CN118460729A - SNP molecular marker for detecting relationship between calving interval of Nile-Lafei buffalo and application thereof - Google Patents
SNP molecular marker for detecting relationship between calving interval of Nile-Lafei buffalo and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker for detecting the relationship between the calving interval of Nile-Lafei buffalo and application thereof. The SNP molecular marker is chr4_827220, can be used for identifying different propagation traits of Nile-Laffy buffalo, has the advantages of high accuracy, good stability, low cost, simple operation, convenience, rapidness and the like, can be used for auxiliary breeding of Nile-Laffy buffalo molecular markers, accelerates breeding of improved variety Nile-Laffy buffalo, can remarkably improve selection efficiency, shortens breeding period, reduces breeding and breeding cost, improves breeding efficiency, and has wide application prospect.
Description
The application relates to a division application of patent application 202410106024.9 for detecting SNP molecular marker combination and application of propagation traits of Nile-Lafei buffalo, and the application date is 2024.01.25.
Technical Field
The invention belongs to the technical field of molecular biology, and relates to SNP molecular marker combination for detecting propagation traits of Nile-Lafei buffalo and application thereof.
Background
The reproductive trait is an important component of the economic trait of buffalo, and the improvement of the reproductive rate of buffalo is a key link for increasing the economic benefit of the breeding industry. Buffalo is a single animal, usually one calf is produced, the breeding efficiency is low, the generation interval is long, and a long time span is required for breeding cattle and improving population genetics. Therefore, improving the fertility of buffalo is one of the main targets for new breed cultivation. While buffalo reproductive traits exhibit low to moderate genetic power, it is often difficult to genetically improve within a single breed in a short period of time using conventional breeding methods. Therefore, how to utilize modern molecular breeding technology to cultivate buffalo varieties with excellent economic characters has profound significance.
The Nile-Lafei buffalo is bred from Bakistan in China for nearly 50 years, and the breed is equivalent to the original place in terms of reproductive performance, growth and development, meat production and milk production performance. Under the environment of subtropical climate in south China, the feed has the characteristics of heat resistance, coarse feeding resistance, strong disease resistance, normal growth, strong adaptability and the like, and has better milk and meat dual-purpose performance compared with local swamp buffalo. The hybrid improvement is carried out by using the Nile-Lafei buffalo and the local buffalo, and the meat and milk production performance of the hybrid offspring can be greatly improved.
At present, the pure Nile-Lafei buffalo has the advantages of less feeding quantity, lower standardization degree, and deficient germplasm resources, and most cattle farms only select and breed seeds by the traditional breeding method, so that the genetic improvement of buffalo is slow to progress. In recent years, with rapid development of technology, molecular marker assisted breeding has become a new method for improving genetic traits. In early research on complex character phenotypes of livestock and poultry, QTL localization was mainly used to find the association between phenotypic variation and genomic variation. Although the method is a classical gene positioning method, the method is more suitable for genetic research of single-gene diseases or single-gene control traits, has limited detection effect on complex traits and traits with low genetic power, obtains a large QTL confidence interval, possibly contains hundreds of genes, is not beneficial to accurate positioning of subsequent functional genes, and has poor repeatability in different groups.
Compared with a QTL linkage analysis method, the whole genome linkage analysis (GWAS) is a method for carrying out overall linkage analysis on common genetic variation (single nucleotide polymorphism and copy number) in the whole genome range, the method takes natural population as a research object, combines the diversity of a target character phenotype and polymorphism of genes (or marker loci) based on Linkage Disequilibrium (LD) among genes (loci) remained after long-term recombination, and can directly identify the gene loci or marker loci which are closely related to the phenotype variation and have specific functions. The GWAS technology is adopted to conduct research in the whole genome range, multiple characters can be located at one time, the method is suitable for research in the aspects of locating character association intervals, functional gene research, development character breeding, functional marking and the like, and the obtained results are more reliable. The GWAS technology is taken as a new method and can be widely applied to the field of livestock breeding.
