Cotton 10K functional site breeding chip and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a cotton 10K functional site breeding chip and application thereof. The breeding chip is named as' Cotton Core No.1 ', and comprises a 10K functional site targeting capture probe group designed based on a reference genome of disease-resistant, high-quality and high-yield modern upland cotton No.8, the probe group comprises 11159 SNP (Single Nucleotide Polymorphism) loci, 3981 of the SNP loci are functional loci associated with 13 important agronomic characters, and 7178 of the SNP loci are background loci reflecting genetic diversity; the SNP site information is shown in the specification table 1. The invention discloses a cotton 10K functional site breeding chip and application thereof, the breeding chip contains functional sites with more index function genetic variation, has higher detection accuracy among different cotton varieties, is applied to cotton molecular marker-assisted selection breeding, whole genome selection breeding and whole genome correlation analysis, and has higher detection accuracy. The breeding efficiency can be obviously improved, and the practical application is wider.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a cotton 10K functional site breeding chip and an application thereof. Background Art

[0002] Cotton, as an important cash crop in agricultural production, has always been a key focus of breeding efforts for its yield and quality. Traditional cotton breeding primarily relies on phenotypic selection, which involves screening and selecting cotton plants in the field based on their appearance, yield, quality, and other traits. However, this approach has significant limitations. Firstly, the traditional breeding cycle is lengthy. Cultivating a single superior cotton variety typically takes 8-10 years or even longer. The breeding process involves multiple generations of selfing, hybridization, and selection. Phenotypic identification must be performed after each generation has grown, flowered, and fruited. This not only consumes significant time and labor, but also results in low breeding efficiency, making it difficult to quickly meet market demand for updated cotton varieties. Secondly, traditional breeding is susceptible to environmental interference. Cotton phenotype is the result of the interaction between genotype and environment. Differences in field conditions, such as soil fertility, climatic conditions, and pest and disease occurrence, can influence cotton phenotype, leading to inaccurate phenotypic identification results. For example, in a certain year, due to abnormal climate, the yield and quality of cotton may be affected, making it difficult for breeders to accurately determine whether the excellent traits of a certain plant are determined by the genotype or caused by environmental factors, thereby increasing the difficulty of selecting excellent genotypes and reducing the accuracy of breeding.

[0003] Furthermore, traditional breeding is less effective at capturing some recessive favorable alleles. Many important agronomic traits, such as disease resistance and stress tolerance, may be controlled by recessive genes, making them difficult to directly observe phenotypically. Traditional breeding methods struggle to accurately identify these recessive favorable alleles early in the breeding process, leading to these desirable genes being easily missed during the breeding process, impacting the quality of new varieties.

[0004] With the advancement of molecular biology techniques, molecular breeding has gradually become an important tool in cotton breeding. Among them, molecular breeding techniques based on gene chips can detect and analyze cotton genetic information at the genome-wide level, providing a more precise tool for cotton breeding. Currently available cotton gene chips are mostly high-density chips that contain a large number of SNP loci. However, these high-density chips are expensive, creating a significant economic burden when testing large-scale breeding populations, limiting their widespread application in practical breeding. Furthermore, existing high-density chips contain a large number of redundant loci. These redundant loci may not be directly associated with the target agronomic traits, increasing testing costs and complicating data analysis, reducing testing efficiency. Furthermore, most existing chips are designed based on ancient reference genomes, resulting in a poor match with the genetic background of modern cotton breeding populations. With the continuous advancement of cotton breeding, the genetic structure of modern breeding populations has undergone significant changes. Ancient reference genomes cannot accurately reflect the genetic information of modern breeding populations, compromising the accuracy and reliability of chip testing results. Genotype-environment interactions are significant, making it difficult to meet the precision and efficiency requirements of modern cotton breeding. Summary of the Invention

[0005] The present invention aims to provide a cotton 10K functional site breeding chip and its application. The breeding chip contains more functional sites that indicate functional genetic variation, has higher detection accuracy among different cotton varieties, and is applied to cotton molecular marker-assisted selection breeding, whole genome selection breeding and whole genome association analysis, which can significantly improve breeding efficiency and has more practical applications.

