A SNP molecular marker combination of alfalfa and its application

Abstract

The present invention discloses a SNP molecular marker combination of alfalfa and its application. The SNP molecular marker combination consists of 61,351 SNP molecular markers. The physical positions of the 61,351 SNP molecular markers are determined by sequence alignment based on the alfalfa reference genome ZM-4 alfalfa genome. The SNP molecular marker sites of the present invention have good site specificity, strong universality, and high polymorphism, and can effectively distinguish alfalfa germplasms from different sources. Preparing the SNP molecular markers of the present invention into a whole-genome liquid gene chip for alfalfa can be used for genetic diversity assessment of alfalfa germplasm resources, construction of genetic maps and functional gene mapping, genome-wide association analysis, etc., providing important tools and technical support for molecular design breeding of alfalfa and identification of variety authenticity, and having important significance for the high-quality development of the alfalfa seed industry in China.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a SNP molecular marker combination of alfalfa and its application. Background Art

[0002] Alfalfa (Medicago sativa L.) is one of the most important perennial leguminous forages in the world, and also an excellent leguminous forage with the earliest cultivation, the widest distribution and the highest utilization efficiency. It has high yield, rich nutritional value, good palatability, drought resistance, salt tolerance, and strong biological nitrogen fixation and soil improvement characteristics. It is known as the "king of forages" and plays an irreplaceable role in promoting the adjustment of agricultural industrial structure, the development of high-efficiency and high-quality animal husbandry, and ecological governance and restoration.

[0003] At present, alfalfa breeding is still in the stage of 1.0 - 2.0. The identification efficiency of germplasm resources is low, the accuracy is not high, the development and utilization of excellent germplasm resources are seriously insufficient, there is a lack of high-throughput genotype identification and analysis technology systems and molecular markers with important breeding value, and genome-wide selection breeding has not been deeply carried out, resulting in low efficiency in the research and development of alfalfa varieties, few excellent alfalfa varieties with outstanding traits, and seriously restricting the development of the alfalfa industry. Therefore, developing a high-density alfalfa whole-genome liquid breeding chip can efficiently, accurately and low-costly genotype and detect large-scale alfalfa germplasm resource populations, break through the bottleneck problems existing in the evaluation of alfalfa germplasm resources and variety cultivation, improve breeding efficiency, and help alfalfa enter intelligent molecular design breeding from conventional 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. For this purpose, the present invention proposes a SNP molecular marker combination of alfalfa.

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

[0006] The present invention also proposes a gene chip having the above-mentioned SNP molecular marker combination of alfalfa.

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

[0008] The present invention also proposes an application of the above-mentioned SNP molecular marker combination of alfalfa, gene chip and kit.

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

[0010] According to one aspect of the present invention, an alfalfa SNP molecular marker combination is proposed, which consists of 61,351 SNP molecular markers. The physical positions of the 61,351 SNP molecular markers are determined by sequence alignment based on the alfalfa reference genome (ZM-4 alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c), and the locus information is shown in Table 1 below.

[0011] Table 1

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[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, where 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 represented in the form of chromosome number: physical position.

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

[0083] According to the 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 by using targeted capture sequencing technology according to the position of the SNP site and the sequences on both sides thereof.

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

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

[0087] The alfalfa whole-genome liquid chip of the present invention is based on the principle of liquid chip and can be efficiently, accurately and low-costly applied to the genotyping detection of large-scale alfalfa populations. (1) In terms of performance, all SNP sites of this chip come from whole-genome resequencing data. Compared with the currently widely used reduced-representation genome sequencing technologies (such as RAD-seq, GBS, etc.), these sites have a wider coverage, stronger representativeness and higher polymorphism in the alfalfa whole genome, and have advantages in detection accuracy and density. (2) In terms of economic cost, compared with the currently widely used resequencing method, the detection cost of this chip for alfalfa genotype is about 1000-1200 yuan / sample, and the price of imported sequencing instruments is higher, while the detection cost of this technology can be as low as 200 yuan / sample, greatly reducing the detection cost, and the chip sites are flexible, and new functional marker sites can be added at any time later, with strong application and promotion. (3) This chip contains 1448 trait-associated sites, which can be directly used for the identification, evaluation, development and utilization of important economic traits such as yield, quality and stress resistance of alfalfa germplasm materials, and has strong practicability.

