A Low-Density Liquid Chromatography Chip for Upland Cotton Based on Targeted Capture Sequencing and Its Application

Abstract

This invention discloses a low-density liquid chromatography-mass spectrometry (LC-MS) chip for upland cotton based on targeted capture sequencing and its applications. The low-density chip includes 908 SNP loci, of which 329 are significantly associated with important agronomic traits such as fiber quality, yield, and disease resistance in upland cotton, 14 are common transgenic detection loci, and 565 are other loci. This chip is suitable for applications in detecting transgenic components in upland cotton varieties, resource evaluation and kinship identification, seed purity identification, and genetic improvement of major agronomic traits in upland cotton varieties.

Description

Technical Field

[0001] This invention relates to the fields of genetics, molecular biology, bioinformatics and cotton molecular breeding, and in particular to a low-density liquid phase chip for upland cotton based on targeted capture sequencing and its application. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) refer to variations in a single nucleotide in the genome, formed by substitutions, transversions, insertions, or deletions of a single nucleotide pair. As a third-generation genetic marker, SNPs are numerous, densely distributed, and easily detected, making them an ideal target for genotyping. Looking at the innovations in molecular marker technology, SNPs based on DNA sequence variations have approached the ultimate standard for variation detection at the molecular level. It is expected that the development of molecular detection technologies and platforms in the coming years will largely focus on improvements and enhancements based on this foundation. Therefore, efficient and low-cost SNP genotyping technologies have become the best choice for developing shared technologies and platforms.

[0003] Genotyping by target sequencing (GBTS) technology selects specific target sites in 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 functionally known sites (such as cloned genes), functionally unknown sites (candidate genes), and neutral sites. The flexibility in the number of markers in the system provides enormous flexibility for various applications, essentially suitable for all scenarios requiring different numbers of markers, such as marker-assisted master gene selection, backcross breeding and background selection, multi-gene aggregation breeding, seed purity detection, and transgenic component identification. Therefore, GBTS will remain the preferred method for genotyping for a considerable period of time.

[0004] Developed cotton SNP chips, such as Illumina 80K and ZJU40K, have been widely adopted by breeding companies and research institutions for genotyping, genome selection, and genetic diversity analysis of germplasm resources. These chips generally have high SNP densities. However, the Illumina 80K solid-phase chip has the drawback of not being customizable. Chip locus design is generally based on loci evenly distributed throughout the genome, without considering their association with traits, resulting in high genotyping costs and limited targeted selection. Traditional breeding has a long cycle, unpredictable phenotypic variation, and relies mainly on breeders' experience for selection. Improving the target trait can easily lead to the loss of other desirable traits. Molecular marker-assisted breeding utilizes the close linkage between molecular markers and genes determining the target trait. By detecting molecular markers, the target gene and locus are identified to achieve the goal of selecting the target trait. It has the advantages of being fast, accurate, and unaffected by environmental conditions. Its key limiting factor is the accurate identification of loci associated with the trait.

[0005] In summary, based on the loci significantly associated with important agronomic traits such as fiber quality, yield, and disease resistance of upland cotton, as well as common transgenic detection loci and some background loci, 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. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by discovering and screening 894 SNP loci, and using these 894 SNP loci of upland cotton and 14 major transgenic species to develop a liquid-phase gene chip for the detection of transgenic components and genotyping of upland cotton.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] A low-density liquid phase chip for upland cotton based on targeted capture sequencing is disclosed. The upland cotton whole-genome low-density SNP chip consists of probes for detecting 894 SNP loci and 14 major transgenes. The 14 major transgenes are: 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 loci are shown in Table 1.

[0009] Furthermore, the probes for detecting the 14 major transgenic organisms are shown in SEQ ID NO.1 to SEQ ID NO.14.

[0010] The gene chip described in this invention is a liquid-phase chip. Based on liquid-phase probe capture technology, probes are designed and a set of 908 loci information is synthesized. It can be used to detect whether upland cotton contains 14 common transgenic components, as well as for upland cotton resource evaluation and kinship identification, seed purity identification, and genetic improvement of the main agronomic traits of upland cotton varieties.

