A set of microsatellite markers, detection primers thereof and application thereof

Abstract

The present application belongs to the field of genetic engineering, and provides a method for inbreeding recession risk assessment or genetic assessment, and a microsatellite marker combination or primer combination used in the method. The present application increases the microsatellite markers of bovine chromosome 23 from the known 6 to 34, basically covers the whole bovine chromosome 23, greatly improves the density of the microsatellite markers, and has good spatial distribution uniformity. The 28 microsatellite markers obtained by screening using the present application can prevent the economic risk caused by inbreeding recession, genetically assess the inbreeding degree of different individuals in advance, more accurately assess the genetic polymorphism of a population, and provide a basis for scientific decision-making in the process of cattle breeding.

Description

Technical Field

[0001] This invention relates to the development and utilization of molecular markers, and in particular to a set of microsatellite markers, their detection primers, and their applications. Background Technology

[0002] Inbreeding depression is a serious problem in modern cattle farming. The main causes are twofold: firstly, the use of artificial insemination techniques, where the semen of one bull is used for tens of thousands of cows, and superovulation further exacerbates the exploitation of cows; secondly, in the breeding process, regardless of whether genomic selection, pedigree selection, optimal linear unbiased estimation, or marker-assisted selection are used, all are elimination selection techniques. As the breeding process progresses, the genetic variance of some important basic and core populations gradually decreases. These factors combined have led to inbreeding depression becoming a very common phenomenon in the cattle industry. Specific manifestations of inbreeding depression include embryonic death in cattle, abortion in cows, calf malformations, and decreased productivity. In the molecular mechanism of habitual abortion in humans, the deletion of heterozygotes at multiple MHC loci has been confirmed to prevent pregnant women from producing protective antibodies against the fetus, thus inducing abortion.

[0003] In the cattle industry, molecular marker-based genetic assessment is applied to many stages, including introduction of new breeds, construction of breeding core populations, construction of propagation populations, inbreeding risk assessment, individual identification, pedigree construction, and genetic diversity assessment. Among these, the Major Histocompatibility Complex (MHC) holds a crucial position. It is the region with the highest genetic polymorphism in the vertebrate genome, making it the most sensitive region for population genetic polymorphism changes in a species. It exhibits tight linkage and haplotype inheritance characteristics. In the bovine genome, the MHC region is located on chromosome 23, and its genetic polymorphism is extensively linked to various economic traits and disease resistance.

[0004] Therefore, utilizing DNA polymorphisms in the MHC region and its extension region has become a hot topic in the development and utilization of bovine molecular markers. Microsatellites are among the most widely used molecular genetic markers. Discovering a single microsatellite that does not require specific genomic location, level of genetic polymorphism, or correlation with biological traits is relatively easy. However, the calculation results for the same genetic indicator vary depending on the combination of different molecular genetic markers, and whether the corresponding genetic assessment effect can meet practical needs is uncertain. Furthermore, due to the large number of potential microsatellites, selecting a set of microsatellite markers that simultaneously meets the requirements of genetic polymorphism level, reasonable marker density, appropriate marker uniformity, sufficient coverage, and genetic assessment effect that meets practical needs is not easy.

[0005] Currently, only six microsatellite markers have been reported in the bovine MHC region: BF1, BM1258, DYMS1, BM1818, BM1443, and BM1905. Bovine chromosome 23 is approximately 60,000,000 bp in length, and the physical location of the MHC region in the bovine genome ranges roughly from 7,000,000 to 28,000,000 bp. Some researchers have used certain microsatellite markers for bovine MHC haplotype typing and genetic polymorphism assessment. In breeding practice, improving selection accuracy is crucial for project success, and achieving selection accuracy requires improving genotype accuracy. Therefore, using MHC region DNA polymorphism as molecular markers for breeding necessitates improving the haplotype resolution of this region. A haplotype refers to the allele combination of different polymorphic markers on a single chromosome. In studies of gene-phenotype relationships, longer-range and higher-resolution haplotypes often demonstrate better performance in constructing gene-phenotype relationships.