[0006] A cotton 10K functional site breeding chip, named "Cotton Core No. 1", includes a 10K functional site targeted capture probe set designed based on the Agricultural University Cotton No. 8 reference genome; the probe set contains 11,159 SNP sites, of which 3,981 are functional sites associated with 13 agronomic traits and 7,178 are background sites reflecting genetic diversity; the SNP site information is shown in Table 1 of the specification.

[0007] Preferably, the Nongda cotton 8 reference genome link is https: / / www.cottongen.org / data / download / genome_tetraploid / AD1.

[0008] Preferably, the 13 agronomic traits include flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, low phosphorus tolerance, low nitrogen tolerance, and salt tolerance.

[0009] The present invention also provides the application of the cotton 10K functional site breeding chip in upland cotton population genotyping, agronomic trait identification, genome-wide association analysis, and genome-wide selection.

[0010] Preferably, the agronomic trait identification includes flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, low phosphorus tolerance, low nitrogen tolerance, and salt tolerance.

[0011] Preferably, the breeding chip is obtained by the following method:

[0012] S1, functional site screening;

[0013] S2. Probe Design: Based on a thermodynamic stability algorithm, liquid-phase capture probes were designed based on the GC content and sequence specificity of the target sites, and a targeted probe set containing 11,159 sites was constructed.

[0014] S3. The breeding chip is developed using liquid phase probe precise positioning sequencing typing technology.

[0015] Preferably, in S1, the functional site screening comprises the following steps:

[0016] S11. Perform high-depth resequencing and variant detection on the cotton core collection to obtain SNP variant data. The filtering parameters are: sequencing depth ≥3X for each variant site, missing data rate ≤20%, and only retaining the minimum allele frequency ≥5%;

[0017] S12. Based on population linkage disequilibrium, heterozygosity polymorphism, non-SNP hotspot regions and non-high copy regions, 40,000 candidate loci were retained;

[0018] S13. Through genome-wide association analysis, functional loci significantly associated with 13 agronomic traits were screened, and the genotype-phenotype relationship was quantified in combination with phenotypic data.

[0019] The present invention also provides a genotyping method for the cotton 10K functional site breeding chip, comprising the following steps:

[0020] T1. DNA sample extraction and quality control: DNA was extracted using the magnetic bead method and the concentration and integrity of the DNA sample were determined;

[0021] T2, Targeted Capture Library Construction: Fragment the DNA into 150-200 bp, ligate with INDEX adapters, and amplify;

[0022] T3, capture the target site by liquid phase hybridization, elute the non-target fragments and perform PCR amplification to construct a targeted sequencing library;

[0023] T4. Library quality inspection and sequencing: PE150 sequencing was performed using the DNBSEQ-T7 platform, and the data were compared and analyzed to obtain genotyping results.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] The present invention discloses a cotton 10K functional locus breeding chip and its application. This breeding chip uses the reference genome of Nongda Cotton No. 8, a superior modern upland cotton variety in my country, and offers numerous significant advantages. It integrates 11,159 loci from Chinese cotton breeding populations that are stable across multiple environments and have large genetic effects, including 3,981 loci for superior traits and 7,178 genomic background loci. These loci comprehensively cover 13 important traits: flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, low-phosphorus tolerance, low-nitrogen tolerance, and salt tolerance. Testing has verified that the chip sample detection rate and repeatability are both greater than 99%, demonstrating excellent performance. Based on these characteristics, the chip is suitable for a variety of applications, including breeding parent identification, trait selection, and variety evaluation. In breeding parent identification, it can accurately determine the target trait genes carried by the parents; in trait selection, it can quickly and accurately screen individuals with the target traits; and in variety evaluation, it provides a basis for comprehensively assessing the genetic potential of the variety. This not only significantly improves the accuracy and efficiency of cotton breeding, reduces resource waste in the breeding process, and shortens the breeding cycle, but also helps in multi-trait aggregation breeding, cultivates better quality, higher yield, and more resistant cotton varieties, enhances the adaptability and stability of varieties in different environments, and reduces breeding costs at the same time. It provides a powerful tool for cotton whole genome selection breeding and excellent trait identification, laying a solid foundation for the sustainable development of the cotton industry.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the distribution density of functional sites of the breeding chip provided in Example 1;

