Gossypium hirsutum low-density liquid phase chip based on targeted capture sequencing and use thereof

Abstract

Disclosed are a Gossypium hirsutum low-density liquid phase chip based on targeted capture sequencing and the use thereof. The low-density chip comprises 908 SNP sites, including 329 sites significantly associated with important agronomic traits such as fiber quality, yield, and disease resistance of Gossypium hirsutum, 14 common sites for plant transgene detection, and 565 other sites. The chip is suitable for the use in transgenic ingredient detection, resource evaluation, genetic identification, and seed purity identification in Gossypium hirsutum varieties, and genetic improvement of major agronomic traits in Gossypium hirsutum varieties.

Description

A low-density liquid phase chip for upland cotton based on targeted capture sequencing and its application Technical Field

[0001] The present invention relates to the fields of genetics, molecular biology, bioinformatics and cotton molecular breeding, and in particular to a low-density liquid phase chip of upland cotton based on targeted capture sequencing and applications thereof. Background Art

[0002] Single Nucleotide Polymorphism (SNP) refers to the variation of a single nucleotide in the genome, which is a form of variation formed by the substitution, transversion, insertion or deletion of a single nucleotide pair. As a third-generation genetic marker, SNPs are numerous, densely distributed, and easy to detect, making them ideal targets for genotyping. Looking at the innovation of molecular marker technology, single nucleotide polymorphisms (SNPs) based on DNA sequence variation are close to the ultimate standard for variation detection at the molecular level. It is expected that the development of molecular detection technology and platforms in the next few years will be roughly based on the improvement and enhancement of this basis. Therefore, efficient and low-cost SNP genotyping technology has become the best choice for the development of shared technologies and platforms.

[0003] SNP-based genotyping by target sequencing (GBTS) technology involves selecting specific target sites from genomic DNA for sequencing and genotyping. This targeted or fixed simplified genome sequencing significantly reduces the amount of DNA sequencing, simplifies bioinformatics analysis and data processing, and improves adaptability to various genotyping platforms. Furthermore, this technology is suitable for detecting all marker sites, including sites with known functions (such as cloned genes), sites with unknown functions (candidate genes), and neutral sites. The system's flexible marker count provides enormous flexibility for various applications, essentially adapting to all scenarios requiring different marker counts, such as marker-assisted major gene selection, backcross breeding and background selection, multi-gene aggregation breeding, seed purity testing, and identification of transgenic components. Therefore, GBTS will be the preferred choice for genotyping testing for a long time to come.

[0004] Developed cotton SNP arrays, such as the Illumina 80K and ZJU40K, have been widely adopted by breeding companies and research institutions for genotyping, genomic selection, and analysis of genetic diversity in germplasm resources. These arrays generally have high SNP densities, and the Illumina 80K solid-phase array suffers from the drawback of being difficult to customize. Array loci are generally designed based on uniform distribution across the genome, without considering associations with traits. This results in high typing costs and limited selection. Traditional breeding cycles are long, phenotypic variation is unpredictable, and selection relies primarily on the breeder's experience. This can easily lead to the loss of other desirable traits when improving a target trait. Molecular marker-assisted breeding leverages the tight linkage between molecular markers and genes that determine the target trait. By detecting molecular markers and target genes and loci, selection for the target trait is achieved. It offers the advantages of rapidity, accuracy, and environmental robustness. However, its key limitation is the accurate identification of loci associated with the trait.

[0005] In summary, based on the loci significantly associated with important agronomic traits of upland cotton such as fiber quality, yield, and disease resistance discovered through genome sequencing and whole-genome association analysis, as well as common plant transgenic detection sites and some background sites, it is urgent to develop a low-density liquid-phase SNP chip for upland cotton genotyping, transgenic detection, germplasm resource evaluation, and molecular marker-assisted selection.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies of the existing technology, discover and screen 894 SNP sites, and develop a liquid gene chip using the 894 SNP sites of upland cotton and 14 major transgenics for the detection and genotyping of transgenic components in upland cotton.

[0008] The technical solutions adopted in the present invention are as follows:

[0009] A low-density liquid phase array of upland cotton based on targeted capture sequencing, wherein the whole-genome low-density SNP array of upland cotton is composed of probes for detecting 894 SNP sites and 14 major transgenics; the 14 major transgenics are specifically: A1Bt, A2CPTI, A3CP4-epsps, A4CP4-epsps, A5bar, A6pat, A7CdP450, A8cp4-epsps, B.1CaMV35S, B.2FMV35S, B.3NOS, B.4NOS, B.5CaMV35S and NPTII; the 894 SNP sites are shown in Table 1.

[0010] Furthermore, the probes for detecting the 14 major transgenic genes are shown in SEQ ID NO. 1 to SEQ ID NO. 14.

