CN116287311A - Microsatellite marker, detection primer and application thereof - Google Patents

Microsatellite marker, detection primer and application thereof Download PDF

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CN116287311A
CN116287311A CN202310263777.6A CN202310263777A CN116287311A CN 116287311 A CN116287311 A CN 116287311A CN 202310263777 A CN202310263777 A CN 202310263777A CN 116287311 A CN116287311 A CN 116287311A
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万君兴
华子昂
刘宝全
张龙雨
乌春莹
张建
朱美瑛
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Abstract

The invention belongs to the field of genetic engineering, and provides a method for performing risk assessment or genetic assessment of inbreeding degradation, and a microsatellite marker combination or a primer combination adopted in the method. The invention increases the microsatellite markers of the 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 can prevent economic risks caused by inbreeding recession, genetic evaluation is carried out on the inbreeding degree of different individuals in advance, more accurate evaluation is carried out on the genetic polymorphism of the population, and a basis is provided for scientific decision of the cattle raising process.

Description

Microsatellite marker, detection primer and application thereof
Technical Field
The invention relates to development and utilization of molecular markers, in particular to a group of microsatellite markers, detection primers and application thereof.
Background
Inbreeding deterioration is a serious problem in modern cattle. The cause of inbreeding decline mainly comprises two aspects, namely the use of an artificial breeding technology, and the semen of one bull is used for tens of thousands of cows during breeding due to the use of the artificial insemination technology, so that the utilization of the cows is further aggravated due to superovulation; on the other hand, in the breeding link, no matter genome selection, pedigree selection, optimal linear unbiased estimation or molecular marker assisted selection is adopted, the genetic variances of some important basic groups and core groups gradually decrease along with the implementation of the breeding process. The combination of these factors results in inbreeding deterioration, a very common phenomenon in the cattle industry. The inbred deterioration is specifically manifested by death of cow embryo, abortion of cow, calf malformation, reduced production level, etc. It has been established on the molecular mechanism of habitual abortion in humans that heterozygous deletions of multiple MHC loci can lead to failure of pregnant women to produce blocking antibodies that protect the fetus, thereby triggering abortion.
In the cattle industry, genetic evaluation using molecular markers is applied to many links such as introduction, breeding core group construction, propagation group construction, inbreeding risk evaluation, individual identification, pedigree construction, genetic diversity evaluation, etc., wherein the major histocompatibility complex (MHC, major Histocompatibility Complex) has a dominant position in all molecular markers, which is a region with highest genetic polymorphism in vertebrate genomes, which also determines that the region is a region with the most sensitive genetic polymorphism changes of populations of one species, which has characteristics of tight linkage and haplotype inheritance. In bovine genome, MHC region is located on chromosome 23, and this region genetic polymorphism has a wide link to various economic traits and disease resistance.
Therefore, how to use the DNA polymorphism of the MHC region and the extension region of MHC has become a hot spot in the development and use of bovine molecular markers. Microsatellites are one of the most widely used molecular genetic markers. It is easy to find a single microsatellite which does not have any requirement on the physical location of the genome, the level of genetic polymorphism and the correlation of biological properties, but the calculation results of the same genetic index by the combination of different molecular genetic markers are different, and whether the corresponding genetic evaluation effect can meet the actual requirements is also not necessarily. In addition, because of the numerous potential microsatellites, it is not easy to screen out a set of microsatellite markers, and meanwhile, the genetic polymorphism level, the reasonable molecular marker density, the proper marker uniformity and the sufficient coverage range can be met, and the genetic evaluation effect can meet the actual demands.
Currently, there are only 6 microsatellite markers reported in the bovine MHC region, namely BF1, BM1258, DYMS1, BM1818, BM1443, BM1905. The total length of chromosome 23 of cattle is about 60000000bp, and the physical location of MHC region in the genome of cattle is approximately located at 7000000 ～ 28000000bp. Bovine MHC haplotypes and genetic polymorphism assessments have been performed by the present scholars using some of these microsatellite markers. In breeding practice, how to improve the selection accuracy is the key to the success of the project, and the accuracy of genotyping must be improved to achieve the selection accuracy, so that molecular markers bred with MHC region DNA polymorphism must improve the haplotype resolution of the region. Haplotype refers to a combination of alleles of different polymorphic markers on a single chromosome. In the study of the relationship between genes and phenotypes, longer range and high resolution haplotypes tend to have better performance in constructing the relationship between genes and phenotypes.