[0011] Furthermore, key agronomic traits include fiber quality traits, yield traits, and disease resistance.

[0012] The liquid-phase chip technology used in this invention is the GenoBaits technology system. Its working principle is based on probes designed with target SNP markers, which bind to the DNA of the test sample through targeted sequence complementarity and sequencing, thereby achieving the purpose of detecting the genotype of the target SNP marker in the test sample. In this invention, any probe capable of accurately detecting 894 SNP loci and 14 major transgenic genes can achieve the same technical effect. The application of this chip can greatly improve the accuracy of genotype detection, enhance detection levels, reduce detection costs, and provide important technical support for cotton molecular breeding. Attached Figure Description

[0013] Figure 1 A statistical graph showing the distribution of SNP markers on chromosomes; the statistical results of the number of SNP markers on different chromosomes: a. The horizontal axis represents the chromosome number; b. The left vertical axis represents the number of loci and segments; c. The right vertical axis represents the chromosome length (in Mb);

[0014] Figure 2 This is a map showing the distribution of SNP sites on chromosomes. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to specific embodiments.

[0016] Example 1: Design and fabrication of a low-density liquid phase chip for upland cotton based on targeted capture sequencing.

[0017] The specific method is as follows:

[0018] 1. Based on the VCF files obtained from the 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 allotetraploidcotton. Nature genetics, 2019, 51(4):739-748.) as a reference, 601 SNP loci (background loci) were screened according to the principles of MAF>0.35, heterozygosity <15%, NA ratio <10%, and uniform chromosome distribution in the population.

[0019] 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. Euthytica, 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 loci (prospect loci) that were significantly associated with agronomic traits and disease resistance were selected.

[0020] 3. A total of 1235 target regions of the upland cotton genome were obtained. After screening, 1148 candidate target regions were obtained by detecting whether the candidate SNP positions and loci genotypes were consistent with the upland cotton reference genome TM-1V2.1 (cotton.zju.edu.cn).

[0021] 4. Based on 1148 candidate target regions, probe design was performed. The specific design principles were as follows: all probes with a length of 100-120 bp that could cover the target region were selected; the GC content of the probes covering the target region was calculated; and the number of homologous regions of the probes was calculated. Specifically, a. probes with a length of 40 bp or more that were completely identical; b. probes with a similarity of 85% and a length of 80 bp; and c. probes with a similarity of 95% and a length of 70 bp were all considered as a single homologous region. Probe selection principles were: probes with a GC content between 30% and 70% were selected; probes with ≤5 homologous regions were selected; and probe regions that did not contain SSR or N regions were selected. Based on these design and selection principles, a total of 1795 probes were designed and selected, capable of detecting 894 target regions. The core SNP loci information corresponding to the 894 target regions is shown in Table 1, and the SNP marker chromosome distribution statistics are as follows. Figure 1 As shown, the distribution is as follows Figure 2 As shown.

[0022] 5. Based on the 14 common detection sequences for cotton transgenic detection published in this embodiment, 14 detection probes were designed. These 14 probes, as shown in SEQ ID NO.1 to SEQ ID NO.14, are used to detect 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 in sequence. The amplification products of this primer pair combination can be used for high-throughput sequencing and analysis to complete the detection of transgenic components in the sample. The probes for detecting the A3CP4-epsps and B.4NOS genes (SEQ ID NO.3 and SEQ ID NO.12) are shown in the table; where I represents hypoxanthine.

[0023]

[0024]

[0025] 6. Probes for detecting 894 SNP sites and 14 major transgenes were used to construct a low-density liquid phase chip for upland cotton based on targeted capture sequencing; among them, 329 sites were significantly associated with important agronomic traits such as fiber quality, yield, and disease resistance of upland cotton, 14 common plant transgene detection sites, and 565 other sites.