[0028] Figure 2 The breeding chip sample detection process provided in Example 1;

[0029] Figure 3 The detection rate of the breeding chip sample test provided in Example 1;

[0030] Figure 4 The genotype consistency rate detected by the breeding chip samples provided in Example 1;

[0031] Figure 5 This is the breeding chip key trait site test provided in Example 1. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0033] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0034] Source of test materials:

[0035] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0036] Example 1

[0037] A cotton 10K functional locus breeding chip, named "Cotton Core No. 1", was obtained by the following method:

[0038] S1. Functional site screening:

[0039] S11. High-depth resequencing and variant detection were performed on 1081 cotton core collections to obtain SNP variant data. The filtering parameters were: sequencing depth ≥3X for each variant site, missing data rate ≤20%, and only the minimum allele frequency ≥5% was retained.

[0040] S12. Based on population linkage disequilibrium, heterozygosity polymorphism, non-SNP hotspot regions and non-high copy regions, 40,000 candidate loci were retained;

[0041] S13. Through genome-wide association analysis, functional loci significantly associated with 13 agronomic traits were screened, and the genotype-phenotype relationship was quantified in combination with phenotypic data.

[0042] S2. Probe design: Based on the GC content and sequence characteristics of the genome near the target region, probe design is carried out based on the principle of thermodynamic stability. The complexity of the target species genome, the location of the target site, the GC content near the target site and other factors are comprehensively considered to ensure 100% capture of conventional areas. For complex areas with high difficulty, the effective coverage of high-difficulty areas is improved by adjusting the probe position and placing multi-layer "shingled" probes. The specific parameters are designed as follows: 1) The GC content is 30% to 80%; the number of sites near a single target site is <10; the specificity on the genome is >50%, the chip is scored, and the site set with the lowest average score is taken; 2) All GWAS-related sites are retained regardless of the score value, and liquid capture probes are designed to construct a targeted probe group containing 11,159 SNP sites. The SNP site information is shown in Table 1, and the functional site distribution density diagram is shown in Table 1. Figure 1The breeding array contains 3,981 GWAS loci associated with 13 important agronomic traits and 7,178 loci reflecting the genetic background of the core germplasm. The number of loci significantly associated with functional loci and phenotypes is shown in Table 2.

[0043] S3. The 11,159 SNPs identified were developed into a cotton 10K liquid-phase breeding array using Huazhi's proprietary liquid-phase probe precision positioning sequencing (cGPS) technology. cGPS uses an optimized thermodynamic stability algorithm model to design specific probes for target interval sequences. Synthesized specific probes are then used to capture and enrich multiple target sequences at different genomic locations through liquid-phase hybridization. Sequencing libraries are then constructed and high-throughput sequenced on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel sites within the target region.

[0044] The 11,159 SNP sites screened in this example are not only rich in genetic information, but also have a clear relationship between traits and genotypes, providing a powerful tool for whole-genome selection breeding and identification of excellent traits in cotton. This innovative breeding chip will significantly improve the accuracy and efficiency of cotton breeding, laying a solid foundation for cultivating higher-quality, higher-yielding, and more resistant cotton varieties.

[0045] A genotyping method for a cotton 10K functional locus breeding chip comprises the following steps:

[0046] T1. DNA Sample Extraction and Quality Control: DNA samples were extracted using a magnetic bead method and quality tested. Quality testing included determining DNA concentration using a Qubit fluorometer and checking DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality testing were used for library preparation.

[0047] T2. Targeted capture library construction:

[0048] T21. Take 200 ng of genomic DNA that has passed the quantitative quality inspection, digest the DNA sample with fragmentase, repair the digested ends, add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis;

[0049] T22, using T4 ligase, ligate the adapter fragments to both ends of the DNA, purify the ligation products using fragment sorting magnetic beads, detect the concentration of the purified products using a Qubit fluorescence quantification instrument, and detect the fragment size by agarose gel electrophoresis;

[0050] T23. Perform PCR amplification on the purified ligation product and screen the amplified product using magnetic beads. The concentration of the screened product is determined using a Qubit fluorescence quantification instrument, and the fragment size is determined by agarose gel electrophoresis.