Disclosure of Invention
The invention aims at providing a SNP molecular marker combination for detecting propagation traits of Nile-Lafei buffalo and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
A SNP molecular marker combination for detecting a nile-raffia buffalo reproductive trait, the SNP molecular marker combination comprising chr24_37929, chr17_2661880, chr24_430958, chr4_827220, chr18_197177, and chr6_135119, wherein:
Chr24_37929 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.1, and the polymorphism is A or G;
chr17_2661880 is located at position 51 of the nucleotide sequence shown as SEQ ID NO.2, and the polymorphism is T or G;
Chr24_430958 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.3, and the polymorphism is A or G;
Chr4_827220 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.4, and the polymorphism is C or T;
chr18_197177 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.5, and the polymorphism is A or G;
chr6_135119 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.6, and the polymorphism is A or C;
the reproductive trait is the first mating month of age and/or calving interval reproductive trait.
It is another object of the present invention to provide a primer set for amplifying a SNP molecular marker combination as described above.
Further, the primer set comprises Forward primer and REVERSE PRIMER; wherein,
The sequences of Forward primer and REVERSE PRIMER primer in the chr24_37929 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.7 and SEQ ID NO. 8;
The sequences of Forward primer and REVERSE PRIMER primer in the chr17_2661880 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.9 and SEQ ID NO. 10;
The sequences of Forward primer and REVERSE PRIMER primer in the chr24_430958 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.11 and SEQ ID NO. 12;
The sequences of Forward primer and REVERSE PRIMER primer in the chr4_827220 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.13 and SEQ ID NO. 14;
The sequences of Forward primer and REVERSE PRIMER primer in the primer group marked by the chr18_197177 molecule are respectively shown as nucleotide sequences SEQ ID NO.15 and SEQ ID NO. 16;
the sequences of Forward primer and REVERSE PRIMER primer in the chr6_135119 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.17 and SEQ ID NO. 18.
It is another object of the present invention to provide a kit comprising the primer set as described above.
The invention also aims to provide an application of the SNP molecular marker combination or the primer set in the auxiliary breeding of the Nile-Lafei buffalo molecular marker.
The invention also aims at providing the application of the SNP molecular marker combination or the primer set in the identification of the propagation traits of the Nile-Latifolia buffalo variety.
It is another object of the present invention to provide a method for detecting the genotype of Nile-Lafei buffalo using molecular biological techniques, comprising the steps of: the nucleotide sequences of two flanks of the 6 SNP molecular marker loci according to claim 1 are respectively designed into amplification primers, the DNA of the Nile-Lafei buffalo individuals is used as a template for amplification, the first generation sequencing is carried out on the amplification products, and the Nile-Lafei buffalo individuals are subjected to genotyping according to the sequencing result, so that the genotypes of the Nile-Lafei buffalo individuals to be detected are identified.
Further described is the application of the method in the molecular marker assisted breeding of Nile-Lafei buffalo.
The invention also provides a method for breeding or assisting in breeding the Nile-Laffy buffalo variety or strain related to the propagation trait of the Nile-Laffy buffalo by using the SNP molecular marker, which comprises the following steps:
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 37929 th site of chromosome 24, measuring the deoxynucleotide at 37929 th site as A or G, determining that the genotype of Nile-Laffy buffalo to be detected is AA type, AG or GG type, selecting Nile-Laffy buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the initial mating month age of Nile-Laffy buffalo with AA type genes is far lower than AG type;
Extracting genome DNA of Nile-Lafei buffalo, detecting deoxynucleotide at 2661880 th site of chromosome 17, detecting that deoxynucleotide at 2661880 th site is T or G, determining that genotype of Nile-Lafei buffalo to be detected is TT type, GT type or GG type, selecting Nile-Lafei buffalo with TT type, GT type or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the first mating month age of Nile-Lafei buffalo with GG type genes is far lower than GT and TT type;
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 430958 th site of chromosome 24, detecting that deoxynucleotide at 430958 th site is A or G, determining that genotype of Nile-Laffy buffalo to be detected is AA type, GA or GG type, selecting Nile-Laffy buffalo with AA type, GA or GG type gene for next seed selection and/or breeding according to breeding requirement, wherein the initial month age of Nile-Laffy buffalo with AA type gene is far lower than GA type;
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 827220 th site of chromosome 4, detecting deoxynucleotide at 827220 th site as T or C, determining that genotype of Nile-Laffy buffalo to be detected is TT type, CT type or CC type, selecting Nile-Laffy buffalo with TT type, CT type or CC type genes for next seed selection and/or breeding according to breeding requirements, wherein calving interval of Nile-Laffy buffalo with TT type genes is far lower than TC and CC type;
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 197177 th site of 18 # chromosome, detecting that deoxynucleotide at 197177 th site is A or G, determining that genotype of Nile-Laffy buffalo to be detected is AA type, AG or GG type, selecting Nile-Laffy buffalo with AA type, AG or GG type gene for further seed selection and/or breeding according to breeding requirement, wherein calving interval of Nile-Laffy buffalo with AA type gene is far lower than AG type;
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 135119 th site of chromosome 6, detecting that deoxynucleotide at 135119 th site is A or C, determining that genotype of Nile-Laffy buffalo to be detected is AA type, CA or CC type, selecting Nile-Laffy buffalo with AA type, CA or CC type gene for further seed selection and/or breeding according to breeding requirement, wherein calving interval of Nile-Laffy buffalo with AA type gene is far lower than that of CA and CC type.
