Buffalo growth trait breeding molecular marker ghrl gene and application thereof

Abstract

The present application relates to the technical field of molecular biology and genetic breeding, and particularly relates to a buffalo growth trait breeding molecular marker GHRL gene, a nucleotide sequence of the GHRL gene is shown as SEQ ID NO.1; the molecular marker is located at the 295th base of a buffalo reference sequence EF583468.1 on a NCBI database, and a polymorphism site is G>C. Compared with the prior art, the present application separates out the GHRL gene, and G or C of the 165th base of the GHRL gene is a SNP site, the SNP site is firstly determined to exist correlation with buffalo growth traits such as body slant length, cross section height and body height, and the SNP site is applied to breeding and breeding process, so that breeding and breeding time can be shortened.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic breeding technology, specifically to a molecular marker for the selection of buffalo growth traits, the GHRL gene, and its application. Background Technology

[0002] Water buffalo are an important livestock in tropical and subtropical regions, playing a crucial role in the agricultural economies of many countries. They provide over 15% of the world's milk supply, and compared to cow's milk, buffalo milk contains richer amounts of fat, lactose, protein, and minerals. Buffalo milk is used to make butter, high-quality cheese, and other products. Compared to beef, buffalo meat has less fat and cholesterol. Furthermore, water buffalo are excellent draft animals, providing 20% ​​to 30% of agricultural labor, and are particularly effective in waterlogged conditions such as rice paddies. Therefore, selecting buffalo with superior growth performance is crucial for buffalo breeding.

[0003] SNP markers, or single nucleotide polymorphisms, refer to DNA sequence polymorphisms caused by nucleotide variations with a mutation frequency higher than 1%; transitions and transversions are the main types of SNP mutations. SNP markers have many advantages compared to other molecular genetic markers: a. They are numerous and have high genome coverage; SNP coverage in the human genome is as high as one in a thousand, and even higher in the genomes of some species. b. They exhibit bimorphism. c. They are representative; due to their high genome coverage, some SNPs located in coding regions may affect protein synthesis or even alter protein function, leading to changes in some traits of the organism; therefore, some SNPs are directly related to biological traits. d. They have strong genetic stability; because various physiological activities in organisms have precise regulatory mechanisms, the mutation rate per base in each generation is extremely low, allowing SNPs to be stably inherited between organisms and their offspring.

[0004] Growth and development capacity is a prerequisite for good production performance in buffaloes and plays a vital role in the economic benefits of the buffalo industry. To improve growth and development and increase the economic benefits of buffaloes, in addition to providing scientific and reasonable feeding and management, it is also necessary to select for and enhance the growth and development capacity of buffaloes based on their genetic makeup. Genetic methods can be used to genetically modify the phenotypic traits of buffaloes, thereby improving their growth and development capacity and production performance. Currently, there is limited research both domestically and internationally on the correlation between buffalo SNP markers and buffalo growth performance, and relevant molecular marker loci have not yet been identified. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for the selection of buffalo growth traits, the GHRL gene, and its application, aiming to obtain new molecular markers for the selection of buffalo growth traits and apply them to buffalo breeding and / or selection.

[0006] To achieve the above objectives, the present invention provides a molecular marker for the selection of buffalo growth traits, namely the GHRL gene, the nucleotide sequence of which is shown in SEQ ID NO.1; the molecular marker is located at the 295th base of the buffalo reference sequence EF583468.1 in the NCBI database, and its polymorphic site is G>C.

[0007] The nucleotide sequence of SEQ ID NO.1 is as follows: GCAGGTGAGACGCCACCCCAGGAGCCCCGCGTCCTGAATGCCCCGAGCCGTGTGAGCTGGGCAGTGGCTCGCCCTGTCTGAGCTTCAGCTTTCTCCCCGAGGCCGAAGGAGGGCTCTGGGTCTGACCGTGGGTCCACACCTCACCCTGCTTCTCGGAGGAGAGGGGGGATTCAGGGCCTAAGGGGAGCACCTCCTCTTTC CTGCAGAGAAAGGAACCTAAGAAGCCATCAGGCAGACTGAAGCCCCGGGCCCTGGAAGGCCAGTTTGACCCGGAGGTGGGAAGTCAGATGGAAGGTGCAGAGGACGAGCTGGAAATCCGGGTGGGTTCCTCTGCAGTAGGAGGTGGGGGTGGGGAGGGCGGCTACCCACAGACACCCACTTAGCAGCTACTCAAGGGACA.

[0008] Preferably, in the above technical solution, the 165th base of the GHRL gene is the molecular marker site.

[0009] Preferably, in the above technical solution, the molecular marker base is G or C, and the body oblique length, body height and cross height of GC type or CC type buffalo individuals are all longer than those of GG type buffalo individuals.

[0010] To achieve the above objectives, the present invention provides a primer set for detecting the GHRL gene, a molecular marker for breeding growth traits in buffalo. The primer set includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0011] The nucleotide sequence of SEQ ID NO.2 is specifically: F5´-GCAGGTGAGACGCCACCCCAGG-3´, and the nucleotide sequence of SEQ ID NO.3 is specifically: R5´-TGTCCCTTGAGTAGCTGCTAAG-3´.

