Molecular marker g.198719C>T in the LUC7L gene and its application in detecting growth traits in goats.

Abstract

The application belongs to the technical field of biology and particularly relates to a molecular marker g.198719 C>T in LUC7L gene and application thereof in detection of goat growth traits. The application discloses a goat molecular marker which is applied to detection of goat growth traits or goat breeding. The goat SNP molecular marker is Chr25:198719 C>T on goat chromosome 25, and the reference genome is Capra hircus ARS1. The application further provides a detection reagent and kit of the molecular marker, and simultaneously provides a method for detecting goat growth traits and a breeding method for improving goat growth traits. The results of examples show that the molecular marker is significantly or extremely significantly related to goat growth traits (3-month chest circumference and pipe circumference, 6-month body height and chest circumference), and can be applied to genotype of a gene related to goat growth traits or correlation analysis related to goat growth traits. The application provides a new molecular marker resource for molecular marker assisted selection of goat growth traits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the molecular marker g.198719 C>T in the LUC7L gene and its application in detecting growth traits in goats. Background Technology

[0002] Goats, as one of the earliest domesticated livestock in ancient my country, have long provided humans with important livestock products such as meat, milk, and fur. In recent years, my country's demand for mutton has continued to grow, requiring large-scale imports of frozen bone-in mutton and frozen whole mutton from countries such as New Zealand and Australia every year. Since 2012, China has become the world's largest importer of mutton, reflecting a certain gap between domestic mutton supply and demand. Therefore, improving the meat production performance of domestic sheep is of great significance.

[0003] Chubao Blackhead Goat is a new breed of meat goat developed in my country. It not only boasts delicious meat with a mild muttony flavor but also inherits the high reproductive performance of its maternal parent, Macheng Black Goat. However, there is still room for improvement in its meat production capacity. The growth traits of goats mainly include indicators such as body weight, body size, daily weight gain, and feed conversion ratio. These traits directly relate to the meat production efficiency of goats and are therefore a key focus of genetic breeding research.

[0004] Single nucleotide polymorphisms (SNPs) refer to DNA sequence differences caused by variations in a single nucleotide (A, T, C, G) at a specific location in the genome. These variations mainly include base transitions, transversions, insertions, and deletions. SNPs are abundant, widely distributed, genetically stable, and easily detected using high-throughput automated methods, making them widely used in molecular breeding, gene mapping, population genetics, and evolutionary research. In modern breeding practices, using SNP markers with significant genetic effects for marker-assisted selection (MAS) and genomic selection (GS) can effectively accelerate the genetic improvement of growth traits in goats, thereby enhancing the meat production performance of offspring and promoting the efficient development of goat farming in my country.

[0005] Currently, several SNP loci associated with growth traits in goats have been reported, such as the c.454C>G locus in the goat NFAT5 gene, the c.1103G>A locus in the RSAD2 gene, and the g.7919G>A locus in the ZBP1 gene. However, existing marker resources are still insufficient to fully meet the needs of efficient breeding. Therefore, discovering new SNP loci associated with growth traits will provide new tools for marker-assisted selection in goats, helping to accelerate the breeding process of superior goats and promote the sustainable development of the goat industry. Summary of the Invention

[0006] The purpose of this invention is to provide a goat SNP molecular marker for use in goat growth trait detection or goat breeding.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides an application of goat SNP molecular markers, the application of which includes: goat growth trait detection or goat breeding; the goat SNP molecular marker is Chr25: 198719 C>T on goat chromosome 25, with Capra hircus ARS1 as the reference genome.

[0009] Furthermore, the SNP site is located at position 51 of the nucleotide sequence shown in SEQ ID NO:1, where position 51 is represented by N.

[0010] The present invention also provides a molecular marker detection reagent, the kit comprising primers or probes for detecting the above-mentioned SNP molecular markers.

[0011] The present invention also provides a molecular marker detection kit, the kit comprising the above-described detection reagents.