By adopting the technical scheme, the invention has the following beneficial effects:
The invention provides a SNP molecular marker combination related to the propagation trait phenotype of Nile-Laffy buffalo, which can be used for molecular marker assisted breeding of Nile-Laffy buffalo, and can be used for molecular marker assisted breeding of Nile-Laffy buffalo and accelerating breeding of improved variety of Nile-Laffy buffalo by screening SNP molecular markers obviously related to the propagation trait of Nile-Laffy buffalo by using whole genome association analysis. The SNP molecular marker is used for identifying the propagation traits of the Nile-Lafei buffalo, so that the selection efficiency can be obviously improved, the breeding period can be shortened, the breeding cost can be reduced, the breeding efficiency can be improved, and the method has wide application prospect.
Drawings
FIG. 1 is a diagram of the early mating of Manhattan for the genome-wide association analysis of propagation traits of Nile-Lafei buffalo of the present invention. Reference numerals illustrate: relates to the early-matched month-old character of Nile-Lafei buffalo, which is higher than 3 SNP molecular markers chr24_37929, chr17_2661880 and chr24_430958 screened by the invention on dotted lines, and the markers are respectively positioned on chromosome 24, chromosome 17 and chromosome 24 of Nile-Lafei buffalo.
FIG. 2 is a diagram of the initial mating month age qq of the Nile-Lafei buffalo reproductive trait whole genome correlation analysis of the present invention.
FIG. 3 is a calving interval Manhattan plot of the invention for a Nile-Lafei buffalo reproductive trait whole genome correlation analysis. Reference numerals illustrate: relates to the calving interval character of Nile-Lafei buffalo, which is higher than 3 SNP molecular markers chr4_827220, chr18_197177 and chr6_135119 screened by the invention on dotted lines, and the markers are respectively positioned on chromosome 4, chromosome 18 and chromosome 6 of Nile-Lafei buffalo.
FIG. 4 is a calving interval qq diagram of the Nile-Lafei buffalo reproductive trait whole genome correlation analysis of the present invention.
Detailed Description
The following is a further description of the specific embodiments of the invention with reference to the accompanying drawings.
Example 1 screening and development of SNP molecular marker combinations for detecting propagation traits of Nile-Lafei buffalo
1. Population selection and reproductive trait phenotype data collection
And selecting 142 NIR-Lafei buffalo with good health and development in the cattle farm of Guangxi buffalo institute, and collecting data related to the reproduction traits of the early matched month old and 1-2 calves at intervals.
2. Collecting, sorting and statistically analyzing propagation character phenotype data
Statistical analysis of the collected phenotypic data, including minimum, maximum, mean, standard deviation, coefficient of variation, was performed using SPSS20 software and the results are shown in table 1.
