Lemon whole genome 50K liquid chip and application thereof

Abstract

The invention provides a lemon whole genome 50K liquid phase chip, which comprises a 50K liquid phase chip designed and synthesized by using SNP and INDEL marker sets and detailed annotation information of each marker site, and is composed of a genome data basis, an SNP site screening strategy, probe design and chip layout. The liquid-phase probe hybridization capture process comprises the following steps: S1, DNA library denaturation; s2, hybridizing the probe with a target spot; s3, capturing a target spot hybridized with the probe; s4, recovering the captured target spot sequence; s5, target spot amplification; the problems that lemon breeding is long in period and low in efficiency are solved, and molecular markers related to important characters of fruit trees are recognized and developed, so that the screening process of excellent fruit tree varieties is accelerated, the breeding period is shortened, and the breeding efficiency is improved through molecular marker-assisted selection in the breeding process; the gene chip solves the problem that the breeding difficulty of disease-resistant / disease-tolerant germplasm resources is high, and the genetic diversity of different fruit tree varieties can be evaluated by applying the gene chip.

Description

Technical Field

[0001] The present invention relates to the technical field of lemon breeding, and in particular to a lemon whole genome 50K liquid phase chip and applications thereof. Background Art

[0002] Lemon is an important commercial plant of the genus Citrus in the Rutaceae family, distributed throughout the world. It is rich in various nutrients and has antioxidant capacity. Lemon alkaloids have a wide range of anti-cancer and antibacterial activities. However, with the large-scale cultivation of lemons, diseases have become more frequent. The main diseases include yellow vein disease, resin disease, scab disease, ulcer disease, etc., which have caused serious losses to the lemon industry. In addition, problems such as the single main cultivated lemon variety, widespread disease of germplasm resources, and insufficient germplasm innovation and evaluation and utilization have become increasingly prominent. There is a large gap between the research and application of resource evaluation, variety breeding, quality improvement and modern production technology and that of developed countries. Therefore, the growth traits and resistance characteristics of lemons improved through breeding technology are crucial to promoting the healthy and green development of the lemon industry, and are of great significance to the high-quality development of Chongqing City's modern and efficient characteristic agricultural demonstration zone. Improving breeding efficiency and shortening breeding cycles are essential for the healthy and rapid development of the lemon industry. This requires a better understanding of the genes and genomic regions involved in agronomically important traits. Third-generation molecular marker technology, primarily SNP markers, demonstrates greater applicability and superiority over microsatellite markers, demonstrating improved genetic stability, efficiency, and automated analysis. A growing number of studies have uncovered significant associations between SNPs and important economic traits, and SNP analysis has identified candidate genes with diverse functions. With the continuous advancement of selective breeding technology, liquid phase microarray technology combined with high-throughput sequencing can sequence large numbers of samples. This highly efficient technology minimizes both human and material resources. However, the long lemon breeding cycle, low efficiency, and difficulty in selecting disease-resistant / tolerant germplasm resources remain unresolved. To solve the above technical problems, a lemon whole-genome 50K liquid phase chip and its application are proposed to meet the needs of commercial lemon breeding. Summary of the Invention

[0003] In view of this, the present invention provides a lemon whole genome 50K liquid phase chip and its application to solve or alleviate the technical problems existing in the prior art and at least provide a beneficial option.

[0004] The technical solution of the present invention is achieved as follows: a lemon whole genome 50K liquid phase chip, including a 50K liquid phase chip designed and synthesized using a set of SNP and INDEL markers and detailed annotation information for each marker site, consisting of a genomic data foundation, a SNP site screening strategy, and probe design and chip layout; The liquid phase probe hybridization capture process is as follows: S1, DNA library denaturation; S2, probe hybridization with target; S3, capture target hybridized with probe; S4, recovering the captured target sequence; S5. Target amplification.

[0005] Further preferably, in said S1, after the genomic DNA is extracted, the genomic DNA is fragmented using ultrasound and sequencing adapters are added.

[0006] Further preferably, in S2, the RNA probe labeled with biotin is combined with the DNA fragment that has been attached with a linker sequence.

[0007] Further preferably, in S3, the streptavidin-coated magnetic beads bind to the double-stranded complex of the biotin-labeled RNA probe and DNA.