[0006] Currently, the six existing microsatellite markers cover a small area on bovine chromosome 23 and have a low marker density. They cannot cover the extended region of the bovine MHC or the entire length of chromosome 23, and therefore cannot identify different haplotypes arising from DNA polymorphisms outside the 7,000,000-28,000,000 base pairs on chromosome 23, leading to confusion between different haplotypes. This means that using only these six microsatellite markers for bovine MHC typing can easily produce false linkage disequilibrium relationships between the covered regions and other regions, which can be misleading in selection processes or other genetic assessments. Summary of the Invention

[0007] To address the aforementioned deficiencies in existing technologies, this invention provides a set of microsatellite markers that can meet the requirements of genetic polymorphism level, reasonable molecular marker density, appropriate marker uniformity, sufficient coverage, and genetic evaluation effect that can meet practical needs.

[0008] Microsatellites are a class of short, highly variable DNA sequences found in the genomes of higher organisms. They typically exist in tandem repeats of 2 to 6 nucleotides, hence they are also known as simple sequence repeats (SSRs) or short tandem repeats (STRs).

[0009] Microsatellite markers are currently the most widely used polymorphic DNA markers, and they have the following significant characteristics:

[0010] 1. They are widely distributed in the genomes of higher organisms. It is estimated that there is one microsatellite marker every 10-50 kb. The bovine genetic map published in 2004 contained 3,802 microsatellite markers.

[0011] 2. They exhibit high polymorphism and rich information content; many microsatellites possess more than 5 alleles.

[0012] 3. It exhibits codominant inheritance, making it easy to distinguish between homozygous and heterozygous types;

[0013] 4. Individual genotypes can be directly obtained through PCR amplification and electrophoresis typing, making detection simple;

[0014] 5. Most microsatellite markers are located in non-coding regions of the genome and are not affected by natural or artificial selection, making them neutral markers.

[0015] Therefore, microsatellite markers play an irreplaceable role in genetic testing such as human forensic identification and animal pedigree confirmation.

[0016] In addition, microsatellite detection requires less sample, has high sensitivity and success rate, and is suitable for biological samples from various sources.

[0017] To this end, the present invention provides a combination of microsatellite markers covering the full-length sequence of bovine chromosome 23.

[0018] In a preferred embodiment of the present invention, the microsatellite marker combination comprises microsatellite markers BANX001-BANX028.

[0019] In a more preferred embodiment of the present invention, the microsatellite markers BANX001-BANX028 respectively comprise the sequences shown in SEQ ID NO.1-28.

[0020] Microsatellite marker detection involves two steps: PCR amplification and genotyping. For applications such as paternity testing, individual identification, or population genetic variation analysis, multiple microsatellite loci typically require amplification and detection, resulting in a large workload and high experimental costs.

[0021] Therefore, another aspect of the present invention provides a primer combination for detecting microsatellite marker combinations covering the full-length sequence of bovine chromosome 23.

[0022] In a preferred embodiment of the present invention, the primer combination comprises the primer sequences shown in SEQ ID NO.29-84.

[0023] Currently, there are relatively few commercially available bovine microsatellite marker detection kits, and two prominent problems exist:

[0024] 1. The marker positions in the kit are relatively fixed, making it impossible to increase the number of markers for specific detection needs;

[0025] 2. The reagent kits are expensive, resulting in high testing costs.

[0026] These drawbacks limit the application of the kit in bovine breeding practices and scientific research.

[0027] Therefore, another aspect of the present invention provides a kit comprising the microsatellite marker combination described in the present invention or the primer combination described in the present invention.

[0028] In the bovine MHC region, only six microsatellite markers have been reported so far: BF1, BM1258, DYMS1, BM1818, BM1443, and BM19056. For bovine chromosome 23, these six microsatellite markers exhibit poor coverage, low density, and uneven spatial distribution. Therefore, using only these six microsatellite markers is insufficient for effective inbreeding depression risk assessment and genetic evaluation.

