Medicago sativa SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof

Abstract

The invention discloses an alfalfa SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The SNP molecular marker combination is composed of 61351 SNP molecular markers, the physical positions of the 61351 SNP molecular markers are determined based on sequence alignment of a medicago sativa reference genome ZM-4alfalfa gene, the SNP molecular markers are good in site specificity, high in universality and high in polymorphism, medicago sativa germplasms of different sources can be effectively distinguished, and the application prospect is wide. The alfalfa whole-genome liquid-phase gene chip prepared from the SNP molecular marker can be used for alfalfa germplasm resource genetic diversity evaluation, genetic map construction, functional gene positioning, whole-genome association analysis and the like, and provides important tools and technical supports for alfalfa molecular design breeding and variety authenticity identification. The method has important significance on high-quality development of alfalfa seed industry in China.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an alfalfa SNP molecular marker combination and application thereof. Background Art

[0002] Alfalfa (Mediacago sativa L.) is one of the most important perennial legume forages in the world. It is also the earliest cultivated, most widely distributed, and most efficiently utilized excellent legume forage. It has high yield, rich nutritional value, good palatability, and is drought-resistant and salt-alkali-tolerant. It also has strong biological nitrogen fixation and soil improvement and fertilization properties. It is known as the "king of forages" and plays an irreplaceable role in promoting agricultural industrial structure adjustment, the development of efficient and high-quality animal husbandry, and ecological governance and restoration.

[0003] At present, alfalfa breeding is still in the 1.0-2.0 stage. The efficiency and accuracy of germplasm resource identification are low, the development and utilization of excellent germplasm resources are seriously insufficient, and there is a lack of high-throughput genotype identification and analysis technology systems and molecular markers of important breeding value. Whole genome selection breeding has not yet been carried out in depth, resulting in low efficiency in alfalfa variety research and development, and few excellent alfalfa varieties with outstanding traits, which seriously restricts the development of the alfalfa industry. Therefore, a high-density alfalfa whole genome liquid phase breeding chip is developed, which can efficiently, accurately and low-costly perform genotyping detection on large-scale alfalfa germplasm resource groups, break through the bottleneck problems in alfalfa germplasm resource evaluation and variety breeding, improve breeding efficiency, and help alfalfa move from conventional breeding to intelligent molecular design breeding. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a combination of SNP molecular markers of alfalfa.

[0005] The present invention also provides a primer set and / or liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker combination.

[0006] The invention also provides a gene chip having the above alfalfa SNP molecular marker combination.

[0007] The present invention also provides a test kit having the above-mentioned alfalfa SNP molecular marker combination.

[0008] The invention also provides an application of the alfalfa SNP molecular marker combination, gene chip and kit.

[0009] The invention also provides a breeding method for alfalfa.

[0010] According to one aspect of the present invention, a combination of SNP molecular markers for alfalfa is proposed, which consists of 61351 SNP molecular markers. The physical positions of the 61351 SNP molecular markers are determined by sequence alignment based on the alfalfa reference genome (ZM-4alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c). The specific site information is shown in Table 1 below.

[0011] Table 1

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[0078] In some embodiments of the present invention, Alt is ".", indicating that it has multiple mutation possibilities.

[0079] In some embodiments of the present invention, when ref is A, Alt is ".", indicating that the mutation can be T, G, C or N; when ref is T, Alt is ".", indicating that the mutation can be A, G, C or N; when ref is G, Alt is ".", indicating that the mutation can be A, T, C or N; when ref is C, Alt is ".", indicating that the mutation can be A, T, G or N, wherein N is a deletion.

[0080] In some embodiments of the present invention, the “-” in the table is “_”.

[0081] In some embodiments of the present invention, the position information of the SNP molecular marker is expressed in the form of chromosome number: physical position.

[0082] In some embodiments of the present invention, the 61351 SNP molecular markers are shown as SNP00001-61351; the specific site information of SNP00001-61351 is arranged from top to bottom and from left to right in the above table.

[0083] According to a second aspect of the present invention, a primer set and / or liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker combination is proposed.

[0084] In some embodiments of the present invention, the liquid phase probe is designed and synthesized using targeted capture sequencing technology based on the location of the SNP site and the sequences on both sides of the SNP site.

[0085] In some embodiments of the present invention, the liquid phase probe is about 100 bp in length and has a GC content between 20% and 80%.