TABLE 1 statistical analysis of propagation trait phenotypes of Nile-Lafei buffalo
| Phenotype of phenotype | Minimum value | Maximum value | Average value of | Standard deviation of | Coefficient of variation |
| First month of the design is month old (month) | 18 | 56 | 31 | 7.66 | 24.71 |
| Calving interval (Tian) | 375 | 527 | 439.86 | 38.42 | 8.73 |
3. DNA extraction and detection
5ML of jugular vein blood of each cattle is collected, heparin sodium is anticoagulated, the mixture is gently shaken to be uniformly mixed, the genomic DNA of the cattle is extracted by adopting a blood genomic DNA extraction kit, the DNA concentration is detected by a nucleic acid concentration detector, and the mass of the DNA sample is determined by agarose gel electrophoresis of 1 percent. Sending the DNA sample which is qualified to Beijing NodeB origin bioinformation technology Co., ltd for whole genome re-sequencing, carrying out double-End (Paired-End) 150 sequencing by utilizing IlluminaHiSeq PE sequencing platforms, filtering sequencing machine data and obtaining effective information, and filtering the original data by the method that: (1) filtering reads containing the linker sequence; (2) When the N content in a single-ended sequencing read exceeds 10% of the length ratio of the strip, the pair PAIRED READS needs to be removed; (3) When the low mass (<=5) base number contained in a single-ended sequencing read exceeds 50% of the length proportion of the strip, the pair PAIRED READS needs to be removed; by stringent filtering of the sequencing data, CLEAN DATA of high quality is obtained. The statistics of the output data, including sequencing data yield, sequencing error rate, Q20 content, Q30 content, GC content are shown in Table 2, finally the high quality CLEAN DATA data size is 102.69Gb.
TABLE 2 Nile-Lafei buffalo genomic DNA sequencing results statistics
4. Genomic data alignment
Efficient high quality sequencing data was aligned to the reference genome by BWA bioinformatics software (parameters: mem-t4-k 32-M). The average comparison rate of the population samples with reference genome download address ：ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/471/725/GCF_000471725.1_UMD_CASPUR_WB_2.0/GCF_000471725.1_UMD_CASPUR_WB_2.0_genomic.fna.gz is 99.39%, the average sequencing depth of the genome is 13.97X, and the average 1X coverage (coverage of at least one base) is 3.62%, and the information is shown in Table 3.
TABLE 3 Nile-Lafei buffalo genome data alignment statistics
5. SNP quality control and filtration
Detection of population SNPs was performed using SAMTOOLS software. Polymorphic sites in the population are detected using a bayesian model. The quality control method comprises the following steps: (1) Filtering SNPs with the quality value of more than 20 (error rate more than 1 percent); (2) If the distance between the two SNPs is detected to be within 5bp, removing the SNPs; (3) The depth of coverage of SNPs is between 1/3 and 5 times the average depth. Quality control is carried out to preliminarily obtain 1972403 SNP loci, the obtained SNPs are filtered to obtain high-quality SNPs, the filtering conditions are dp2, miss0.2 and Maf0.01, and finally 205936 SNP loci are obtained in total for subsequent association analysis.
6. Whole genome association analysis
And carrying out association analysis by adopting GEMMA software and combining phenotype information and genome SNP information, and screening potential candidate SNPs when the-log 10 (P) >5 time difference is different and obvious through the associated significance (P-value). The results are shown in FIGS. 1 to 4, wherein Manhattan diagrams are graphs in which the effect value of genetic markers, namely the P value of the whole genome subjected to F test, is ordered according to the physical position on the chromosome, the abscissa is the physical position on the chromosome of the genome, the ordinate is-log 10P, and the smaller the P value, the stronger the correlation is, and the larger the ordinate is. The horizontal dashed line in the Manhattan plot represents the level of significance, and when-log 10 (P) >5, the SNP is considered significantly associated with the trait. qq plot (Quantile-quantileplot) representing a distribution of actual P values and unassociated zero hypothesis expected P values for detecting the impact of population stratification and individual affinity on association analysis, if observed P values and expected P values occur only at the far right end of the distribution, indicating that the trait is not caused by population stratification. QQplot are mainly used to estimate the difference between quantitative trait observations and predictions. Generally, quantitative trait data obtained by us are normal distribution data. The X and Y axes of QQplot in the GWAS study were predominantly-lgPvalues representing the individual SNPs. The predicted line is a dashed line at a 45 angle from the origin. In the GWAS analysis process, individual relationships and population stratification are the main factors responsible for false associations. Therefore, the mixed linear model is adopted to carry out the character association analysis, the population genetic structure is used as a fixed effect, the individual relationship is used as a random effect, and the influence of the population structure and the individual relationship is corrected:
y＝Xα+Zβ+Wμ+e
Wherein y is a phenotypic trait, X is an indication matrix of a fixed effect, and alpha is an estimated parameter of the fixed effect; z is an indication matrix of SNP, and beta is the effect of SNP; w is an indicator matrix of random effects, μ is the predicted random individuals, e is the random residual, subject to e to (0, δe2).
