Medicago sativa SNP molecular marker combination and use thereof

Abstract

Provided are a Medicago sativa SNP molecular marker combination and a use thereof. The SNP molecular marker combination consists of 61351 SNP molecular markers. The physical positions of the 61351 SNP molecular markers are determined by sequence alignment on the basis of the Medicago sativa reference genome ZM-4 alfalfa genome. The SNP molecular markers have good site specificity, strong versatility, and high polymorphism and can effectively discriminate Medicago sativa germplasms from different sources. A Medicago sativa genome-wide liquid-phase gene chip is prepared from the SNP molecular markers, and can be used for genetic diversity assessment of Medicago sativa germplasm resources, genetic map construction and functional gene mapping, genome-wide association study, etc., providing an important tool and technical support for Medicago sativa molecular design breeding and variety authenticity identification, and achieving important significance for high-quality development of the alfalfa seed industry in China.

Description

A combination of SNP molecular markers in alfalfa and its application Technical Field

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

[0002] Alfalfa (Medicago 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 fertility improvement 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] Currently, alfalfa breeding is still in the 1.0-2.0 stage. The efficiency and accuracy of germplasm identification are low, the development and utilization of excellent germplasm resources are seriously insufficient, and there is a lack of high-throughput genotyping and analysis technology systems and molecular markers with important breeding value. Whole-genome selective breeding has not yet been carried out in depth, resulting in low efficiency in alfalfa variety research and development and a small number of excellent alfalfa varieties with outstanding traits, which seriously restricts the development of the alfalfa industry. Therefore, the development of a high-density alfalfa whole-genome liquid-phase breeding chip can efficiently, accurately, and low-costly genotype detection of large-scale alfalfa germplasm resource populations, breaking through the bottleneck problems in alfalfa germplasm resource evaluation and variety breeding, improving breeding efficiency, and helping alfalfa to move from conventional breeding to intelligent molecular design breeding.

[0004] Summary of the Invention

[0005] 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 an alfalfa SNP molecular marker combination.

[0006] The present invention also provides a primer set and / or liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker combination.

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

[0008] The present invention also provides a kit having the above-mentioned alfalfa SNP molecular marker combination.

[0009] The present invention also provides an application of the alfalfa SNP molecular marker combination, gene chip and kit.

[0010] The invention also provides a breeding method for alfalfa.

[0011] According to one aspect of the present invention, a combination of 61,351 SNP molecular markers is proposed for alfalfa. The physical positions of the 61,351 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.

[0012] Table 1

[0013] In some embodiments of the present invention, Alt is ".", indicating that it has multiple mutation possibilities.

[0014] 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.

[0015] In some embodiments of the present invention, the “-” in the table is “_”.

[0016] 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.

[0017] 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 in sequence from top to bottom and from left to right as shown in the above table.

[0018] According to a second aspect of the present invention, a primer set and / or liquid phase probe for detecting the aforementioned alfalfa SNP molecular marker combination is proposed.

[0019] 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.

[0020] 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%.

[0021] According to a third aspect of the present invention, an alfalfa whole-genome liquid phase chip is provided. The alfalfa whole-genome liquid phase chip comprises a primer set and / or liquid phase probe for detecting the above-mentioned alfalfa SNP molecular marker combination.

[0022] The alfalfa whole genome liquid phase chip of the present invention is based on the targeted capture principle and can be applied to genotyping detection of large-scale alfalfa populations with high efficiency, accuracy and low cost. (1) In terms of performance, all the SNP sites of this chip are derived from whole-genome resequencing data. Compared with the currently widely used simplified genome sequencing technology (RAD-seq, GBS, etc.), 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 genotype detection of alfalfa by this chip is mostly 1000-1200 yuan / sample. The price of using imported sequencers (Illumina, Thermo Fisher, etc.) is even higher. The detection cost of this technology can be as low as 200 yuan / sample, which greatly reduces the detection cost. In addition, the chip sites are flexible, and new functional marker sites can be added at any time in the future, which has strong application and promotion. (3) This chip contains 1448 trait-associated sites, 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.