[0008] Further preferably, in said S4, the DNA in the target region is cleaned, the purpose of which is to remove non-specific hybridization and improve the capture efficiency.

[0009] Further preferably, in S5, the eluted DNA product is amplified by PCR to construct an Illumina sequencing library.

[0010] The embodiment of the present invention adopts the above technical solution, which has the following advantages: First, the present invention solves the problems of long lemon breeding cycles and low efficiency by identifying and developing molecular markers related to important fruit tree traits, thereby accelerating the screening process of excellent fruit tree varieties through molecular marker-assisted selection during the breeding process, shortening the breeding cycle, and improving breeding efficiency; 2. The present invention solves the problem of difficulty in breeding disease-resistant / tolerant germplasm resources. The application of gene chips can evaluate the genetic diversity among different fruit tree varieties, provide a basis for selecting excellent germplasm, and can quickly identify genes related to resistance traits and cultivate new resistant varieties.

[0011] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0013] Figure 1 This is a capture flow chart of the liquid phase chip in the present invention; Figure 2 Design a flow chart for the liquid phase probe of the present invention; Figure 3 This is a gel image of the test sample DNA of the present invention; Figure 4 This is a flow chart of the probe synthesis of the present invention; Figure 5 This is a distribution map of 114,338 sites of the present invention; Figure 6 The distribution map of 53,366 sites selected for the present invention. DETAILED DESCRIPTION

[0014] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0015] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0016] Example 1 like Figure 1-4 As shown, the embodiment of the present invention provides a lemon whole genome 50K liquid phase chip, which is composed of a haplotype analysis genome based on Pacific Biosciences HiFi sequencing, ONT ultra-long read sequencing and Hi-C technology, a SNP site screening strategy and probe design and chip layout. The liquid phase probe design process is as follows Figure 2 As shown; The liquid phase probe hybridization capture process is as follows: S1. Denature the DNA library, extract the genomic DNA, fragment it using ultrasound, and add sequencing adapters. S2, probe hybridization with the target, combining the biotin-labeled RNA probe with the DNA fragment that has been attached with the adapter sequence; S3, capture the target hybridized with the probe, and the streptavidin-coated magnetic beads will bind to the double-stranded complex of the biotin-labeled RNA probe and DNA; S4, recover the target sequence, wash the target region DNA, and remove the non-specific hybridization to improve the capture efficiency; S5, target amplification, PCR amplification of the eluted DNA product, and construction of Illumina sequencing library.

[0017] Example two The embodiment of the present application also provides a 50K liquid chip designed and synthesized by using a SNP and INDEL marker set and detailed annotation information of each marker site, and a target region capture scheme, which comprises the following steps of preparing a Block blocking agent, extracting and testing a DNA sample, and hybridizing and capturing a liquid probe. When the Block blocking agent is prepared, the DNA concentration requirement is 1 μg / μl, the required volume is 50 ml, the sample needs to be collected from multiple representative strains, and some fragments with high repetitive sequences are combined with the target region in advance by the Block blocking agent before hybridization. These fragments combined in advance by the Block cannot be combined with the RNA hybridization probe, thereby improving the specific hybridization and capture efficiency. After ultrasonic disruption of the DNA, a linker sequence is added at both ends of the fragment. Since the similarity of these universal linker / primer sequences is very high, they are very easy to form complementary pairs and become Daisy-Chains, which affects subsequent sequencing. The principle of the Block is to block the linker sequences at the front and rear ends to prevent self-connection or connection. When the DNA sample is extracted and tested, fresh leaves are preferably used for subsequent target capture samples, and the samples stored at 4° and -80° are prone to degradation during the experiment. The CTAB method or magnetic bead extraction is used for DNA extraction, and the quality control standards are as follows: nanodrop quality control, OD260 / 280≥1.8, OD260 / 230≥1.5; Qbit quantification, the initial amount of DNA is 1 μg, the minimum is not less than 200 ng, and the concentration is not less than 1 ng / μL; DNA electrophoresis band, the main band is complete, and there is no obvious degradation; the DNA gel map of the test sample is as shown in Figure 3 ; When the liquid probe hybridization and capture are performed, the probe synthesis is performed, the probe synthesis flow chart is as shown in Figure 4 , and the capture process is as follows: S1, selecting 200 samples of different lemon varieties and hybridization types for resequencing; S2, after extracting the genomic DNA, the genomic DNA is fragmented by enzyme digestion and a sequencing adapter is added; the biotin-labeled RNA probe is combined with the DNA fragments with the adapter sequence; S3, the streptavidin-coated magnetic beads are combined with the double-stranded complex of the biotin-labeled RNA probe and the DNA (the probe is excessive); S4: Clean the DNA of the target region, perform PCR amplification on the eluted DNA product, construct an Illumina sequencing library, and then identify high-quality SNPs; S5. Design and synthesize a 50K liquid phase chip using the SNP and INDEL marker set and detailed annotation information for each marker site, and evaluate the information content of the SNPs on the chip.