7. Screening and extracting SNP molecular markers related to initial mating month age and 1-2 calving interval propagation traits of Nile-Lafei buffalo
The number of independent SNP sites on the genome is obtained by using the parameters of PLINK, -indep-parilwise 2550.2, and the number of 1/SNP site is used as a multiple detection threshold. The polymorphic sites reaching significant association level are extracted by R software, preferably 6 SNP molecular marker sites respectively associated with the initial mating month age and 1-2 calving intervals of the Nile-Lafei buffalo, and can be used for breeding the propagation characters of the Nile-Lafei buffalo at the initial mating month age and 1-2 calving intervals. Details are shown in Table 4.
TABLE 4 information of 6 molecular markers selected
Example 2: application of primer group of 6 SNP (Single nucleotide polymorphism) marker loci in detection of propagation traits of Nile-Lafei buffalo
The Nile-Lafei buffalo 52 heads were selected. The jugular vein collects blood and extracts genomic DNA by using a blood genomic DNA extraction kit, and detects DNA concentration by using a nucleic acid concentration measuring instrument, and the mass of the DNA sample is measured by agarose gel electrophoresis of 1%. Amplification primers were designed for the 6 candidate SNP sites selected (see Table 5). The primer sequences are as follows:
Table 5 6 primer design for candidate SNP loci
Note that: f in the table is an upstream primer, forward primer, R is a downstream primer REVERSE PRIMER
PCR amplification was performed using the above primers and the Nile-Lafei buffalo blood genomic DNA as a template. A50. Mu.L PCR reaction system was used: ddH2O 19. Mu.L, premix Taq TM. Mu.L, DNA template 2.0. Mu.L, primers (10. Mu. Mol/L for both upstream and downstream primers) 2.0. Mu.L each.
PCR reaction conditions:
Pre-denaturation at 95 ℃ for 4min; denaturation at 94℃for 10s, annealing for 30s (60 ℃) and extension at 72℃for 1min for 35 cycles; extending at 72℃for 5min.
The PCR amplified product was purified by Gel Extraction Kit kit from Shanghai Biotechnology Co., ltd, and specific steps are shown in the kit instruction. The PCR purified product obtained above is recovered and directly sent to Huada gene (Shenzhen) Biotech company for first generation sequencing. And (5) genotyping the individual according to the sequencing result. Genotyping results are shown in Table 6.
TABLE 66 candidate SNP loci at Nile-Larphine buffalo genotype frequencies and allele frequencies
It can be seen from Table 6 that the A allele frequencies of mutation sites chr24_37929 and chr18_197177 are significantly greater than the G allele frequencies; the G allele frequencies at chr17_2661880 and chr24_430958 sites are significantly greater than the T and a allele frequencies; the C allele frequencies of the chr4_827220 and chr6_135119 sites are significantly greater than T and A.
TABLE 7 verification of significant association SNP for early matched month age of Nile-Lafei buffalo
The initial month-old table type value is expressed by 'least square mean value +/-standard deviation', and different letters of the same-column data shoulder marks show that the difference is obvious (P < 0.05); the same letters of the shoulder marks or no letter labels indicate that the difference is not significant (P > 0.05); the SNP locus genotypes are sequentially arranged according to mutant type, heterozygous type and reference type.
The results show that the candidate SNPchr24_37929, chr17_2661880 and chr24_430958 have obvious differences from the initial mating month ages of the niry-raffmos (table 7), the AA type initial mating month ages are obviously smaller than the GA type, the GG type initial mating month ages are obviously smaller than the GT type, the TT type and the AA type initial mating month ages are obviously smaller than the GA type, the initial mating month ages are small, the propagation interval can be shortened, and the method can be used for molecular marker assisted breeding for identifying the initial mating month ages of the propagation characteristics of the niry-raffmos and is applied to production.