The current 6 microsatellite markers have small coverage range on the bovine chromosome 23, low marker density, can not cover the extension region of bovine MHC and the full length range of the chromosome 23, and can not identify different haplotypes generated by DNA polymorphism except 7000000-28000000 base of the bovine genome chromosome 23, thereby causing the condition of confusion of different haplotypes. This results in the easy occurrence of false linkage disequilibrium between the region covered by bovine MHC and other regions using only the 6 microsatellite markers available, which can be misleading to the selection process or other genetic evaluation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a set of microsatellite markers which can meet the level of genetic polymorphism, reasonable molecular marker density, proper marker uniformity and sufficient coverage, and the genetic evaluation effect can meet the actual demands.
Microsatellites (microsatellites) are a class of DNA sequences of relatively short length and extremely high variability within the genome of higher organisms. Usually in the form of tandem repeats from 2 to 6 nucleotide repeat units, and are therefore also referred to as simple repeats (Simple Sequence Repeat, SSR) or tandem repeats (Short Tandem Repeat, STR).
Microsatellite markers are polymorphic DNA markers which are extremely widely used at present and have the following remarkable characteristics:
1. widely distributed within the genome of higher organisms, it is estimated that there is one microsatellite marker at about 10-50kb intervals, and the bovine genetic map published in 2004 already contains 3802 microsatellite markers;
2. the polymorphism is high, the information content is rich, and a plurality of microsatellites have more than 5 alleles;
3. is co-dominant, and is easy to distinguish homozygous and heterozygous;
4. the individual genotype can be directly obtained through PCR amplification and electrophoresis typing, and the detection is simple and convenient;
5. most microsatellite markers are located in non-coding regions of the genome, are not affected by natural and artificial selection, and are neutral markers.
Therefore, microsatellite markers have an irreplaceable role in genetic testing such as human forensic identification, animal pedigree confirmation, and the like.
In addition, the microsatellite detection has the advantages of less detection material quantity, high sensitivity and success rate, and suitability for biological detection materials from various sources.
To this end, in one aspect, the invention provides a microsatellite marker combination covering the full length sequence of bovine chromosome 23.
In a preferred embodiment of the invention, the microsatellite marker combination comprises the microsatellite markers BANX001-BANX028.
In a more preferred embodiment of the invention, the microsatellite markers BANX001-BANX028 comprise the sequences shown in SEQ ID No.1-28, respectively.
The detection of microsatellite markers comprises two steps of PCR amplification and genotyping. Aiming at the conditions of paternity test, individual identification or population genetic variation analysis, a plurality of microsatellite loci are usually required to be amplified and detected, and the workload and the test cost are high.
To this end, in another aspect, the invention provides a primer combination for detecting a microsatellite marker combination covering the full length sequence of bovine chromosome 23.
In a preferred embodiment of the invention, the primer combination comprises the primer sequences shown in SEQ ID NOS.29-84.
At present, commercial bovine microsatellite marker detection kits are few, and two prominent problems exist:
1. the marking seats of the kit are relatively fixed, so that the marking quantity can not be increased according to the special detection requirement;
2. the kit is high in price and high in detection cost.
These disadvantages limit the use of the kit in bovine breeding practices and scientific research.
To this end, the invention provides in a further aspect a kit comprising a microsatellite marker combination according to the invention or a primer combination according to the invention.
In the bovine MHC region, there are only 6 microsatellite markers currently reported, BF1, BM1258, DYMS1, BM1818, BM1443, BM 19056. For bovine chromosome 23, the coverage of these 6 microsatellite markers was poor, the density of microsatellite markers was low, and the spatial distribution was also poor. Therefore, if only these 6 microsatellite markers are used, the risk assessment of inbreeding failure and genetic assessment cannot be effectively performed.
For this purpose, the invention further provides the application of the microsatellite marker combination, the primer combination or the kit in the risk assessment of inbreeding degradation.
In a preferred embodiment of the invention, the above-mentioned use is in the performance of risk assessment of bovine inbreeding disease.
In another aspect, the invention provides the use of a microsatellite marker combination, primer combination or kit according to the invention for genetic evaluation.
