amplification product of SEQ ID NO.1 in a specific embodiment of the present invention;

[0030] Figure 2 It is a sequencing peak diagram of the CC genotype of SEQ ID NO.1 in a specific embodiment of the present invention;

[0031] Figure 3 is a sequencing peak diagram of the CT genotype of SEQ ID NO.1 in a specific embodiment of the present invention;

[0032] Figure 4 It is a sequencing peak diagram of the TT genotype of SEQ ID NO.1 in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0033] The specific implementation modes of the present invention are described in detail below in conjunction with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0034] 1. Extraction of genomic DNA from bovine blood

[0035] The experimental cattle of the present invention are 60 24-month-old buffaloes from the Guangxi Buffalo Research Institute. The TIANGEN blood genome extraction kit is used to extract the bovine genomic DNA according to the kit instructions. The extracted DNA is tested for concentration and quality and stored in a -20°C refrigerator for later use.

[0036] 2. Obtaining the detection fragment of buffalo SNP genetic marker

[0037] (1) PCR amplification

[0038] A pair of primers was designed based on the SNP genetic marker detection sequence of the buffalo reference sequence XM_044935926 (shown in SEQ ID NO.1) to amplify the fragment of the polymorphic site. The primers are as follows:

[0039] Forward primer: F 5′-AGGCACCTCCTCTTCATCT-3′

[0040] Reverse primer: R 5′-TGGAAGGCGAAATCAGTGG-3′

[0041] PCR amplification was performed using the above primers and genomic DNA from the blood of 24-month-old buffalo as a template;

[0042] The PCR reaction system is:

[0043] TaqMix 25μL SensePrimer 1μL Anti-sense Primer 1μL Genomic DNA 1μL <![CDATA[ddH2O]]> 22μL TotalVolume 50μL

[0044] The PCR reaction conditions were:

[0045]

[0046] The PCR products were detected by 1.5% agarose gel electrophoresis. Figure 1 As shown, lane M is DL1000 Marker, lanes 1-5 are bovine amplified fragments, and the size of the amplified fragment is 687 bp.

[0047] (2) PCR purification

[0048] The PCR amplification product was purified using the Gel Extraction Kit from Shanghai Shenggong Biotechnology Co., Ltd. For specific steps, see the kit instructions.

[0049] 3. Detection of molecular markers using direct sequencing of PCR products

[0050] The PCR purified products obtained above were directly sent to Qingke Bio for sequencing. According to the sequencing results, the genotypes of this locus in the test population were CC genotype, CT genotype and TT genotype. The results were analyzed using Chromas software as shown in the attached figure. Figure 2 As shown, there is a C>T base mutation at 247bp in the sequence, and the above mutation causes polymorphism at the base site.

[0051] 4. Detection of polymorphic distribution of molecular marker loci in buffalo populations

[0052] This molecular marker locus was used to detect the polymorphism distribution pattern of the 247th base C>T site of the buffalo reference sequence XM_044935926 in a population of 60 buffaloes. The detection results are shown in Table 1.

[0053] Table 1 Base genotype frequencies and allele frequencies of 60 buffaloes

[0054]

[0055] Note: The numbers outside the brackets in Table 1 are the number of individuals, and the numbers inside the brackets are the genotype frequencies.

[0056] It can be seen from Table 1 that the frequency of the C allele is significantly greater than the frequency of the T allele, indicating that there is a C>T base mutation at the 247bp position of the buffalo reference sequence XM_044935926.

[0057] Table 2 Association analysis between polymorphism of molecular marker loci and growth traits

[0058]

[0059]

[0060] Note: The values ​​in the table are mean ± standard error. The values ​​marked with a and b in the same row are significant differences at the level of P < 0.05

[0061] As can be seen from Table 2, among 24-month-old buffaloes, the cross height of the TT genotype individuals at the 247th base C>T site was significantly higher than that of the CC genotype individuals (P<0.05), the body oblique length of the TT genotype individuals was significantly higher than that of the CT genotype individuals (P<0.05), and the body height and chest width of the individuals at the 247th base C>T site were not significantly different. Therefore, the TT genotype individuals are dominant genotype individuals, and individuals carrying this dominant allele should be retained in breeding, which is beneficial to improving the growth rate of the buffalo population and serving as a potential molecular marker to accelerate breeding progress.

[0062] Example 1

[0063] A method for molecular marker site-assisted detection of buffalo growth traits, the method comprising the following steps:

[0064] S1, designing amplification primers according to the nucleotide sequence SEQ ID NO.1 of the molecular marker, wherein the amplification primers consist of a forward primer and a reverse primer, wherein the forward primer is shown in SEQ ID NO.2, and the reverse primer is shown in SEQ ID NO.3;

[0065] S2, using the amplification primers to perform PCR amplification with the blood genomic DNA of the buffalo to be bred as a template to obtain an amplification product;

[0066] S3, sequencing the amplified products, and genotyping the molecular markers according to the sequencing results, and dividing them into CC genotype, CT genotype and TT genotype. According to the correlation analysis between the polymorphism and the growth traits of buffaloes, it is obtained that the cross height of buffaloes with TT genotype individuals is better than that of buffaloes with CT genotype individuals, and the body oblique length of buffaloes with TT genotype individuals is better than that of buffaloes with CC genotype individuals. It is concluded that the TT genotype individuals are the dominant genotype individuals, so the buffalo individuals carrying the TT genotype are selected and retained to carry out seed selection and / or breeding work.

[0067] The advantages of the present invention are that it provides a new locus existing in the Chinese buffalo population for the first time, and performs correlation analysis between the polymorphism of the locus and the growth traits of the buffalo, and obtains that the cross height and body oblique length of the buffalo individuals with the TT genotype of the locus are superior to those of the buffalo individuals with other genotypes. Furthermore, the detection method of the present invention is simple, low-cost, and the detection results are direct and reliable. It is suitable for large-scale screening and diagnosis of gene loci and can be applied to buffalo selection and / or breeding.

[0068] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A molecular marker associated with growth traits of buffalo, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, the 247th base of SEQ ID NO.1 is C or T, the buffalo growth traits associated with the molecular marker are the height of the cross section and the oblique length of the body of the buffalo, the dominant genotype individual of the molecular marker is the TT genotype individual, the height of the cross section of the buffalo of the TT genotype individual is better than the height of the cross section of the buffalo of the CT genotype individual, and the oblique length of the buffalo of the TT genotype individual is better than the oblique length of the buffalo of the CC genotype individual.

2. Application of a reagent for detecting a molecular marker associated with a growth trait of a buffalo in the selection and / or breeding of growth traits of a buffalo, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, the 247th base of the SEQ ID NO.1 is C or T, the buffalo growth traits associated with the molecular marker are the cross height and body oblique length of the buffalo, the dominant genotype individual of the molecular marker is the TT genotype individual, the cross height of the buffalo of the TT genotype individual is better than the cross height of the buffalo of the CT genotype individual, the body oblique length of the buffalo of the TT genotype individual is better than the body oblique length of the buffalo of the CC genotype individual, and the buffalo individuals carrying the TT genotype are selected and retained to carry out seed selection and / or breeding work.

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