TABLE 8 verification of significant correlation SNP at calving intervals for Nile-Lafei buffalo
Calving interval phenotype values are expressed by 'least square mean value +/-standard deviation', and different letters of the same-column data shoulder marks show significant differences (P < 0.05); the same letters of the shoulder marks or no letter labels indicate that the difference is not significant (P > 0.05); the SNP locus genotypes are sequentially arranged according to mutant type, heterozygous type and reference type.
The results show that the candidate chr4_827220, chr18_197177 and chr6_135119 have significant differences between different genotypes and the calving interval of the nii-raffia buffalo (see table 8), the TT-type calving interval of chr4_827220 is significantly shorter than the TC and CC types, and the TC and CC types are not significantly different; the AA calving interval of chr18_197177 is significantly shorter than AG and the AA calving interval of chr6_135119 is significantly shorter than CA and CC types, with no significant CA and CC type differences. The method shows that the calving interval is short, the reproduction period can be obviously shortened, and the method can be used for molecular marker assisted breeding for identifying the calving interval of the propagation characteristics of the Nile-Lafei buffalo and applied to production.
Example 4:
When the Nile-Lafei buffalo is bred, the 6 SNP molecular markers can be utilized, amplification primers are respectively designed on nucleotide sequences of two lateral wings of the Nile-Lafei buffalo, jugular vein blood of the Nile-Lafei buffalo is collected at 3-6 months of age, a blood genome DNA extraction kit is adopted to extract genome DNA, PCR amplification is carried out by taking individual DNA of the Nile-Lafei buffalo as a template, first generation sequencing of amplified products is carried out, genotyping is carried out according to a sequencing result, individuals with target character genotypes are reserved, the breeding period can be shortened, and the breeding speed is accelerated.
The SNP molecular marker breeding/auxiliary breeding method of the Nile-Laffy buffalo variety or strain related to the propagation trait of the Nile-Laffy buffalo can be applied, and the method comprises the following steps:
Extracting genome DNA of Nile-Lafei buffalo, detecting deoxynucleotide at 37929 th site of 24 # chromosome, measuring the deoxynucleotide at 37929 th site as A or G, determining that the genotype of Nile-Lafei buffalo to be detected is AA type, AG or GG type, selecting the Nile-Lafei buffalo with AA type, AG or GG type gene for further seed selection and/or breeding according to breeding requirements, wherein the initial mating month age of the Nile-Lafei buffalo with AA type gene is far lower than AG. And/or
Extracting genome DNA of Nile-Lafei buffalo, detecting deoxynucleotide at 2661880 th site of chromosome 17, detecting that deoxynucleotide at 2661880 th site is T or G, determining that genotype of Nile-Lafei buffalo to be detected is TT type, GT type or GG type, selecting Nile-Lafei buffalo with TT type, GT type or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the first mating month age of Nile-Lafei buffalo with GG type genes is far lower than GT and TT type. And/or
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 430958 th site of chromosome 24, measuring the deoxynucleotide at 430958 th site as A or G, determining that the genotype of Nile-Laffy buffalo to be detected is AA type, GA or GG type, selecting the Nile-Laffy buffalo with AA type, GA or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the initial mating month age of the Nile-Laffy buffalo with AA type genes is far lower than that of the GA type. And/or
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 827220 th site of chromosome 4, detecting deoxynucleotide at 827220 th site as T or C, determining that genotype of Nile-Laffy buffalo to be detected is TT type, TC or CC type, selecting Nile-Laffy buffalo with TT type, TC or CC type genes for next seed selection and/or breeding according to breeding requirements, wherein calving interval of Nile-Laffy buffalo with TT type genes is far lower than TC and CC type. And/or
Extracting genome DNA of Nile-Lafei buffalo, detecting deoxynucleotide at 197177 th site of 18 # chromosome, measuring the deoxynucleotide at 197177 th site as A or G, determining that the genotype of Nile-Lafei buffalo to be detected is AA type, AG or GG type, selecting Nile-Lafei buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the calving interval of Nile-Lafei buffalo with AA type genes is far lower than AG type. And/or
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 135119 th site of chromosome 6, detecting that deoxynucleotide at 135119 th site is A or C, determining that genotype of Nile-Laffy buffalo to be detected is AA type, CA or CC type, selecting Nile-Laffy buffalo with AA type, CA or CC type gene for further seed selection and/or breeding according to breeding requirement, wherein calving interval of Nile-Laffy buffalo with AA type gene is far lower than that of CA and CC type.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (9)
1. The SNP molecular marker for detecting the relationship between the calving interval of the Nile-Lafei buffalo is characterized in that: the SNP molecular marker is chr4_827220, wherein: chr4-827220 is located at position 51 of the nucleotide sequence shown as SEQ ID NO.4, and the polymorphism is C or T.