[0011] The gene chip described in the present invention adopts a liquid phase chip and is based on liquid phase probe capture technology to design and synthesize a set of probes containing 908 site information, which can be used to detect whether upland cotton contains 14 common transgenic components, as well as upland cotton resource evaluation and kinship identification, seed purity identification and genetic improvement of the main agronomic traits of upland cotton varieties.

[0012] Furthermore, the main agronomic traits include fiber quality traits, yield traits, and disease resistance.

[0013] The liquid phase chip technology system used in the present invention is the GenoBaits technology system. The working principle is that the probe designed based on the target SNP marker performs targeted sequence complementary binding and sequencing with the DNA of the test sample, thereby achieving the purpose of detecting the target SNP marker genotype of the test sample. In the present invention, all probes that can accurately detect 894 SNP sites and 14 major transgenics can achieve the same technical effect. The application of the chip of the present invention can greatly improve the accuracy of genotype detection, improve the detection level, reduce the detection cost, and provide important technical support for cotton molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the distribution of SNP markers on chromosomes; the statistical results of the number of SNP markers on different chromosomes: a. The horizontal axis is the chromosome number; b. The left vertical axis represents the number of sites and segments; c. The right vertical axis represents the chromosome length (unit: Mb);

[0015] Figure 2 shows the distribution of SNP sites on chromosomes. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to specific embodiments.

[0017] Example 1. Design and preparation of a low-density liquid phase chip of upland cotton based on targeted capture sequencing.

[0018] The specific method is:

[0019] 1. Based on the VCF files obtained from resequencing of three populations (Fang et al. Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits. Nature genetics, 2017, 49(7): 1089; Liu et al. Association mapping of seed oil and protein contents in upland cotton. Euphytica, 2015, 205(2): 637-645); and with the cotton genome TM-1_V2.1 (Hu et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton. Nature genetics, 2019, 51(4): 739-748.), 601 SNP sites (background sites) were screened out according to the principles of MAF>0.35, heterozygosity rate<15%, NA ratio<10%, and uniform distribution on chromosomes in the population.

[0020] 2. Based on the GWAS association results of major agronomic traits (Fang et al. Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits. Nature genetics, 2017, 49(7): 1089; Liu et al. Association mapping of seed oil and protein contents in upland cotton. Euphytica, 2015, 205(2): 637-645; Li et al. Genomic Insights into the Genetic Basis of Cotton Breeding in China, Molecular Plant 16, 662-677), 634 SNP sites (prospect sites) significantly associated with agronomic traits and disease resistance were selected.

[0021] 3. A total of 1235 target segments of the upland cotton genome were obtained above. By testing whether the candidate SNP positions and locus genotypes were consistent with the upland cotton reference genome TM-1V2.1 (cotton.zju.edu.cn), 1148 candidate target segments were obtained after screening.

[0022] 4. Based on the 1148 candidate target segments, probe design was performed. The specific design principles were as follows: select all probes with a length of 100-120bp that can cover the region; calculate the GC content of the probes that can cover the target region; and calculate the number of homology regions of the probes. Among them, a. completely identical over 40bp; b. 85% similarity and 80bp length; c. 95% similarity and 70bp length are all considered as one homology region. Probe selection principles: select probes with a GC content between 30% and 70%; select probes with a number of homology regions ≤ 5; select probe regions that do not contain SSR or N regions. Based on the above design and selection principles, a total of 1795 probes were designed and selected, which can detect 894 target segments. The core SNP site information corresponding to the 894 target segments is shown in Table 1. The chromosome distribution statistics of SNP markers are shown in Figure 1, and the distribution is shown in Figure 2.

[0023] 5. Based on the 14 common detection sequences for cotton GMO detection published by the Ministry of Agriculture, 14 detection probes were designed. The 14 detection probes provided in this example are shown in SEQ ID NOs. 1 to 14, and detect, in order, the A1Bt, A2CPTI, A3CP4-epsps, A4CP4-epsps, A5bar, A6pat, A7CdP450, A8cp4-epsps, B.1CaMV35S, B.2FMV35S, B.3NOS, B.4NOS, B.5CaMV35S, and NPTII genes. The amplification products of this primer pair combination can be subjected to a single high-throughput sequencing and analysis to complete the detection of GMO components in the sample. Among them, the probes for detecting the A3CP4-epsps gene and the B.4NOS gene (SEQ ID NOs. 3 and 12) are shown in the table. Wherein, I is hypoxanthine.

[0024] 6. A low-density liquid phase array of upland cotton based on targeted capture sequencing will be composed of probes for detecting 894 SNP sites and 14 major transgenics; among them, 329 sites are significantly associated with important agronomic traits such as upland cotton fiber quality, yield, and disease resistance, 14 common plant transgenic detection sites, and 565 other sites.