[0012] It also includes the application of the aforementioned detection reagents or kits in the detection of growth traits in goats or in goat breeding.

[0013] The present invention also provides a method for detecting growth traits in goats, the method comprising: detecting the above-mentioned molecular markers in goats, genotyping goats, and finding that the growth traits of CC-type goats are superior to those of CT and TT-type goats.

[0014] The present invention also provides a breeding method for improving the growth traits of goats, wherein the breeding method is to increase the frequency of the above-mentioned SNP molecular marker gene as C in the core goat herd.

[0015] The present invention also provides a breeding method for improving the growth traits of goats, the method comprising: detecting the above-mentioned molecular markers of goats, performing genotyping of goats based on the molecular markers, and selecting individuals with the genotype CC.

[0016] Preferably, the detection method includes one or more of the following: PCR amplification, gene sequencing, and molecular probes.

[0017] Preferably, the growth trait is any one or more of the following: chest circumference and cannon bone circumference at 3 months of age, body height and chest circumference at 6 months of age.

[0018] The present invention also provides a breeding method for improving the growth traits of goats, wherein the breeding method is to increase the frequency of the above-mentioned SNP molecular marker gene as C in the core goat herd.

[0019] The present invention has the following beneficial effects:

[0020] This invention uses the SNP locus Chr25: 198719 C>T for goat growth trait detection or goat breeding, selects genotypes that are beneficial to improving goat growth traits for breeding, thereby increasing the gene frequency of dominant alleles generation by generation, accelerating the process of breeding and improving goat breeds, and bringing economic benefits to goat farming.

[0021] The SNP molecular markers described in this invention are associated with chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference performance at 6 months of age. They can be applied to the genetic improvement of breeding sheep to enhance chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference performance at 6 months of age, thereby improving the chest circumference and cannon bone circumference performance of offspring at 3 months of age, and body height and chest circumference performance at 6 months of age, and thus increasing the market competitiveness of breeding enterprises. Detailed Implementation

[0022] This invention provides an application of a goat SNP molecular marker, including its use in goat growth trait detection or goat breeding; the goat SNP molecular marker is Chr25: 198719 C>T on goat chromosome 25, with the reference genome being Capra hircus ARS1. The SNP site is located at position 51 of the nucleotide sequence shown in SEQ ID NO:1, where position 51 is represented by N, and the base at this site is either C or T, where T at position 51 represents the nucleotide of the allelic mutation. The specific nucleotide sequence is as follows:

[0023] AAGCTGCACAGCATACCCTAAAATAAATTAACAAAATAAAGTGACCAAATNGTGGATTCCCAATGGGGACAGGAATGAAAACTGGTCTCAAAGAGTGAAGA

[0024] Specifically, the gene containing the molecular marker described in this invention is the LUC7L gene.

[0025] The present invention also provides a detection method and a detection reagent for detecting the molecular marker.

[0026] The method for detecting molecular markers in this invention can be performed using conventional methods in the field, including gene sequencing, molecular probes, liquid phase capture or mass spectrometry, etc.

[0027] The primers used in this invention can be primers designed by those skilled in the art according to primer design principles to amplify the sequence shown in SEQ ID NO:1, in order to detect the SNP marker genotypes associated with goat growth traits in this invention.

[0028] The detection reagents of the present invention also include reagents conventionally used in the art.

[0029] The breeding methods described in this invention include: molecular marker-assisted breeding, goat breed improvement, germplasm resource improvement, and genome selection.

[0030] This invention does not limit the breed of goat, but may include breeds such as Chubao Blackhead Goat, Macheng Black Goat, Boer Goat, Yichang White Goat, and Matou Goat.