2. A primer set for amplifying the SNP molecular marker according to claim 1.
3. The primer set according to claim 2, wherein: the primer set comprises Forwardprimer and REVERSE PRIMER; wherein, the sequences of Forwardprimer and REVERSE PRIMER primers in the primer group marked by the chr4_827220 molecule are respectively shown as the nucleotide sequences SEQ ID NO.13 and SEQ ID NO. 14.
4. A kit comprising the primer set of claim 2 or 3.
5. Use of the SNP molecular marker of claim 1 or the primer set of any one of claims 2 or 3 in niri-raffia buffalo molecular marker assisted breeding.
6. Use of the SNP molecular marker of claim 1 or the primer set of any one of claims 2 or 3 in the identification of the propagation trait of niri-raffia buffalo.
7. A method for detecting the genotype of niry-raffia buffalo by using a molecular biological technique, which is characterized by comprising the following steps: the SNP molecular marker locus according to claim 1, wherein the nucleotide sequences of both flanks of the SNP molecular marker locus are respectively designed into amplification primers, the DNA of the Nile-Lafei buffalo individual is used as a template for amplification, the first generation sequencing is carried out on the amplification products, and the Nile-Lafei buffalo individual is subjected to genotyping according to the sequencing result, so that the genotype of the Nile-Lafei buffalo individual to be detected is identified.
8. Use of the method of claim 7 in the molecular marker assisted breeding of niri-raffia buffalo.
9. A method for breeding/assisting in breeding a variety or strain of niri-raffia buffalo associated with calving intervals using the SNP molecular marker of claim 1, characterized in that the method is:
Extracting genome DNA of Nile-Laffy buffalo, detecting deoxynucleotide at 827220 th site of chromosome 4, detecting deoxynucleotide at 827220 th site as T or C, determining that genotype of Nile-Laffy buffalo to be detected is TT type, TC or CC type, selecting Nile-Laffy buffalo with TT type, TC or CC type genes for next seed selection and/or breeding according to breeding requirements, wherein calving interval of Nile-Laffy buffalo with TT type genes is far lower than TC and CC type.
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| CN202410106024.9A CN117904317B (en) | 2024-01-25 | 2024-01-25 | SNP molecular marker combination for detecting propagation traits of Nile-Lafei buffalo and application |
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| CN202410616177.8A Pending CN118308500A (en) | 2024-01-25 | 2024-01-25 | SNP molecular marker for detecting related to early matched month age of Nile-Lafei buffalo and application thereof |
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| CN202410616318.6A Pending CN118813809A (en) | 2024-01-25 | 2024-01-25 | SNP molecular markers associated with first mating age of Nili-Lafite buffalo and their application |
| CN202410616785.9A Pending CN118345177A (en) | 2024-01-25 | 2024-01-25 | SNP molecular marker related to propagation traits of Nile-Lafei buffalo and application thereof |
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| CN202410616177.8A Pending CN118308500A (en) | 2024-01-25 | 2024-01-25 | SNP molecular marker for detecting related to early matched month age of Nile-Lafei buffalo and application thereof |
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| CN202410616785.9A Pending CN118345177A (en) | 2024-01-25 | 2024-01-25 | SNP molecular marker related to propagation traits of Nile-Lafei buffalo and application thereof |
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| CN112662789B (en) * | 2021-01-28 | 2024-04-23 | 武汉市农业科学院 | SNP (Single nucleotide polymorphism) marker related to birth age of Holstein cows in south China and application thereof |
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