[0026] In this embodiment, a capture probe chip was constructed using technology synthesized by Bored Biotechnology Co., Ltd. This chip is based on GenoBaits, a liquid-phase probe hybridization targeted genotyping (GBTS) technology. GenoBaits works by capturing targets at specific sites based on the complementary binding of the target probe and the target sequence. First, a gDNA library is constructed from the material to be tested. Simultaneously, based on the principle of DNA complementarity, probes covering the target SNP are designed at each test site and labeled with biotin. Then, the biotin-labeled target probes hybridize with the target genomic region in a liquid state to form double strands. Subsequently, streptavidin-coated magnetic beads are used to adsorb the biotin-labeled target probes, thereby capturing the target sites that hybridize with the probes. Finally, the captured target sequences are eluted, amplified, library constructed, and sequenced to obtain the genotype of the target SNP.

[0027] Table 1 Information on 894 core SNP sites

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] Where 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.

[0040] Example 2: Application of upland cotton low-density liquid phase chip based on targeted capture sequencing in upland cotton genotyping and transgenic component detection.

[0041] The chip was tested using 200 upland cotton varieties. The results showed that the average site detection rate of the cotton functional marker chip was 94.17%, which meets the requirements for upland cotton genotyping.

[0042] The entire experimental procedure was completed based on the GenoBaits technology system. The specific experimental procedure is as follows:

[0043] 1. Extract genomic DNA from the sample to be tested and construct a sample library;

[0044] 1.1 Sample DNA Extraction

[0045] DNA was extracted from the samples using the CTAB method.

[0046] 1.2 Sample DNA quality inspection

[0047] The DNA concentration of the test samples was determined using a Qubit Fluorometric Quantitation (Thermo Fisher) instrument, and the integrity of the DNA was detected by 1 wt% agarose gel electrophoresis. Samples that passed the tests were stored at 4°C for future use.

[0048] 1.3 Sample DNA Fragmentation

[0049] Take 12 μL of qualified DNA and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic disruptor to randomly break the DNA into fragments of 200-400 bp.

[0050] 1.4 Sample end repair

[0051] Add 4 μL of GenoBaits End Repair Buffer (GenoBaits, i.e., Shijiazhuang Borui Biotechnology Co., Ltd.) and 2.7 μL of GenoBaits End Repair Enzyme to the tube, add water to make up to 20 μL, and incubate at 37°C for 20 minutes in an ABI 9700 PCR instrument to complete the end repair and A addition process of the fragmented fragments.

[0052] 1.5 Sample sequencing adapter connection

[0053] Remove the tube from the PCR instrument and add 2 μL of GenoBaits Ultra DNA ligase, 8 μL of GenoBaits Ultra DNA Ligase Buffer, and 2 μL of GenoBaits Adapter. Add water to a final volume of 40 μL, then place the tube on an ABI 9700 PCR instrument at 22°C for 30 minutes to complete the ligation of the sequencing adapter.

[0054] 1.6 Sample DNA Purification

[0055] 48 μL of Beackman AMPure XP Beads (Beackman Corporation) was added to the ligation product to purify it. After purification, the fragments were screened using magnetic beads, and ligation products with insert fragments of 200–300 bp were retained.

[0056] 1.7 Sample Library Amplification

[0057] Add 5 μL of sequencing adapter with barcode sequence, 1 μL of LP5 adapter, and 10 μL of LevoBaits PCR Master Mix to the PCR tube from the previous step, and bring the volume to 20 μL with pure water. Amplify using an ABI 9700 PCR instrument. The amplification program is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s; repeat the denaturation, annealing, and extension cycles for a total of 8 cycles; final extension at 72℃ for 5 min. Different barcodes are used to distinguish different samples.

[0058] 1.8 Sample Library Purification

[0059] Add 24 μL of Beckmen AMPure XP Beads (Beackman) to the second round of PCR products, pipette and mix well, then place the 0.2 μL PCR tube on a magnetic rack until the solution is clear. Discard the supernatant and wash the magnetic beads once with 75 vol% ethanol. Elute the library DNA with Tris-HCl at pH 8.0.

[0060] 2. Genotyping of target samples at 894 SNP loci and 14 genes was determined using a low-density liquid chromatography-mass spectrometry (LC-MS) chip based on targeted capture sequencing of upland cotton.