[0031] In this embodiment of the invention, the Chubao black-headed sheep was used as a sample for the experiment.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0034] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0035] Example 1

[0036] Whole genome resequencing

[0037] 1. Blood sample collection and leukocyte separation

[0038] A 5 mL blood sample was collected from the goat's jugular vein using a veterinary lancet and placed in an EDTA anticoagulant tube. The anticoagulant tube was then placed in an ice box with plenty of ice packs and brought back to the laboratory. These samples were stored in a 4°C freezer. White blood cells were extracted from the blood sample, following these steps:

[0039] (1) Take 2-3 mL of blood sample into a 10 mL EP tube.

[0040] (2) Add ultrapure water to EP to make the total liquid volume 9 mL.

[0041] (3) Slowly invert the EP tube up and down 20 times and let it stand for 10 minutes.

[0042] (4) Place the EP tube into the centrifuge and centrifuge at 5000 rpm for 10 min.

[0043] (5) Slowly pour out the supernatant from the EP tube.

[0044] (6) Add ultrapure water again to make the total liquid volume 9 mL.

[0045] (7) Repeat steps (3), (4), and (5).

[0046] (8) After the separated white blood cells are numbered, they are placed in a -80℃ refrigerator.

[0047] 2. Genomic DNA extraction and whole-genome resequencing

[0048] DNA extraction from leukocytes was performed using the Tianmo Biotechnology Genomic DNA Mini-Extraction Kit (catalog number: d3024), with specific methods described in the kit's instruction manual. The quality-tested genomic DNA was sent to Beijing Novogene Technology Co., Ltd. for secondary quality control and library construction, followed by PE150 whole-genome resequencing on the BGI Genomics platform. Raw data was obtained in FASTQ format. High-depth whole-genome resequencing was performed on 34 samples, with an average sequencing depth of approximately 19.72X and a total data volume of 1.4T; low-depth whole-genome resequencing was performed on 466 samples, with an average sequencing depth of approximately 1.65X and a total data volume of 1.6T.

[0049] Example 2

[0050] Genome alignment, genetic variation detection and genotype filling

[0051] 1. Analysis of raw sequencing data and genome alignment

[0052] High-depth sequencing data and low-depth sequencing data are subjected to the same quality control process.

[0053] (1) The raw data was filtered using Fastp software. The filtering criteria were as follows: reads with a base quality value below 20 exceeding 30% were removed; reads with n bases greater than 5% were removed. Clean reads were obtained after the above quality control steps.

[0054] (2) Use BWA software to align cleanreads to the goat reference genome (Capra_hircus.ARS1).

[0055] (3) Use Samtools software to sort the compared BAM files.

[0056] (4) Use Picard to mark repeated reads.

[0057] (5) Use Samtools software to build indexes.

[0058] 2. Detection of variant sites and genotyping

[0059] (1) GATKHaploytypeCaller generates gvcf files for each sample according to the autosomal number.

[0060] (2) GATKCombineGVCFs merges the gvcf files of each sample of a single chromosome.

[0061] (3) GATKGenotypeGVCFs were used for population SNP calling based on chromosomes.

[0062] (4) GATKMergeVcfs merges the vcf files of autosomal populations.

[0063] (5) GATKSelectVariants filters the SNPs in the vcf files of the population.

[0064] (6) GATK Variant Filtration to mark false positive SNP sites.

[0065] (7) The grep command filters the marked SNP sites.

[0066] (8) Plink software filters SNP sites (geno0.1--maf0.05--hwe1e-06).

[0067] (9) Beagle software fills in the missing sites.

[0068] (10) Use Sentieon Haplotyper and GVCFtyper modules to detect and genotype population genome genetic variations.

[0069] (11) Genotyping was performed using Beagle to obtain the genotypes of all individuals at the SNP site Chr25: 198719 C>T.