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

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

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

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

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

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

[0094] In some embodiments of the present invention, the application is for 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 gene selection, molecular marker-assisted breeding for directional improvement, genome-wide selection breeding, alfalfa variety authenticity identification, genetic map construction, gene mapping, species evolution analysis, and germplasm resource identification.

[0096] According to a fifth aspect of the present invention, an alfalfa breeding method is provided, which includes the following steps: detecting the DNA of a test sample using at least one of the above-mentioned alfalfa SNP molecular markers, kit or alfalfa whole-genome liquid-phase chip, and selecting suitable alfalfa materials for subsequent breeding.

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

[0098] The present invention has at least the following beneficial effects: The SNP molecular markers of the present invention have good site specificity, strong universality, and high polymorphism, and can accurately and effectively distinguish alfalfa germplasm resources from different sources and with different characteristics. Preparing the alfalfa whole-genome liquid-phase gene chip with the SNP molecular markers of the present invention can be used for alfalfa germplasm resource population genetic structure analysis, genetic diversity assessment, genetic map construction and functional gene mapping, genome-wide association analysis, etc., providing important tools and technical support for alfalfa molecular design breeding and variety authenticity identification, and having important significance for the high-quality development of the alfalfa seed industry in China. Brief Description of the Drawings

[0099] The present invention will be further described below in conjunction with the drawings and embodiments, where:

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

[0101] Figure 2 It is the chromosome distribution map of Medicago sativa 60KcGPS liquid-phase chip loci in Embodiment 1 of the present invention;

[0102] Figure 3 It is the schematic diagram of the cGPS liquid-phase chip process detection in Embodiment 2 of the present invention;

[0103] Figure 4 It is the cluster analysis diagram of Medicago sativa in Embodiment 4 of the present invention. Detailed Embodiments

[0104] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with the embodiments 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 the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0105] Embodiment 1

[0106] This embodiment provides a Medicago sativa SNP molecular marker combination, including 61,351 SNP loci. The physical positions of the 61,351 SNP loci are determined by sequence alignment based on the Medicago sativa reference genome (ZM-4 alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c). The 61,351 SNP molecular markers are as shown in the content of the invention part (specific information is shown in the content table of the invention). The specific process of its screening is as follows:

[0107] 1. Obtain a set of SNP candidate loci from the source of whole-genome resequencing data

[0108] To obtain genome-wide loci rich in genetic information, we collected the resequencing data of 358 core alfalfa germplasm resources from around the world published by the National Genomics 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 libraries were sequenced on the MGI DNBSEQ-T7 high-throughput sequencing platform. For SNP calling, we sequentially used fastp (v0.20.0) for filtering, and aligned the reads to the alfalfa reference genome (ZM-4 alfalfa genome, https: / / figshare.com / s / fb4ba8e0b871007a9e6c) using Burrows-Wheeler Aligner (0.7.12-r1039). The picard1.107 software (http: / / www.psc.edu / index.php / user-resources / software / picard) was used to sort the sam file and convert it to a bam file, and PCR duplicates were removed. The GATK software was used for SNP detection and filtering. According to the genomic data, the following filtering criteria were adopted: 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 variant information for all samples was obtained, with a total of 4,052,804 SNP loci.

[0109] 2. Locus Screening

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

[0111] Example 2

[0112] This example provides an alfalfa 60K whole-genome liquid chip.