In a preferred embodiment of the invention, the above-mentioned use is in the performance of genetic assessment of cattle.
Haplotypes are short for haploid genotypes, genetically referring to combinations of alleles at multiple loci that are co-inherited on the same chromosome.
Haplotype diversity is an important indicator for measuring the degree of variation of a population. Haplotype diversity refers to the frequency at which two different haplotypes are randomly drawn in a sample. The group with high haplotype diversity shows that the genetic diversity is high and the genetic resources are rich.
To this end, the present invention provides a method for determining a genetic polymorphism in an MHC region and an MHC extension region, comprising detecting and analyzing a biological sample using the microsatellite marker combination, the primer combination, or the kit of the present invention.
In a preferred embodiment of the present invention, the above assay is an assay performed on a sample from a cow.
Inbreeding failure occurs because inbreeding increases the probability of homozygosity of the deleterious allele, resulting in a decrease in the ability of the individual to adapt, and this is one of the reasons for the pathogenic genes produced by inbreeding. Population diminution is a main reason for inbreeding recession, and the inbreeding recession is not necessarily obviously shown in practical research, which suggests that the inbreeding recession is closely related to ecology and genetics.
To this end, a further aspect of the invention provides a method for performing risk assessment or genetic assessment of inbred degradation comprising genotyping a biological sample using a microsatellite marker combination, primer combination, or kit of the invention.
In a preferred embodiment of the invention, the genotyping assay described above is performed on a sample from a cow.
By adopting the technical scheme, the invention has the following beneficial effects:
1. according to the invention, the microsatellite markers of the bovine chromosome 23 are increased from 6 known microsatellite markers to 34 microsatellite markers, so that the density of microsatellite markers in the bovine MHC region is increased to 15, and 13 microsatellite markers are covered with 2000000-58500000 bases of the bovine chromosome 23 outside the MHC region, so that the whole bovine chromosome 23 is basically covered, and the density of the microsatellite markers is greatly increased. In addition, the physical interval of different microsatellite markers is about 500000-2000000bp, and the space distribution uniformity of the microsatellite markers is good.
2. The primers for detecting the microsatellite markers obtained by screening on the basis of 28 microsatellite markers have good specificity and good amplification stability.
3. Compared with the existing 6 microsatellite markers, the 28 microsatellite markers obtained by screening are used for carrying out haplotype measurement on bovine MHC and an expansion region, so that higher haplotype resolution can be realized, the degree of inbreeding at individual level can be predicted more effectively, individuals with serious degree of inbreeding can be screened in advance, the genetic polymorphism of the population can be estimated more accurately, and further, the basis is provided for scientific decision in the cattle raising process.
Drawings
FIG. 1 is a partial sequencing map of the sequencing of the seat BANX001 using 1-2G-1 as a sample.
FIG. 2 is a partial sequencing map of the sequencing of seat BANX001 using 1-4G-1 as a sample.
FIG. 3 is a partial sequencing map of the sequencing of seat BANX001 using 1-3G-1 as a sample.
FIG. 4 is a partial sequencing map of the sequencing of seat BANX005 using 5-1G-1 as a sample.
FIG. 5 is a partial sequencing map of the sequencing of seat BANX005 using 5-10M-1 as a sample.
FIG. 6 is a partial sequencing map of the sequencing of the seat BANX006 using 6-1G-1 as a sample.
FIG. 7 is a partial sequencing map of the sequencing of the seat BANX006 using 6-2G-2 as a sample.
FIG. 8 is a partial sequencing map of the sequencing of the locus BANX007 using 7-2G-1 as a sample.
Detailed Description
The present application is further described in detail below by way of the accompanying drawings and examples. The features and advantages of the present application will become more apparent from the description.
Example 1: screening of microsatellite markers
The bovine chromosome 23 fasta sequence with the library number CM024457.1 was downloaded in NCBI database and microsatellite site selection was performed using SSR_pipeline 0.951 (https:// pubs. Usgs. Gov/ds/778/downloads /) software, with the selected site repeat units being 2, 3 and 4 nucleotides in length, respectively. Then, according to the uniformity of the physical position distribution of the genome, 28 SSR markers are screened out and respectively named as: 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, BANX028.