[0061] 2.1 DNA hybridization

[0062] Take 500 ng of the constructed genomic DNA sequencing library, add 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II, and place it on an Eppendorf Concentrator plus vacuum concentrator (Eppendorf) to evaporate to dryness at ≤70℃. Add 8.5 μL of GenoBaits 2×Hyb Buffer, 2.7 μL of GenoBaits Hyb Buffer Enhancer, and 2.8 μL of Nuclease-Free Water to the dry powder tube, mix well with a pipette, and incubate at 95℃ for 10 minutes on an ABI 9700 PCR instrument. Then, remove the PCR tube and add 3 μL of the synthesized probe (probe concentration of 60 ng / μL), vortex to mix, and incubate at 65℃ for 2 hours on an ABI 9700 PCR instrument to complete the probe hybridization reaction.

[0063] 2.2 DNA capture

[0064] Add 100 μL of GenoBaits DNA Probe Beads to the reaction mixture from the previous hybridization step, pipette up and down 10 times, and incubate at 65°C for 45 minutes on an ABI 9700 PCR instrument to allow the magnetic beads to bind to the probe. Wash the probe-bound magnetic beads with 100 μL of GenoBaits Wash Buffer I and 150 μL of GenoBaits Wash Buffer II at 65°C, then wash them again 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. Resuspend the washed magnetic beads in 20 μL of Nuclease-Free Water.

[0065] Add 13 μL of resuspended DNA (with magnetic beads) to a new 0.2 mL PCR tube, then add 15 μL GenoBaits PCR Master Mix and 2 μL GenoBaits Primer Mix to prepare a post-PCR system. Use an ABI 9700 PCR instrument for library amplification. The amplification program is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s; repeat denaturation, annealing, and extension for a total of 15 cycles; 72℃ extension for 5 min.

[0066] Add 45 μL of Beckmen AMPure XP Beads (Beackman) to the post-PCR product and mix thoroughly using a pipette. Then place a 0.2 mL PCR tube on a magnetic rack until the solution becomes clear. Discard the supernatant and wash the magnetic beads twice with 75 vol% ethanol. Elute the library DNA with Tris-HCl at pH 8.0. This completes the probe hybridization capture process.

[0067] 2.3 Quality Inspection of DNA Hybridization Capture Library

[0068] The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and then the fragment size of the library DNA was detected by agarose gel electrophoresis to determine whether it was between 300-400 bp.

[0069] 2.4 DNA hybridization capture library sequencing

[0070] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0071] 2.5 Genotype Data Analysis

[0072] After the sequencing data underwent quality control by FastQC (www.bioinformatics.babraham.ac.uk / project), the sequencing data was aligned to the reference genome using the default parameters of BWA (bio-bwa.sourceforge.net). SNP identification of the sequencing data was performed using GATK (software.broadinstitute.org / gatk) software. A self-written Perl script was used to extract the genotyping information from the probe-captured sequencing data, forming the final genotyping results, as shown in Tables 2 and 3.

[0073] Table 2. Detection rate of low-density liquid phase gene chip in 200 cotton varieties.

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Table 3. Detection of transgenic components in 200 cotton varieties using low-density liquid phase gene chip.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] The above results confirm that the low-density SNP liquid phase chip achieved a detection rate of 90.7%-96.5% in 200 upland cotton materials, with an average locus detection rate of 94%. It exhibits high polymorphism among upland cotton varieties, making it highly suitable for upland cotton germplasm resource evaluation, genetic improvement, and transgenic component detection, with high accuracy. Compared to existing solid-phase chips and developed high-density liquid phase chips, cotton breeding research can be conducted at a lower cost.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A low-density liquid phase chip for upland cotton based on targeted capture sequencing, characterized in that, Using the cotton genome TM-1_V2.1 as a reference genome, the upland cotton low-density liquid phase chip consists of probes for detecting 894 SNP sites and 14 major transgenes; the probes for the 14 major transgenes are shown in SEQ ID NO.1~SEQ ID NO.14; the 894 SNP sites are shown in Table 1.

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

3. The application according to claim 2, characterized in that, The main agronomic traits include fiber quality traits, yield traits, and disease resistance.

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