[0086] According to a third aspect of the present invention, an alfalfa whole genome liquid phase chip is provided, wherein the alfalfa whole genome liquid phase chip comprises a primer set and / or a liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker combination.

[0087] The alfalfa whole genome liquid phase chip of the present invention is based on the principle of liquid phase chip, and can be applied to the genotyping detection of large-scale alfalfa populations in an efficient, accurate and low-cost manner. (1) In terms of performance, the SNP sites of this chip are all from the whole genome resequencing data. Compared with the simplified genome sequencing technology (RAD-seq, GBS, etc.) that is currently widely used, these sites have wider coverage, stronger representativeness, higher polymorphism in the whole genome of alfalfa, and have advantages in detection accuracy and density. (2) In terms of economic cost, compared with the currently widely used resequencing method, the cost of this chip for alfalfa genotype detection is mostly 1000-1200 yuan / sample, and the price of imported sequencers is higher, while the detection cost of this technology can be as low as 200 yuan / sample, which greatly reduces the detection cost, and the chip site is flexible, and new functional marker sites can be added at any time in the later stage, which has strong application promotion. (3) This chip contains 1,448 trait-associated loci, which can be directly used for the identification, evaluation, and development and utilization of important economic traits such as yield, quality, and stress resistance of alfalfa germplasm materials, and is highly practical.

[0088] According to a fourth aspect of the present invention, a kit is provided, which comprises a primer set and / or a liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker.

[0089] According to a fifth aspect of the present invention, the application of the above-mentioned alfalfa SNP molecular marker combination, kit or alfalfa whole genome liquid phase chip is proposed.

[0090] In some embodiments of the present invention, the application is application in alfalfa genotyping.

[0091] In some embodiments of the present invention, the application is application in genome-wide association analysis of alfalfa.

[0092] In some embodiments of the present invention, the application is application in alfalfa cluster analysis or phylogenetic identification.

[0093] In some embodiments of the present invention, the application is application in the evaluation of genetic diversity of alfalfa.

[0094] In some embodiments of the present invention, the application is application in alfalfa breeding or assisted breeding.

[0095] In some embodiments of the present invention, the breeding or assisted breeding includes at least one of assisted major effect gene selection, molecular marker-assisted breeding directed improvement, whole genome selection breeding, alfalfa variety authenticity identification, genetic map construction, gene positioning, species evolution analysis and germplasm resource identification.

[0096] According to a fifth aspect of the present invention, a method for breeding alfalfa is proposed, comprising the following steps: using at least one of the above-mentioned alfalfa SNP molecular markers, kits or alfalfa whole genome liquid phase chips to detect the DNA of the sample to be tested, and selecting suitable alfalfa materials for subsequent breeding.

[0097] In some embodiments of the present invention, the detection is performed based on liquid phase probe capture sequencing typing technology.

[0098] The present invention has at least the following beneficial effects: the SNP molecular marker site of the present invention has good specificity, strong versatility, and high polymorphism, and can accurately and effectively distinguish alfalfa germplasm resources of different sources and different characteristics. The SNP molecular marker of the present invention is used to prepare an alfalfa whole-genome liquid-phase gene chip, which can be used for genetic structure analysis of alfalfa germplasm resource populations, genetic diversity assessment, genetic map construction and functional gene positioning, whole-genome association analysis, etc., providing important tools and technical support for molecular design breeding of alfalfa and authenticity identification of varieties, which is of great significance to the high-quality development of my country's alfalfa seed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0100] Figure 1 is the MAF distribution histogram in Example 1 of the present invention;

[0101] Figure 2 The chromosome distribution map of the alfalfa 60KcGPS liquid phase chip loci in Example 1 of the present invention;

[0102] Figure 3 This is a schematic diagram of the cGPS liquid chip process detection in Example 2 of the present invention;

[0103] Figure 4 This is a cluster analysis diagram of alfalfa in Example 4 of the present invention. DETAILED DESCRIPTION

[0104] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0105] Example 1

[0106] This embodiment provides a combination of SNP molecular markers for alfalfa, including 61,351 SNP sites, the physical locations of which are determined by sequence alignment based on the alfalfa reference genome (ZM-4alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c), and the 61,351 SNP molecular markers are shown in the invention content section (specific information is shown in the invention content table). The screening and obtaining process is as follows:

[0107] 1. Obtain the SNP candidate site set from whole genome resequencing data

[0108] In order to obtain whole-genome loci rich in genetic information, we collected resequencing data of 358 core alfalfa germplasm resources from around the world published by the National Genome Science Data Center (https: / / ngdc.cncb.ac.cn / ), and collected 380 alfalfa materials from different countries such as China, the United States, Russia, and Spain for whole-genome resequencing. The library was sequenced on the MGI's DNBSEQ-T7 high-throughput sequencing platform. SNP calling was filtered using fastp (v0.20.0) according to the process, aligned to the alfalfa reference genome (ZM-4alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c) using Burrows-Wheeler Aligner (0.7.12-r1039), and picard1.107 software (http: / / www.psc.edu / index.php / user-resources / software / picard) was used to sort and convert sam files into bam files, and PCR duplicates were removed. GATK software was used to detect and filter SNPs. According to the genomic data, the filtering criteria were as follows: QD<2.0||FS>60.0||MQ<35.0||MQRankSum<-12.5||Read PosRankSum<-8.0||DP>6950. After filtering, a .vcf file containing SNP variation information of all samples was obtained, totaling 4,052,804 SNP sites.

[0109] 2. Site screening

[0110] The quality indicators of all sites were calculated, and 1,739,982 SNP sites were screened based on the parameters of Maf ≥ 0.05, detection rate ≥ 85%, heterozygosity ≤ 0.5, and Depth ≥ 5, and these sites were used for probe design. The principle of probe design is to design probes within 100 bp of all target sites. The probe length is generally 100 bp, and the GC content is between 20% and 80%. According to the probe design results, probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences are removed. Based on the principle of uniform distribution on chromosomes, 59,903 highly polymorphic SNP sites were finally obtained, such as Figure 1As shown. The functional genes reported to be related to important economic traits of alfalfa such as growth and development, quality and stress resistance were collected, and the related SNP sites on these genes were screened as candidate sites, totaling 1448; including 295 trait sites related to growth and development, 1129 sites related to resistance (including resistance to biotic stress and resistance to abiotic stress), 11 sites related to quality traits, and 12 other unknown functional sites. Finally, 61351 candidate SNP sites were obtained. The site information is shown in Table 1, with an average spacing of 41Kb, and the chromosome distribution map is shown in Figure 2 shown.

[0111] Example 2

[0112] This embodiment provides an alfalfa 60K whole genome liquid phase chip.

[0113] According to the upstream and downstream sequences of 61,351 SNP polymorphic sites of alfalfa, based on the design principle of the probe in Example 1, a liquid phase capture probe was synthesized by Huazhi Biotechnology Co., Ltd., and a system of alfalfa whole genome liquid phase chip was formed using the precise positioning sequencing typing technology (cGPS) based on liquid phase capture of the target region genome sequence. cGPS is based on an optimized thermodynamic stability algorithm model, and probes are designed for the genome sequences of different target regions. Synthesized specific probes are used to perform liquid phase hybridization capture enrichment on multiple different target sequences located at different genomic positions, and then the captured and enriched target genome sequences are sequenced. Library construction and high-throughput sequencing are performed to obtain the genotypes of all SNP / InDel sites in the target region. The cGPS liquid phase chip process detection schematic diagram is shown below. Figure 3 shown.

[0114] Example 3 Application of Alfalfa 60K Whole Genome Liquid Phase Chip in Detecting Alfalfa DNA Samples

[0115] 1. Extraction and detection of gDNA from alfalfa

[0116] 25 alfalfa germplasm materials were selected as validation samples for the alfalfa 60K whole genome liquid phase chip development system. Fresh leaves of 25 alfalfa materials were collected and gDNA was extracted from the tissues using the magnetic bead method. The integrity and purity of gDNA were analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.

[0117] 2. cGPS Experiment Process

[0118] (1) Take 200 ng of gDNA that has passed the quantitative quality inspection, cut the DNA into 100-500 bp fragments using an enzyme digestion reagent, and then add Taq enzyme for end repair;

[0119] (2) Use T4 ligase to connect the adapter fragments to both ends of the DNA, use fragment sorting magnetic beads to purify the ligation products and amplify the library to complete the library construction;

[0120] (3) Place the library that has passed the quality inspection, the blocking reagent, the RNase inhibitor RNase Block, and the 60K liquid phase chip probe on a PCR instrument for hybridization reaction, and incubate the hybridization at 55°C overnight (16-24h);

[0121] (4) The hybridization product was captured using streptavidin, the captured library was amplified and enriched, and PE150 sequencing was performed using the BGI sequencing platform;

[0122] (5) The raw data after high-throughput sequencing is processed by quality control filtering, and fastp software is used to remove reads containing adapter contamination and low-quality reads. BWA software is used to compare with the target genome, and then GATK software is used to analyze the variant sites of the sequencing results to obtain the genotyping results of the target sites. The process diagram is shown in the figure. Figure 2 shown.