[0012] To achieve the above objectives, the present invention also provides a kit for detecting the GHRL gene, a molecular marker for the selection of growth traits in buffalo, the kit comprising the aforementioned primer set.

[0013] To achieve the above objectives, the present invention also provides the application of a reagent for detecting the GHRL gene, a molecular marker for buffalo growth traits, in buffalo assisted breeding and / or selection. The molecular marker site is located at the 165th base of the GHRL gene, and the base is G or C. The breeding and / or selection is for obtaining buffaloes with better growth traits, including body length, body height, and cross height. The body length, body height, and cross height of GC or CC type buffalo individuals are all longer than those of GG type buffalo individuals.

[0014] To achieve the above objectives, the present invention also provides a method for using the GHRL gene, a molecular marker for selecting growth traits in buffalo, for assisted breeding and / or selection. The method involves extracting genomic DNA from buffaloes of the candidate breed and / or breeding stock, amplifying it using forward and reverse primers, detecting that the 51st base of SEQ ID NO.1 is G or C, genotyping the candidate breed and / or breeding stock according to GG, GC, or CC types, and selecting GC or CC type buffaloes for further selection and / or breeding based on the fact that the body length, body height, and cross height of GC or CC type buffaloes are all longer than those of GG type buffaloes. This results in obtaining buffalo breeds with superior growth traits.

[0015] Preferably, in the above technical solution, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0016] Preferably, in the above technical solution, the amplification conditions are: 95℃ for 3 minutes, cycled once; 95℃ for 30 seconds, 59℃ for 20 seconds, 72℃ for 30 seconds, cycled 34 times in total; and 72℃ for 10 minutes.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The GHRL gene was isolated, and the G or C at the 165th base of the isolated GHRL gene was identified as a SNP site. For the first time, it was clearly established that this SNP site is associated with buffalo growth traits such as body length, cross height, and body height. Applying this SNP site to the breeding and selection process can shorten the breeding and selection time. Furthermore, the detection method of this invention is simpler, has lower content, and provides more direct and reliable detection results, making it suitable for large-scale gene screening and diagnosis. Attached Figure Description

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0019] Figure 1 This is an electrophoresis image of the PCR products of this invention detected by 1.5% agarose gel electrophoresis; Figure 2 This is the sequencing diagram of the CC genotype of this invention; Figure 3 This is the sequencing diagram of the GC genotype of this invention; Figure 4 This is the sequencing diagram of the GG genotype of this invention. Detailed Implementation

[0020] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example 1

[0021] I. Extraction of bovine blood genomic DNA: The experimental cattle breed used in this invention are 60 24-month-old water buffaloes from the Guangxi Water Buffalo Research Institute.

[0022] Genomic DNA was extracted from buffalo blood using the TIANGEN Blood Genomic DNA Extraction Kit, following the kit's instructions. The extracted DNA was then tested for concentration and quality and stored at -20°C for later use.

[0023] II. Obtaining the GHRL gene, a molecular marker for selecting growth traits in buffalo. (1) Amplification to obtain PCR products Based on the SNP genetic marker detection sequence of buffalo reference sequence EF583468.1 from the NCBI database (as shown in SEQ ID NO. 1), a pair of primers was designed to amplify fragments at polymorphic sites. The primers are as follows: Forward primer: F5´-GCAGGTGAGACGCCACCCCAGG-3´; Reverse primer: R5´-TGTCCCTTGAGTAGCTGCTAAG-3´.

[0024] Using the primers described above, PCR amplification was performed using genomic DNA from the blood of a 24-month-old buffalo as a template.

[0025] PCR reaction system: Taq Mix 25 μL Sense Primer 1 μL Anti-sense Primer 1 μL Genomic DNA 1 μL ddH2O 22 μL Total Volume 50 μL PCR reaction conditions:

[0026] PCR products were detected by 1.5% agarose gel electrophoresis. The results are shown in the attached figure. Figure 1 As shown, lane M is the DL1000 Marker, and lanes 1-5 are amplified fragments of buffalo, with a fragment size of 400bp.

[0027] The specific nucleotide sequence of the GHRL gene is as follows: GCAGGTGAGACGCCACCCCAGGAGCCCCGCGTCCTGAATGCCCCGAGCCGTGTGAGCTGGGCAGTGGCTCGCCCTGTCTGAGCTTCAGCTTTCCCCGAGGCCGAAGGAGGGCTCTGGGTCTGACCGTGGGTCCACACCTCACCCTGCTTCTCGGAGGAGAGGGGGGATTCAGGGCCTAAGGGGAGCACC TCCTCTTTCCTGCAGAGAAAGGAACCTAAGAAGCCATCAGGCAGACTGAAGCCCCGGGCCCTGGAAGGCCAGTTTGACCCGGAGGTGGGAAGTCATGGAAGGTGCAGAGGACGAGCTGGAAATCCGGGTGGGTTCCTCTGCAGTAGGAGGTGGGGGTGGGGAGGGCGGCTACCCACAGACACCCACTTAGCAGCTACTCAAGGGACA.