[0023] According to a fourth aspect of the present invention, a kit is provided, comprising a primer set and / or a liquid phase probe for detecting the aforementioned alfalfa SNP molecular marker.

[0024] 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.

[0025] In some embodiments of the present invention, the application is application in alfalfa genotyping.

[0026] In some embodiments of the present invention, the application is an application in genome-wide association analysis of alfalfa.

[0027] In some embodiments of the present invention, the application is application in alfalfa cluster analysis or phylogenetic relationship identification.

[0028] In some embodiments of the present invention, the application is application in the evaluation of alfalfa genetic diversity.

[0029] In some embodiments of the present invention, the application is application in alfalfa breeding or assisted breeding.

[0030] In some embodiments of the present invention, the breeding or assisted breeding includes at least one of major effect gene selection, molecular marker-assisted breeding directed improvement, whole genome selection breeding, alfalfa variety authenticity identification, genetic map construction, gene positioning and germplasm resource genetic diversity evaluation.

[0031] 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 chip to detect the DNA of a sample to be tested, and selecting suitable alfalfa germplasm materials for subsequent breeding.

[0032] In some embodiments of the present invention, the detection is performed based on liquid phase probe capture sequencing typing technology.

[0033] 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 variety authenticity identification, which is of great significance to the high-quality development of my country's alfalfa seed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0035] FIG1 is a MAF distribution histogram in Example 1 of the present invention;

[0036] FIG2 is a chromosome distribution diagram of the alfalfa 60K cGPS liquid phase chip loci in Example 1 of the present invention;

[0037] FIG3 is a schematic diagram of the cGPS liquid chip process detection process in Example 2 of the present invention;

[0038] FIG4 is a cluster analysis diagram of alfalfa in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] The following will be clearly and completely described with reference to the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, features 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 fall within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially.

[0040] Example 1

[0041] This example provides a combination of SNP molecular markers for alfalfa, including 61,351 SNP sites. The physical locations of the 61,351 SNP sites were determined by sequence alignment based on the alfalfa reference genome (ZM-4 alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c). The 61,351 SNP molecular markers are shown in the Summary of the Invention section (detailed information is shown in the Summary of the Invention table). The screening process is as follows:

[0042] 1. Obtain a set of SNP candidate sites from whole genome resequencing data

[0043] To obtain genome-wide 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 / ). We also collected 380 alfalfa germplasm materials from different countries, including China, the United States, Russia, and Spain, for whole-genome resequencing. The libraries were sequenced using MGI's DNBSEQ-T7 high-throughput sequencing platform. SNP calling was performed according to the workflow using fastp (v0.20.0) for filtering and Burrows-Wheeler Aligner (0.7.12-r1039) for alignment to the ZM-4 alfalfa reference genome (https: / / figshare.com / s / fb4ba8e0b871007a9e6c). Picard 1.107 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 for SNP detection and filtering. Based on the genomic data, the following filtering criteria were used: 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.

[0044] 2. Site screening

[0045] Quality metrics were calculated for all loci, and 1,739,982 SNPs were screened based on the following parameters: Maf ≥ 0.05, detection rate ≥ 85%, heterozygosity ≤ 0.5, and Depth ≥ 5. These loci were then used for probe design. The principle of probe design is to design probes within 100 bp of each target locus. Probes are generally 100 bp in length and have a GC content between 20% and 80%. Based on the probe design results, probes that could not be uniquely aligned on the genome or contained repetitive sequences in the flanking sequences were removed. Based on the principle of uniform distribution across the chromosome, 59,903 highly polymorphic SNPs were ultimately obtained, as shown in Figure 1. We collected reported functional genes associated with important economic traits of alfalfa, such as growth and development, quality, and stress resistance. We then screened these genes for relevant single-nucleotide polymorphisms (SNPs) as candidate loci. A total of 1,448 SNPs were identified, including 295 loci related to growth and development, 1,129 loci associated with resistance (including resistance to biotic and abiotic stresses), 11 loci associated with quality traits, and 12 other loci of unknown function. Ultimately, 61,351 candidate SNPs were identified. The loci are shown in Table 1, with an average spacing of 41 kilobases. The chromosome distribution is shown in Figure 2.