[0123] Table 2

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[0125] Table 3

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[0127] 3. Evaluation of 60K liquid phase chip genotyping effect of alfalfa

[0128] The 60K liquid phase chip obtained in Example 1 was used to perform genotyping detection on 25 alfalfa materials (the specific operation method is shown in Example 2), and a total of 28 data were generated (including 3 repeated samples). The results are shown in Tables 2 and 3. As can be seen from Table 2, the site detection rate of the 28 samples is between 90.00% and 96.75%, and the average detection rate is 92.11%; as can be seen from Table 3, the consistency rate of the genotype comparison results of the three repeated samples is between 97.03% and 97.13%, and the average consistency rate is 97.08%.

[0129] Example 4 Analysis of Alfalfa Population Structure Using 60K Whole Genome Liquid Phase Chip

[0130] The alfalfa 60K whole genome liquid phase chip prepared in Example 2 was used to test 457 samples of alfalfa (known source), extract genotyping, and use Plink software to calculate the genetic distance matrix based on the IBS method and perform cluster analysis. The phylogenetic tree diagram was constructed to determine the genetic relationship, evolutionary relationship and composition structure of different materials. The results are as follows Figure 4 As shown in the figure, it can be seen that alfalfa germplasms from the same origin or the same continent are clustered together, indicating that alfalfa from different regions has large genetic variation. This liquid phase chip can perform good genetic diversity and population structure analysis through alfalfa 60K whole genome liquid phase chip detection.

[0131] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A combination of SNP molecular markers for alfalfa, characterized in that: The SNP molecular marker combination consists of 61,351 SNP molecular markers, and the physical positions of the 61,351 SNP molecular markers are determined based on sequence alignment of the alfalfa reference genome ZM-4alfalfa genome. The site information is specifically shown in Table 1 in the specification.

2. A primer set and / or liquid phase probe for detecting the alfalfa SNP molecular marker combination according to claim 1.

3. A liquid phase chip of alfalfa whole genome, characterized in that: The alfalfa whole genome liquid phase chip comprises the primer set and / or liquid phase probe according to claim 2.

4. A kit, characterized in that: The kit comprises the primer set and / or liquid phase probe according to claim 2.

5. Use of at least one of the alfalfa SNP molecular marker combination according to claim 1, the primer set and / or liquid phase probe according to claim 2, the alfalfa whole genome liquid phase chip according to claim 3, and the kit according to claim 4 in alfalfa genotyping.

6. Use of at least one of the alfalfa SNP molecular marker combination according to claim 1, the primer set and / or liquid phase probe according to claim 2, the alfalfa whole genome liquid phase chip according to claim 3, and the kit according to claim 4 in alfalfa whole genome association analysis.

7. Use of at least one of the alfalfa SNP molecular marker combination according to claim 1, the primer set and / or liquid phase probe according to claim 2, the alfalfa whole genome liquid phase chip according to claim 3, and the kit according to claim 4 in alfalfa cluster analysis or kinship identification.

8. Use of at least one of the alfalfa SNP molecular marker combination according to claim 1, the primer set and / or liquid phase probe according to claim 2, the alfalfa whole genome liquid phase chip according to claim 3, and the kit according to claim 4 in the identification of alfalfa genetic diversity.

9. Use of at least one of the alfalfa SNP molecular marker combination described in claim 1, the primer set and / or liquid phase probe described in claim 2, the alfalfa whole genome liquid phase chip described in claim 3, and the kit described in claim 4 in alfalfa breeding or assisted breeding; preferably, the breeding or assisted breeding includes at least one of assisted major effect gene selection, molecular marker-assisted breeding directed improvement, whole genome selection breeding, alfalfa variety identification, genetic map construction, gene positioning, species evolution analysis and germplasm resource genetic diversity evaluation.

10. A method for breeding alfalfa, characterized in that: The method comprises the following steps: using at least one of the alfalfa SNP molecular marker combination described in claim 1, the primer set and / or liquid phase probe described in claim 2, the alfalfa whole genome liquid phase chip described in claim 3, and the kit described in claim 4 to detect the DNA of the sample to be tested, and selecting suitable alfalfa materials for subsequent breeding.

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