The nucleotide sequence of the microsatellite marker BANX001-BANX028 is shown in Table 1.
TABLE 1 nucleotide sequence listing of microsatellite marker BANX001-BANX028
Figure BDA0004132328790000061
Figure BDA0004132328790000071
Figure BDA0004132328790000081
Figure BDA0004132328790000091
Figure BDA0004132328790000101
Figure BDA0004132328790000111
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Figure BDA0004132328790000121
The physical location of the microsatellite marker BANX001-BANX028 on chromosome 23 of the genome is shown in Table 2.
TABLE 2 physical location of the microsatellite markers BANX001-BANX028 in the genome
Figure BDA0004132328790000122
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Figure BDA0004132328790000131
As can be seen from Table 2, the microsatellite markers BANX001-BANX025 can cover the 23 rd chromosome of bovine genome uniformly from beginning to end.
Example 2: screening of microsatellite-labeled amplification primers
The amplification primers are designed in batches for 28 microsatellite marked BANX001-BANX028 by Primer5.0 software, the length of PCR amplification products is within 500bp, the content of the primer GC is 40% -60%, and no palindromic structure exists.
The screened amplification primers were specifically verified in NCBI database using primer BLAST. The amplified PCR products are suitable for genotyping on a capillary electrophoresis apparatus. The sequence of the microsatellite marked amplification primer is shown in Table 3.
TABLE 3 amplification primer nucleotide sequence Listing of microsatellite marker BANX001-BANX028
Figure BDA0004132328790000132
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Figure BDA0004132328790000141
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Figure BDA0004132328790000151
PCR experiments and capillary electrophoresis typing verification were performed on the amplification stability and polymorphism of 28 microsatellite marker primers using 16 Siemens cattle genomic DNA samples. Wherein the PCR amplification procedure was 95℃for 5min,95℃for 30s,52℃for 30s, 63℃for 30s,72℃for 30s,35 cycles, 72℃for 7min, and the PCR reaction system was as shown in Table 4.
TABLE 4 microsatellite loci PCR amplification reaction System
Figure BDA0004132328790000152
To verify the reliability of the sequence information of the sites screened from NCBI database, we performed a generation of sequencing of the PCR products, sequencing service was Shanghai Bioengineering Co., ltd, and the partial sequencing results are shown in FIGS. 1-8.
The results showed that the amplification was stable and the polymorphism was good at each site, and the size range of PCR products at each site was shown in Table 5.
TABLE 5 PCR product size for 28 microsatellite loci
Figure BDA0004132328790000153
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Figure BDA0004132328790000161
Example 3: genetic assessment of risk of inbreeding failure
362 samples were screened according to the current 6 bovine MHC microsatellite markers, wherein 172 samples were Qinghai-fadazuku, 160 samples were beef cattle in the sinus store of beijing hua Niu Tianjun, the breeding base angust, 27 samples were siemens cattle autotrophic by farmers in the inner mongolian tong Liaoning region, and 3 samples were yellow cattle.
10 samples were screened from the 362 samples, and haplotypes of the 10 samples were constructed using the existing 6 microsatellite markers, and the haplotype results were identical. Then, the 28 microsatellite markers obtained by screening according to the invention are subjected to capillary electrophoresis genotyping, and haplotype inference is carried out by adopting Arlequin3.5 software, and the results are shown in Table 6.
TABLE 6 genotyping results for 10 samples
Figure BDA0004132328790000162
Note that: only 1 allele in Table 6 indicates that the locus is homozygous and that the microsatellite markers not present in the table are of the same genotype in all samples.
As can be seen from Table 6, when these 10 samples were genotyped using the existing 6 bovine MHC microsatellite markers (BF 1, BM1258, DYMS1, BM1818, BM1443, BM 1905), all 10 samples had only 1 allele and were identified as homozygous. When the 28 microsatellite markers obtained by screening are used for genotyping, a sample with a sample ID of 10 shows heterozygosity at positions BANX001, BANX0019, BANX022 and BANX025 4, and the heterozygosity is highest; next, the sample with sample ID of 6 showed heterozygosity at positions BANX022 and BANX025 2. It was verified that the sample with sample ID 10 and the sample with sample ID 6 both belong to cattle. The sample ID 3 had the lowest heterozygosity at BANX001, BANX0019, BANX022, BANX025 4 loci. Verified as belonging to the sieboldii. Samples with sample IDs 1, 2, 7, 8 were fadak, samples with sample IDs 5 and 9 were angust, and sample with sample ID 4 were siemens, which were heterozygous at the BANX001 locus, respectively.