[0018] Example 3 In this example, whole-genome resequencing analysis was performed using 100 lemon samples from different sources, initially obtaining a total of 1,412,202 highly polymorphic marker loci. Subsequently, data were filtered stepwise based on the stringent screening criteria of the chip design: (1) First, we removed multi-allelic sites and retained biallelic sites, obtaining 1,387,752 effective sites; (2) We further extracted 100 bp of flanking sequences upstream and downstream of the SNP from the selected sites and evaluated the GC content of the flanking sequences. Finally, we retained 660,794 sites with a GC content between 0.35 and 0.65. (3) Sites with repetitive sequences in the flanking sequences were removed, and the list was further streamlined to 650,915 effective sites; (4) Then, single copy evaluation was performed, and a total of 114,338 sites with sequence alignment consistency ≥90% and length ≥90 bp were selected, such as Figure 5 shown.

[0019] The above sites present a certain blank area in the lemon genome. To improve the coverage of the chip, the copy number limit of the blank area was further relaxed to 5 times. Finally, 53,366 marker sites suitable for chip development were selected to meet the design requirements of the 50K chip, such as Figure 6 shown.

[0020] The specific distribution of the 53,366 chip marker sites finally determined on each chromosome is shown in the following table:

[0021] The above data is the site information actually used by the lemon 50K chip involved in the present invention.

[0022] When the present invention is working, different lemon varieties and hybrid type samples are selected for resequencing. After extracting genomic DNA, the genomic DNA is fragmented by enzyme digestion and sequencing adapters are added. The RNA probe with biotin labeling is combined with the DNA fragments with the adapter sequence. The magnetic beads coated with streptavidin will be combined with the double-stranded complex of the RNA probe with biotin labeling and DNA. The DNA of the target area is cleaned, and the eluted DNA product is PCR amplified to construct an Illumina sequencing library, thereby identifying high-quality SNPs. The 50K liquid phase chip is designed and synthesized using the SNP and INDEL marker set and the detailed annotation information of each marker site, and the information content of the SNP on the chip is evaluated. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or replacements within the technical scope disclosed by the present invention, which should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A lemon whole genome 50K liquid phase chip, comprising a 50K liquid phase chip designed and synthesized using a set of SNP and INDEL markers and detailed annotation information for each marker site, characterized in that: It consists of genomic data foundation, SNP site screening strategy, probe design and chip layout; The liquid phase probe hybridization capture process is as follows: S1, DNA library denaturation; S2, probe hybridization with target; S3, capture target hybridized with probe; S4, recovering the captured target sequence; S5. Target amplification.

2. The lemon whole genome 50K liquid phase chip according to claim 1, characterized in that: In the S1, after the genomic DNA is extracted, the genomic DNA is fragmented using ultrasound and sequencing adapters are added.

3. The lemon whole genome 50K liquid phase chip according to claim 1, characterized in that: In the S2, the RNA probe labeled with biotin is combined with the DNA fragment that has been attached with the linker sequence.

4. The lemon whole genome 50K liquid phase chip according to claim 1, characterized in that: In the above-mentioned S3, the streptavidin-coated magnetic beads bind to the double-stranded complex of the biotin-labeled RNA probe and DNA.

5. The lemon whole genome 50K liquid phase chip according to claim 1, characterized in that: In said S4, the DNA of the target region is cleaned and obtained. Its purpose is to remove non-specific hybridization and improve capture efficiency.

6. The lemon whole genome 50K liquid phase chip according to claim 1, characterized in that: In the S5, the eluted DNA product is amplified by PCR to construct an Illumina sequencing library.

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