[0025] In this embodiment, a capture probe is synthesized by Boridi Biotechnology Co., Ltd. to construct a chip, which is based on the targeted genotyping detection (GBTS) technology of liquid phase probe hybridization: GenoBaits. The working principle of GenoBaits is to perform fixed-point capture based on the complementary binding of the target probe and the target sequence. First, a gDNA library is constructed for the material to be tested. At the same time, according to the principle of DNA complementarity, a probe covering the target SNP is designed at each site to be tested, and the target probe is labeled with biotin. Then, the biotin-labeled target probe is hybridized with the target region of the genome in a liquid state to form a double chain. Subsequently, streptavidin-coated magnetic beads are used to molecularly adsorb the target probe carrying the biotin label, thereby capturing the target hybridized with the probe. Finally, the captured target sequence is eluted, the target amplified, the library is constructed and sequenced, and the genotype of the target SNP is finally obtained.

[0026] Table 1 Information of 894 core SNP loci

[0027] Among them, Ref represents the base type of the SNP site in the reference genome, and Alt represents the base type of the SNP site in the population.

[0028] Example 2: Application of a low-density liquid phase array of upland cotton based on targeted capture sequencing in upland cotton genotyping and transgenic component detection.

[0029] The array was tested on 200 upland cotton varieties. The results showed that the cotton functional marker array had an average locus detection rate of 94.17%, meeting the requirements for upland cotton genotyping.

[0030] The entire experimental process is based on the GenoBaits technology system. The specific experimental process is as follows:

[0031] 1. Extract genomic DNA from the sample to be tested and construct a sample library;

[0032] 1.1 Sample DNA extraction

[0033] The sample DNA was extracted using the CTAB method.

[0034] 1.2 Sample DNA quality inspection

[0035] The DNA concentration of the test sample DNA was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and the integrity of the DNA was checked using 1 wt% agarose gel electrophoresis. The qualified samples were placed in a 4°C refrigerator for storage and future use.

[0036] 1.3 Sample DNA fragmentation

[0037] Take 12 μL of DNA that has passed the quality inspection and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic disruptor to randomly physically disrupt the DNA until the fragments are broken into 200-400 bp.

[0038] 1.4 Sample end repair

[0039] Add 4 μL of GenoBaits End Repair Buffer (GenoBaits, Shijiazhuang Boruidi Biotechnology Co., Ltd.) and 2.7 μL of GenoBaits End Repair Enzyme to the tube, add water to 20 μL, and place it in an ABI 9700 PCR instrument and incubate at 37°C for 20 minutes to complete the end repair and A addition process of the broken fragments.

[0040] 1.5 Sample Sequencing Adapter Connection

[0041] Remove the tube from the PCR instrument and add 2μL GenoBaits Ultra DNA ligase, 8μL GenoBaits Ultra DNA Ligase Buffer and 2μL GenoBaits Adapter, add water to 40μL, and then place it on the ABI9700 PCR instrument at 22℃ for 30 minutes to complete the connection of the sequencing adapter.

[0042] 1.6 Sample DNA purification

[0043] 48 μL of Beackman AMPure XP Beads (Beackman) were added to the ligation product to purify the ligation product. After purification, magnetic beads were used for fragment screening, and ligation products with insert fragments between 200 and 300 bp were retained.

[0044] 1.7 Sample library amplification

[0045] To the PCR tube from the previous step, add 5 μL of sequencing adapters with barcode sequences, 1 μL of L5 adapters, and 10 μL of GenoBaits PCR Master Mix. Fill to 20 μL with purified water. Amplify using an ABI 9700 PCR instrument using the following program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Repeat denaturation, annealing, and extension for a total of 8 cycles, followed by extension at 72°C for 5 minutes. Different barcodes are used to distinguish different samples.

[0046] 1.8 Sample library purification

[0047] Add 24 μL of Beckmen AMPure XP Beads (Beackman) to the second-round PCR product, pipette up and down to mix evenly, place a 0.2 μL PCR tube on a magnetic stand until the solution is clear, discard the supernatant, wash the magnetic beads once with 75 vol% ethanol, and elute the library DNA with Tris-HCl (pH 8.0).