In bovine farming practice, a higher degree of inbreeding means a higher risk of inbreeding deterioration. Thus, according to the typing results of Table 6, if genotyping is performed according to the existing 6 bovine MHC microsatellite markers, any sample that may cause risk of inbreeding deterioration cannot be eliminated in advance. The 28 microsatellite markers obtained by screening are subjected to genotyping, and samples with the sample ID of 3 can be eliminated in advance in the cultivation practice, so that the risk of inbreeding degradation is avoided in advance, and economic loss is avoided.
The haplotype diversity is defined as the ratio of a group of sequences to 2 different haplotype sequences simultaneously, and is a common index capable of evaluating genetic diversity of a group sample, and a group with high haplotype diversity shows that the genetic diversity is high and the genetic resource is rich. According to this definition, if haplotype diversity calculation is performed on these 10 samples using the existing 6 microsatellite markers, its value is 0; if 28 microsatellite markers screened by the invention are combined, the haplotype diversity is 0.667, and obviously, the set of microsatellite markers adopted by the invention can reasonably evaluate the genetic diversity of the bovine chromosome 23, so that more abundant genetic resources are provided for group construction in cattle raising practice, and the evaluation requirement of group construction in auxiliary cattle raising practice is met.
Further analysis of the typing results of Table 6 from the attenuation characteristics of linkage disequilibrium, when 28 microsatellite markers are used in combination, in addition to the sample with sample ID 3, the attenuation characteristics of linkage disequilibrium which cannot be detected by using only the existing 6 microsatellite markers can be detected, and the sample varieties with sample ID 6 and 10 are cattle, the linkage disequilibrium characteristics of which show a shorter Block structure, and the characteristics conform to the genetic characteristics of local varieties, so that abundant genetic resources are provided for further variety improvement and trait optimization.
The present application has been described in connection with the preferred embodiments, but these embodiments are merely exemplary and serve only as illustrations. On the basis of this, many alternatives and improvements can be made to the present application, which fall within the scope of protection of the present application.

Claims (14)

1. A microsatellite marker combination covering the full length sequence of bovine chromosome 23.
2. The combination of microsatellite markers according to claim 1 comprising the microsatellite markers BANX001-BANX028.
3. The combination of microsatellite markers according to claim 1 or 2 wherein the microsatellite markers BANX001-BANX028 comprise the sequences shown in SEQ ID nos. 1-28, respectively.
4. A primer combination for detecting microsatellite marker combinations covering the full-length sequence of bovine chromosome 23.
5. The primer combination of claim 4, comprising the primer sequences set forth in SEQ ID NOS.29-84.
6. A kit comprising the microsatellite marker combination of any one of claims 1-3 or the primer combination of claim 4 or 5.
7. Use of a microsatellite marker combination according to any one of claims 1-3, a primer combination according to claim 4 or 5, or a kit according to claim 6 for performing an inbred fade risk assessment.
8. The use according to claim 7, wherein the use is in the assessment of risk of bovine inbreeding failure.
9. Use of a microsatellite marker combination according to any one of claims 1-3, a primer combination according to claim 4 or 5, or a kit according to claim 6 for genetic evaluation.
10. The use according to claim 9, wherein the use is in the performance of genetic assessment of cattle.
11. A method for determining a genetic polymorphism in an MHC region and an MHC extension region, comprising performing a detection assay on a biological sample using the microsatellite marker combination of any one of claims 1-3, the primer combination of claim 4 or 5, or the kit of claim 6.
12. The method of claim 11, wherein the assay is performed on a sample from a cow.
13. A method of performing an inbred fade risk assessment or genetic assessment comprising genotyping a biological sample using the microsatellite marker combination of any one of claims 1-3, the primer combination of claim 4 or 5, or the kit of claim 6.
14. The method of claim 13, wherein the genotyping is performed on a sample from a cow.
CN202310263777.6A 2023-03-13 2023-03-13 Microsatellite marker, detection primer and application thereof Pending CN116287311A (en)

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