[0029] Therefore, another aspect of the present invention provides the application of the microsatellite marker combination, primer combination or kit described in the present invention in the assessment of inbreeding depression risk.

[0030] In a preferred embodiment of the present invention, the above application is used in assessing the risk of bovine inbreeding depression.

[0031] Another aspect of the present invention provides the application of the microsatellite marker combinations, primer combinations, or kits described herein in genetic evaluation.

[0032] In a preferred embodiment of the present invention, the above application is in the performance of bovine genetic evaluation.

[0033] Haplotype is short for haploid genotype, which in genetics refers to the combination of alleles at multiple loci on the same chromosome that are inherited together.

[0034] Haplotype diversity is an important indicator for measuring the degree of variation in a population. Haplotype diversity refers to the frequency of randomly selecting two different haplotypes from a sample. A population with high haplotype diversity indicates high genetic diversity and abundant genetic resources.

[0035] Therefore, another aspect of the present invention provides a method for determining the genetic polymorphism of the MHC region and the MHC extension region, including using the microsatellite marker combination, primer combination, or kit described in the present invention to detect and analyze biological samples.

[0036] In a preferred embodiment of the present invention, the above-mentioned detection and analysis is performed on a sample from a cow.

[0037] Inbreeding depression occurs because inbreeding increases the homozygous probability of harmful alleles, leading to decreased individual adaptability. It is also one of the reasons why inbreeding produces disease-causing genes. Population shrinkage is the main cause of inbreeding depression. However, actual research has shown that inbreeding depression is not always clearly manifested, suggesting that it is closely related to ecology and genetics.

[0038] Therefore, in another aspect, the present invention provides a method for assessing the risk of inbreeding depression or for genetic evaluation, including performing genotyping analysis on biological samples using the microsatellite marker combination, primer combination, or kit described in the present invention.

[0039] In a preferred embodiment of the present invention, the above-mentioned genotyping analysis is performed on samples from cattle.

[0040] By adopting the above technical solution, the present invention achieves the following beneficial effects:

[0041] 1. This invention increases the number of microsatellite markers on bovine chromosome 23 from the known 6 to 34, thereby increasing the microsatellite marker density in the bovine MHC region to 15. Simultaneously, outside the MHC region, an additional 13 microsatellite markers cover 2,000,000 to 58,500,000 bases of bovine chromosome 23, essentially covering the entire chromosome and significantly improving the microsatellite marker density. Furthermore, the physical spacing between different microsatellite markers is approximately 500,000-2,000,000 bp, indicating good spatial uniformity of the microsatellite marker distribution.

[0042] 2. The primers obtained by the present invention for detecting the microsatellite markers based on 28 microsatellite markers have good specificity and good amplification stability.

[0043] 3. Compared with using only the existing 6 microsatellite markers, using the 28 microsatellite markers obtained by screening in this invention to determine the haplotype of bovine MHC and extended regions can achieve higher haplotype resolution, more effectively predict the degree of inbreeding at the individual level, screen individuals with severe inbreeding in advance, and more accurately assess the genetic polymorphism of the population, thereby providing a basis for scientific decision-making in the cattle breeding process. Attached Figure Description

[0044] Figure 1 This is a partial sequencing map of BANX001 sequenced using 1-2G-1 as a sample.

[0045] Figure 2 This is a partial sequencing map of BANX001 sequenced using samples 1-4G-1.

[0046] Figure 3 This is a partial sequencing map of BANX001 sequenced using samples 1-3G-1.

[0047] Figure 4 This is a partial sequencing map of BANX005 sequenced using sample 5-1G-1.

[0048] Figure 5 This is a partial sequencing map of the BANX005 locus, which was sequenced using 5-10M-1 samples.

[0049] Figure 6 This is a partial sequencing map of the BANX006 locus sequenced using sample 6-1G-1.

[0050] Figure 7 This is a partial sequencing map of BANX006 sequenced using sample 6-2G-2.