[0113] According to the upstream and downstream sequences of 61351 SNP polymorphic loci of alfalfa, based on the probe design principle in Example 1, liquid-phase capture probes are synthesized by Huazhi Biotechnology Co., Ltd., and a system for the alfalfa whole-genome liquid chip is formed by using the precise positioning sequencing genotyping technology (cGPS) based on the liquid-phase capture of the genomic sequence in the target region. cGPS designs probes for the genomic sequences of different target regions based on an optimized thermodynamic stability algorithm model, uses the synthesized specific probes to perform liquid-phase hybridization capture and enrichment on multiple different target sequences located at different genomic positions, and then constructs a sequencing library and performs high-throughput sequencing on the captured and enriched target genomic sequences, so as to obtain the genotypes of all SNP / InDel loci in the target region. The schematic diagram of the cGPS liquid chip process detection is as Figure 3 shown.

[0114] Example 3 Application of the alfalfa 60K whole-genome liquid chip in detecting alfalfa DNA samples

[0115] 1. Extraction and detection of alfalfa gDNA

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

[0117] 2. cGPS experimental procedure

[0118] (1) Take 200 ng of quantitatively qualified gDNA after quality inspection, use the restriction enzyme reagent to cut the DNA into fragments of 100 - 500 bp in size, and then add Taq enzyme for end repair;

[0119] (2) Use T4 ligase to ligate 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 library construction;

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

[0121] (4) Capture the hybridization products using streptavidin, amplify and enrich the captured library, and perform PE150 sequencing using the BGI sequencing platform;

[0122] (5) The raw data after high-throughput sequencing is processed through quality control filtering, etc. Use the fastp software to remove Reads contaminated with adapters and low-quality Reads. Align with the target genome using the BWA software, and then use the GATK software to analyze the variant sites of the sequencing results to obtain the genotype typing results of the target sites. The flow schematic diagram is as Figure 2 shown.

[0123] Table 2

[0124]

[0125] Table 3

[0126]

[0127] 3. Evaluation of the genotype typing effect of Medicago sativa 60K liquid chip

[0128] Use the 60K liquid chip obtained in Example 1 to perform genotyping detection on 25 Medicago sativa materials (the specific operation method is shown in Example 2), and a total of 28 data are generated (including 3 duplicate samples). The results are shown in Table 2 and Table 3. It can be seen from Table 2 that the site detection rate of the 28 samples is between 90.00% and 96.75%, and the average detection rate is 92.11%; it can be seen from Table 3 that for the genotype comparison results of the 3 duplicate samples, the consistency rate is between 97.03% and 97.13%, and the average consistency rate is 97.08%.

[0129] Example 4 Analysis of the population structure of Medicago sativa using the Medicago sativa 60K whole-genome liquid chip

[0130] Use the Medicago sativa 60K whole-genome liquid chip prepared in Example 2 to detect 457 Medicago sativa (with known sources) samples, extract genotyping, use the Plink software, calculate the genetic distance matrix based on the IBS method and perform cluster analysis, and construct a phylogenetic tree diagram to judge the genetic relationship, evolutionary relationship, and compositional structure population structure analysis among different materials. The results are asFigure 4 As shown, it can be seen from the figure that alfalfa germplasms from the same place of origin or from the same continent are clustered together, indicating that there are significant genetic variations among alfalfa from different regions. This liquid-phase chip can perform very good genetic diversity and population structure analysis through the detection of the 60K whole-genome liquid-phase chip of alfalfa.

[0131] The above has made a detailed description of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. An application of a reagent for detecting alfalfa SNP molecular marker combination in alfalfa genetic diversity evaluation, cluster analysis and kinship identification, 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 alfalfagenome. The site information is specifically shown in Table 1 in the specification.

2. Use of a reagent for detecting alfalfa SNP molecular marker combination in alfalfa genotyping, 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.

3. Use of a reagent for detecting alfalfa SNP molecular marker combination in alfalfa genome-wide association analysis, 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.

Images