[0028] (2) Purification of PCR products The PCR amplification products were purified using the Gel Extraction Kit from Shanghai Sangon Biotech Co., Ltd. Specific steps are detailed in the kit's instruction manual.

[0029] III. Sequencing methods for detecting molecular markers The purified PCR product obtained above was directly sent to Qingke Biotechnology for sequencing. Based on the sequencing results, the genotype of this locus in the test population was determined. Analysis was performed using Chromas software, and the results are shown in the attached figure. Figure 2 As shown, a G>C base mutation was found at 165bp in the sequence, which caused polymorphism at the base site.

[0030] Detection of polymorphic distribution of molecular marker sites in buffalo population The polymorphism distribution of the 295th base G>C site in the buffalo reference sequence EF583468.1 was detected in a population of 60 buffaloes using this molecular marker. The results are shown in Table 1.

[0031] Table 1. Base Genotype Frequency and Allele Frequency

[0032] As can be seen from Table 1, the frequency of the C allele is significantly greater than that of the G allele.

[0033] Table 2. Association analysis between the polymorphism of the 295th base of the reference sequence EF583468.1 and growth traits.

[0034] The advantages of this invention are that it provides, for the first time, a novel locus existing in the Chinese buffalo population and clarifies the correlation between the polymorphism of this locus and buffalo growth traits. The detection method of this invention is simple, low-cost, and provides direct and reliable results, making it suitable for large-scale gene screening and diagnosis.

[0035] As shown in Table 2, in 24-month-old buffalo, individuals with the GC and CC genotypes at the 295th base site of the buffalo reference sequence EF583468.1 had significantly higher body slant length than individuals with the GG genotype (P<0.01). There were no significant differences in body height and cross height between individuals with the GG, GC, and CC genotypes. Therefore, individuals with the GC and CC genotypes are the dominant genotypes, and individuals carrying these dominant alleles should be retained in breeding to improve the growth traits of the buffalo population, serving as potential genetic markers.

[0036] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A molecular marker for selecting growth traits in buffalo, the GHRL gene, characterized in that, The nucleotide sequence of the GHRL gene is shown in SEQ ID NO.1; the molecular marker is located at the 295th base of the buffalo reference sequence EF583468.1 in the NCBI database, and its polymorphic site is G>C.

2. The GHRL gene, a molecular marker for selecting buffalo growth traits as described in claim 1, is characterized in that... The 165th base of the GHRL gene is the molecular marker site.

3. The GHRL gene, a molecular marker for selecting buffalo growth traits as described in claim 2, is characterized in that... The molecular marker has a base of G or C. The body oblique length, body height and cross height of GC or CC type buffalo individuals are all longer than those of GG type buffalo individuals.

4. A primer set for detecting the GHRL gene, a molecular marker for breeding buffalo growth traits, characterized in that... The primer set includes a forward primer and a reverse primer, the nucleotide sequence of which is shown in SEQ ID NO.2 and the nucleotide sequence of which is shown in SEQ ID NO.

3.

5. A kit for detecting the GHRL gene, a molecular marker for breeding growth traits in buffalo, characterized in that, The kit comprises the primer set as described in claim 4.

6. The application of a reagent for detecting the GHRL gene, a molecular marker for buffalo growth traits, in buffalo assisted breeding and / or selection, characterized in that... The molecular marker site is located at the 165th base of the GHRL gene, and the base is G or C. The breeding and / or selection is to obtain buffaloes with better growth traits. The growth traits include body length, body height, and cross height. Among them, the body length, body height, and cross height of GC or CC type buffalo individuals are all longer than those of GG type buffalo individuals.

7. A method for using the GHRL gene, a molecular marker for selecting growth traits in buffalo, in assisted breeding and / or selection, characterized in that, The method involves extracting genomic DNA from buffaloes of the candidate breed and / or breeding stock, amplifying it using forward and reverse primers, detecting whether the 51st base of SEQ ID NO.1 is G or C, genotyping the candidate breed and / or breeding buffaloes according to GG, GC, or CC types, and selecting GC or CC type buffaloes for further selection and / or breeding based on the fact that the body length, body height, and cross height of GC or CC type buffaloes are all longer than those of GG type buffaloes. This results in obtaining buffalo breeds with superior growth traits.

8. The method for using the GHRL gene, a molecular marker for selecting buffalo growth traits as described in claim 7, in assisted breeding and / or selection, characterized in that... The nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

9. The method for using the GHRL gene, a molecular marker for selecting buffalo growth traits as described in claim 7, in assisted breeding and / or selection, characterized in that... The amplification conditions were: 95℃ for 3 minutes, cycled once; 95℃ for 30 seconds, 59℃ for 20 seconds, 72℃ for 30 seconds, cycled 34 times in total; and 72℃ for 10 minutes.

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