[0051] Figure 8 This is a partial sequencing map of BANX007 sequenced using sample 7-2G-1. Detailed Implementation

[0052] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0053] Example 1: Screening of microsatellite markers

[0054] Download the FASTA sequence of bovine chromosome 23 (document number CM024457.1) from the NCBI database. Use SSR_pipeline 0.951 (https: / / pubs.usgs.gov / ds / 778 / downloads / ) software to screen for microsatellite loci. The repeat unit lengths of the screened loci are 2 nucleotides, 3 nucleotides, and 4 nucleotides. Based on the uniformity of the physical location distribution of the genome, 28 SSR markers were selected and named as follows: BANX001, BANX002, BANX003, BANX004, BANX005, BANX006, BANX007, BANX008, BANX009, BANX010, BANX011, BANX012, BANX013, BANX014, BANX015, BANX016, BANX017, BANX018, BANX019, BANX020, BANX021, BANX022, BANX023, BANX024, BANX025, BANX026, BANX027, and BANX028.

[0055] The nucleotide sequences of microsatellite markers BANX001-BANX028 are shown in Table 1.

[0056] Table 1. Nucleotide sequence of microsatellite markers BANX001-BANX028

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] The physical locations of microsatellite markers BANX001-BANX028 on chromosome 23 of the genome are shown in Table 2.

[0065] Table 2 Physical locations of microsatellite markers BANX001-BANX028 in the genome.

[0066]

[0067]

[0068] As shown in Table 2, the microsatellite markers BANX001-BANX025 can uniformly cover bovine chromosome 23 from head to tail.

[0069] Example 2: Screening of microsatellite marker amplification primers

[0070] Primers for amplification of 28 microsatellite markers BANX001-BANX028 were designed in batches using Primer 5.0 software. The PCR amplification products were less than 500 bp in length, and the primers had a GC content of 40%-60% and no palindromic structures.

[0071] The selected amplification primers were specifically validated using primer BLAST in the NCBI database. The amplified PCR products were suitable for genotyping using capillary electrophoresis. The microsatellite marker amplification primer sequences are shown in Table 3.

[0072] Table 3. Nucleotide sequence of amplification primers for microsatellite markers BANX001-BANX028

[0073]

[0074]

[0075]

[0076] The amplification stability and polymorphism of 28 microsatellite marker primers were verified by PCR experiments and capillary electrophoresis using genomic DNA samples from 16 Simmental cattle. The PCR amplification program consisted of 35 cycles: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 52℃-63℃ annealing for 30 s, 72℃ extension for 30 s, followed by a final extension at 72℃ for 7 min. The PCR reaction system is shown in Table 4.

[0077] Table 4. PCR amplification reaction system for microsatellite loci

[0078]

[0079] To verify the reliability of the sequence information of the sites selected from the NCBI database, we performed first-generation sequencing on the PCR products. The sequencing service provider was Shanghai Sangon Biotech Co., Ltd. Some sequencing results are attached. Figure 1-8 As shown.

[0080] The results showed that the amplification at each site was stable and the polymorphism was good. The size range of the PCR products at each site is shown in Table 5.

[0081] Table 5. Sizes of PCR products from 28 microsatellite loci

[0082]

[0083]

[0084] Example 3: Genetic assessment of inbreeding depression risk

[0085] Based on the existing 6 bovine MHC microsatellite markers, 362 samples were screened, including 172 samples of Qinghai Chaidamu Fu cattle, 160 samples of Angus cattle from Beijing Huaniu Tianjun Doudian beef cattle breeding base, 27 samples of Simmental cattle raised by farmers in Tongliao area of ​​Inner Mongolia, and 3 samples of yellow cattle.

[0086] Ten samples were selected from the 362 samples mentioned above, and their haplotypes were constructed using six existing microsatellite markers. The haplotype results showed that all samples were identical. Then, capillary electrophoresis genotyping was performed using the 28 microsatellite markers obtained in this invention, and haplotype inference was performed simultaneously using Arlequin 3.5 software. The results are shown in Table 6.