[0070] Example 3

[0071] Association analysis of C>T molecular markers with growth traits in goats (chest circumference and cannon bone circumference at 3 months of age, body height and chest circumference at 6 months of age): Chr25: 198719

[0072] (1) Phenotyps used for the association analysis of genotype and growth traits shall be measured by professional technicians in strict accordance with the measurement specifications. During the measurement, the ewe being tested shall be standing upright on a solid and flat ground. The results shall be retained to one decimal place and expressed in centimeters (cm): body height is the vertical distance from the highest point of the mantle to the ground as measured by the measuring stick; chest circumference is the length of the posterior edge of the scapula around the chest as measured by the measuring tape; cannon circumference is the horizontal circumference of the thinnest part of the cannon bone of the left forelimb as measured by the measuring tape.

[0073] (2) The association analysis between genotype and growth traits was performed using a linear model in R. After screening for significant fixed effects (such as birth year, measured age, etc.) through stepwise regression, the following model was established for analysis:

[0074] Y = μ + G + F + e

[0075] Where Y is the observed trait value; μ is the population mean; G is the genotypic fixed effect of the SNP locus to be tested; F is the set of other fixed effects screened out by the above steps; and e is the random residual.

[0076] Association analysis results showed that Chr25: 198719 C>T was significantly or extremely significantly associated with goat growth traits (chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference at 6 months of age). The effects of different genotypes of this marker on goat growth traits are shown in Table 1.

[0077] Table 1. Differences in growth traits of goats with different genotypes of the molecular marker Chr25: 198719 C>T.

[0078]

[0079] Note: The sample sizes for the CC, CT, and TT genotypes were 200, 58, and 3 at 3 months of age, respectively, and 184, 60, and 3 at 6 months of age, respectively. Data in the same column with the same letter in the superscript indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0080] As shown in Table 1, for chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference at 6 months of age, individuals with genotype CC were significantly or extremely significantly higher than individuals with CT, indicating that C is an allele that is beneficial to the improvement of growth traits.

[0081] As demonstrated by the above examples, the molecular marker Chr25: 198719 C>T is significantly or highly significantly correlated with goat growth traits (chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference at 6 months of age). Individuals with the genotype CC have significantly higher chest circumference and cannon bone circumference at 3 months of age and body height at 6 months of age than individuals with the genotype CT. Individuals with the genotype CC have a highly significantly higher chest circumference at 6 months of age than individuals with the genotype CT, indicating that C is an allele that is beneficial for improving growth traits. This molecular marker can be used to detect molecular markers related to goat growth traits. Furthermore, when the 51st nucleotide of the sequence shown in SEQ ID NO:1 is C, it is beneficial for goats to have higher chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference at 6 months of age, which is of great significance for goat breeding.

[0082] The molecular markers provided by this invention can be applied to genotypic analysis of genes related to goat growth traits or association analysis of goat growth traits, providing a new molecular marker resource for marker-assisted selection of goat growth traits.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An application of goat SNP molecular markers, characterized in that, The applications include: goat growth trait detection or goat breeding; The goat SNP molecular marker is Chr25: 198719 C>T on goat chromosome 25, with the reference genome being Capra hircus ARS1; the SNP site is located at position 51 of the nucleotide sequence shown in SEQ ID NO:1, where position 51 is represented by N; the growth traits of CC-type goats are superior to those of CT and TT-type goats; The growth traits are any one or more of the following: chest circumference and cannon circumference at 3 months of age, body height and chest circumference at 6 months of age; the goat breeding is based on the selection of any one or more of the following: chest circumference and cannon circumference at 3 months of age, body height and chest circumference at 6 months of age.

2. A breeding method for improving the growth traits of goats, characterized in that, The method is as follows: detect molecular markers in goats, perform genotyping on goats based on molecular markers, and select individuals with the CC genotype; the growth traits are any one or more of the following: chest circumference and cannon bone circumference at 3 months of age, and body height and chest circumference at 6 months of age. The molecular marker is Chr25: 198719 C>T on goat chromosome 25, and the reference genome is Caprahircus ARS1.

3. The method according to claim 2, characterized in that, The detection methods include one or more of the following: PCR amplification, gene sequencing, and molecular probes.

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