[0048] 2. Using a low-density liquid phase array based on targeted capture sequencing to determine the genotypes of target samples at 894 SNP sites and 14 genes

[0049] 2.1 DNA hybridization

[0050] 500 ng of the constructed genomic DNA sequencing library was added to 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II. The mixture was evaporated to dryness at ≤70°C on an Eppendorf Concentrator plus (Eppendorf). 8.5 μL of GenoBaits 2×Hyb Buffer, 2.7 μL of GenoBaits Hyb Buffer Enhancer, and 2.8 μL of Nuclease-Free Water were added to the powder tube. Mix thoroughly by pipetting and incubate the tube at 95°C for 10 minutes on an ABI 9700 PCR instrument. Then, 3 μL of the synthesized probe (at a concentration of 60 ng / μL) was added to the tube. After vortexing and mixing, the tube was incubated at 65°C for 2 hours on an ABI 9700 PCR instrument to complete the probe hybridization reaction.

[0051] 2.2 DNA capture

[0052] Add 100 μL of GenoBaits DNA Probe Beads to the reaction system after hybridization in the previous step, pipette up and down 10 times, and incubate at 65°C in an ABI 9700 PCR instrument for 45 minutes to allow the magnetic beads to bind to the probe. Wash the probe-bound magnetic beads at 65°C with 100 μL of GenoBaits Wash Buffer I and 150 μL of GenoBaits Wash Buffer II, respectively. Then, wash the magnetic beads at room temperature with 100 μL of GenoBaits Wash Buffer I, 150 μL of GenoBaits Wash Buffer II, and 150 μL of GenoBaits Wash Buffer III, respectively. Resuspend the washed magnetic beads in 20 μL of Nuclease-Free Water.

[0053] Take 13 μL of resuspended DNA (with magnetic beads) and add it to a new 0.2 mL PCR tube. Then add 15 μL of GenoBaits PCR Master Mix and 2 μL of GenoBaits Primer Mix to configure the post-PCR system. Use ABI 9700 PCR instrument for library amplification. The amplification program is as follows: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; repeat denaturation, annealing, and extension for a total of 15 cycles; extension at 72°C for 5 minutes.

[0054] Add 45 μL of Beckman AMPure XP Beads (Beackman) to the post-PCR product and pipette up and down to mix thoroughly. Then, place the 0.2 mL PCR tube on a magnetic stand until the solution is clear. Discard the supernatant, wash the beads twice with 75 vol% ethanol, and elute the library DNA with Tris-HCl (pH 8.0). This completes the probe hybridization capture process.

[0055] 2.3 DNA hybridization capture library quality control

[0056] The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and then agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 300 and 400 bp.

[0057] 2.4 DNA hybridization capture library sequencing

[0058] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0059] 2.5 Genotype data analysis

[0060] After quality control by FastQC (www.bioinformatics.babraham.ac.uk / project), the sequencing data were aligned to the reference genome using the default parameters of BWA (bio-bwa.sourceforge.net). SNPs were identified in the sequencing data using GATK (software.broadinstitute.org / gatk) software, and genotyping information from probe capture sequencing was extracted using a self-written Perl script to generate the final genotyping results, as shown in Tables 2 and 3.

[0061] Table 2 Detection rate of low-density liquid phase gene chip in 200 cotton varieties

[0062] Table 3 Detection of genetically modified components in 200 cotton varieties using low-density liquid phase gene chip

[0063] These results demonstrate that the low-density SNP liquid-phase array has a detection rate of 90.7%-96.5% across 200 upland cotton accessions, with an average locus detection rate of 94%. This array exhibits high polymorphism across upland cotton varieties, making it highly suitable for evaluating upland cotton germplasm resources, genetic improvement, and detecting transgenic components, with high accuracy. Compared to existing solid-phase arrays and developed high-density liquid-phase arrays, this array can enable cotton breeding research at a lower cost.

[0064] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications derived therefrom remain within the scope of protection of the present invention.

Claims

1. A low-density liquid phase chip of upland cotton based on targeted capture sequencing, characterized in that: The upland cotton whole genome low-density SNP chip is composed of probes for detecting 894 SNP sites and 14 major transgenics; the 14 major transgenics are specifically: A1Bt, A2CPTI, A3CP4-epsps, A4CP4-epsps, A5bar, A6pat, A7CdP450, A8cp4-epsps, B.1CaMV35S, B.2FMV35S, B.3NOS, B.4NOS, B.5CaMV35S and NPTII; the 894 SNP sites are shown in Table 1.

2. The low-density liquid phase chip of upland cotton based on targeted capture sequencing according to claim 1, characterized in that: The probes for detecting the 14 major transgenic genes are shown in SEQ ID NO.1 to SEQ ID NO.

14.

3. The use of the low-density liquid phase chip of upland cotton based on targeted capture sequencing according to claim 1, characterized in that: include: Detection and genotyping of transgenic components of upland cotton, evaluation of upland cotton resources and kinship identification, seed purity identification and genetic improvement of major agronomic traits of upland cotton varieties.

4. The use according to claim 3, characterized in that: The main agronomic traits include fiber quality traits, yield traits, and disease resistance.

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