[0046] Example 2

[0047] This embodiment provides an alfalfa 60K whole-genome liquid phase chip.

[0048] Based on the upstream and downstream sequences of 61,351 SNP polymorphic sites in alfalfa and the probe design principles of Example 1, a liquid phase capture probe was synthesized by Huazhi Biotechnology Co., Ltd., and a precise positioning sequencing typing technology (cGPS) based on liquid phase capture of target region genomic sequences was used to form an alfalfa whole-genome liquid phase chip system. Based on an optimized thermodynamic stability algorithm model, cGPS designs probes for genomic sequences in different target regions. Synthesized specific probes are used to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic locations. The captured and enriched target genomic sequences are then subjected to library construction and high-throughput sequencing to obtain the genotypes of all SNP / InDel sites in the target region. A schematic diagram of the cGPS liquid phase chip detection process is shown in Figure 3.

[0049] Example 3 Application of Alfalfa 60K Whole Genome Liquid Phase Chip in Detecting Alfalfa DNA Samples

[0050] 1. Extraction and detection of alfalfa gDNA

[0051] Twenty-five alfalfa accessions were selected as validation samples for the 60K whole-genome liquid phase array development system. Fresh leaves were collected from each of the 25 alfalfa accessions, and gDNA was extracted from the tissues using a magnetic bead method. The integrity and purity of the gDNA were analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using a Qubit assay.

[0052] 2. cGPS Experiment Process

[0053] (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;

[0054] (2) Use T4 ligase to connect the adapter fragments to both ends of the DNA, and use fragment sorting magnetic beads to purify the ligation products and amplify the library to complete the library construction;

[0055] (3) Place the library that has passed the quality inspection with the blocking reagent, RNase inhibitor RNase Block, and 60K liquid phase chip probe on a PCR instrument for hybridization reaction, and incubate the hybridization at 55°C overnight (16-24 hours);

[0056] (4) The hybridization products were captured using streptavidin, the captured library was amplified and enriched, and PE150 sequencing was performed using the BGI sequencing platform;

[0057] (5) The raw data after high-throughput sequencing was processed through quality control filtering and other processes. Reads containing adapter contamination and low-quality reads were removed using fastp software. BWA software was used to align the data with the target genome. The sequencing results were then analyzed for variant sites using GATK software to obtain the genotyping results of the target loci. The process flow is shown in Figure 3.

[0058] Table 2

[0059] Table 3

[0060] 3. Evaluation of 60K liquid phase array genotyping results for alfalfa

[0061] Genotyping of 25 alfalfa samples was performed using the 60K liquid phase chip obtained in Example 1 (see Example 2 for the specific operation method), generating a total of 28 data sets (including 3 replicate samples). The results are shown in Tables 2 and 3. As can be seen from Table 2, the locus detection rates of the 28 samples ranged from 90.00% to 96.75%, with an average detection rate of 92.11%. As can be seen from Table 3, the genotype comparison results of the three replicate samples had a consistency rate of 97.03% to 97.13%, with an average consistency rate of 97.08%.

[0062] Example 4 Analysis of Alfalfa Population Structure Using a 60K Whole-Genome Liquid Microarray

[0063] The alfalfa 60K whole-genome liquid phase chip prepared in Example 2 was used to perform sample testing on 457 alfalfa germplasms (known sources). Genotyping was extracted and the genetic distance matrix was calculated using Plink software based on the IBS method, and cluster analysis was performed to construct a phylogenetic tree. The relationship, evolutionary relationship, and population structure between different materials can be determined. The results are shown in Figure 4. As can be seen from the figure, 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.

[0064] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A SNP molecular marker combination 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-4 alfalfa 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 phylogenetic relationship 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 identifying alfalfa genetic diversity.

9. 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 breeding or assisted breeding; preferably, the breeding or assisted breeding includes at least one of major effect gene selection, molecular marker-assisted breeding directed improvement, whole genome selection breeding, alfalfa variety identification, genetic map construction, gene mapping, and genetic diversity evaluation of germplasm resources.

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 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 to detect DNA of a sample to be tested, and selecting suitable alfalfa germplasm materials for subsequent breeding.

Images