[0087] Table 6 Genotyping results of 10 samples

[0088]

[0089] Note: In Table 6, only one allele indicates that the locus is homozygous. Microsatellite markers that do not appear in the table have the same genotype in all samples.

[0090] As shown in Table 6, when the existing six bovine MHC microsatellite markers (BF1, BM1258, DYMS1, BM1818, BM1443, and BM1905) were used for genotyping of these 10 samples, all 10 samples had only one allele and were identified as homozygous. However, when the 28 microsatellite markers obtained by screening in this invention were used for genotyping, sample ID 10 showed the highest heterozygosity at four loci (BANX001, BANX0019, BANX022, and BANX025); followed by sample ID 6, which showed heterozygosity at two loci (BANX022 and BANX025). It was verified that samples ID 10 and 6 both belong to the Yellow Cattle family. Sample ID 3 showed the lowest heterozygosity at four loci (BANX001, BANX0019, BANX022, and BANX025). Upon verification, the cattle belong to the Simmental breed. Samples with IDs 1, 2, 7, and 8 are Qaidam Fu cattle, samples with IDs 5 and 9 are Angus cattle, and sample with ID 4 is a Simmental cattle. They all show heterozygosity at the BANX001 locus.

[0091] In cattle breeding practices, higher inbreeding levels imply a higher risk of inbreeding depression. Therefore, based on the genotyping results in Table 6, genotyping using the existing six bovine MHC microsatellite markers cannot eliminate any samples that might cause inbreeding depression in advance. However, by using the 28 microsatellite markers screened in this invention for genotyping, samples with ID 3 can be eliminated early in breeding practices, thus avoiding the risk of inbreeding depression and preventing economic losses.

[0092] Haplotype diversity is defined as the ratio of two different haplotype sequences extracted from a set of sequences. It is a commonly used indicator for assessing the genetic diversity of a population sample. A population with high haplotype diversity indicates high genetic diversity and abundant genetic resources. According to this definition, if the haplotype diversity of these 10 samples is calculated using the existing 6 microsatellite markers, the value is 0; however, if the 28 microsatellite markers screened in this invention are used in conjunction, the haplotype diversity is 0.667. Obviously, the set of microsatellite markers used in this invention can more reasonably assess the genetic diversity of bovine chromosome 23, thereby providing richer genetic resources for population construction in cattle farming practices and meeting the assessment needs for assisting population construction in cattle farming practices.

[0093] Further analysis of the genotyping results in Table 6 revealed that when 28 microsatellite markers were used in combination, linkage disequilibrium attenuation characteristics that could not be detected by using only the existing 6 microsatellite markers were observed, except for sample ID 3. Samples IDs 6 and 10, which are of the yellow cattle breed, exhibited linkage disequilibrium characteristics with shorter block structures. This characteristic is consistent with the genetic characteristics of local breeds and provides rich genetic resources for further breed improvement and trait optimization.

[0094] The present application has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present application based on these embodiments, all of which fall within the protection scope of the present application.

Claims

1. A primer combination for detecting microsatellite marker combinations covering the full-length sequence of bovine chromosome 23, characterized in that, It contains the primer sequences shown in SEQ ID NO.29-84.

2. A kit comprising the primer combination of claim 1.

3. The application of the primer combination of claim 1 or the kit of claim 2 in the risk assessment of bovine inbreeding depression.

4. The application of the primer combination of claim 1 or the kit of claim 2 in the assessment of bovine genetic diversity.

5. A method for determining genetic polymorphism in bovine MHC regions and MHC extension regions, characterized in that, This includes using the primer combination described in claim 1 or the kit described in claim 2 to detect and analyze bovine biological samples.

6. A method for assessing the risk of bovine inbreeding depression or bovine genetic diversity, comprising performing genotyping analysis on bovine biological samples using the primer combination of claim 1 or the kit of claim 2.

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