[0051] T24. Place the library that has passed the quality inspection, the blocking reagent, and the 10K liquid-phase breeding chip probe on a PCR instrument for hybridization reaction. Incubate the hybridization at 55°C for a minimum of 16 hours and a maximum of 24 hours.

[0052] T3. Use streptavidin to capture the hybridization products, elute the non-target fragments, and amplify them by PCR to construct a targeted sequencing library;

[0053] T4. Library quality inspection and sequencing: PE150 sequencing is performed on the DNBSEQ-T7 platform. The raw data after high-throughput sequencing is processed by quality control filtering and other processes. The adapter fragments and low-quality reads are removed using FASTP software to obtain high-quality Clean Reads. The obtained Clean reads are aligned with the reference genome using BWA software, and the positions are sorted to obtain the bam file after the sample is sorted. The sequencing results are analyzed for variant sites using GATK software to obtain the genotyping results of the target sites. The process flow of the genotyping method is as follows: Figure 2 .

[0054] Table 1 SNP site information

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[0094] As shown in Table 1, the chromosome number information, base position information, reference genotype information and variant genotype information of the 11,159 loci described in the cotton 10K chip are shown.

[0095] Depend on Figure 1 It can be seen that the 11,159 loci described in the cotton 10K chip are evenly distributed on the genome of Nongda cotton No. 8.

[0096] Table 2 Statistics of the number of functional sites significantly associated with phenotypes

[0097] Trait count DI (disease resistance) 148 BW (bell weight) 62 FL (fiber length) 795 FM (fiber fineness) 16 FS (fiber strength) 58 DI (insect resistance) 29 LP (clothing points) 9 LNR (Low Nitrogen Tolerance) 5 SI (sub-index) 14

[0098] Table 2 shows the number of some functional loci in the cotton 10K array, including 148 loci related to disease resistance, 62 loci related to cotton boll weight, 795 loci related to fiber length, 16 loci related to fiber fineness, 58 loci related to fiber strength, 29 loci related to insect resistance, 9 loci related to cotton lint percentage, 5 loci related to low nitrogen tolerance, and 14 loci related to cotton seed index.

[0099] The effect of the breeding chip provided in Example 1 was verified:

[0100] 1. Evaluate the genotyping effect of the breeding chip provided in Example 1 above:

[0101] 82 samples were selected for production test and verification of the cotton 10K chip system and 13 samples were randomly selected from the 82 samples for a technical repeat. The technical repeat samples were named original sample number -re, generating a total of 95 sample data. Figure 3 and Table 3.

[0102] Table 3 Detection rate of breeding chip samples

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[0107] From Table 3 and Figure 3The results show that the site detection rate of the 95 samples ranged from 98.22% to 99.83%, with an average detection rate of 99.26%. The heterozygosity rate ranged from 1.36% to 20.72%, with an average heterozygosity rate of 5.42%. The site detection rate of the reference genome samples ranged from 99.74% to 99.83%, with an average detection rate of 99.79%.

[0108] During the test, 13 technical repetition experiments were set up, and the results are as follows Figure 4 and Table 4.

[0109] Table 4. Consistency rate of genotype detection using breeding chip

[0110] Sample name Number of consistent sites Number of inconsistent sites Genotype consistency rate 24 10954 123 98.89％ 1 11095 22 99.80％ 40 10920 145 98.69％ 48 10966 117 98.94％ 56 10940 111 99.00％ 58 10933 115 98.96％ 64 10962 106 99.04％ 72 10913 127 98.85％ 2 11102 27 99.76％ 13 10936 111 99.00％ 80 10965 81 99.27％ 86 10935 118 98.93％ 87 10943 116 98.95％

[0111] As shown in Table 4, the genotype concordance rates of repeated samples with sample names such as 1, 2, 13, 24, 40, 48, 56, 58, 64, 72, 80, 86, and 87 ranged from 98.69% to 99.80% (Note: in the consistency comparison, if one of the two results for a certain site was missing, the site was not included in the consistency statistics), with an average concordance rate of 99.08%. The average genotype concordance rate of repeated samples of reference genome samples 1 and 2 was 99.78%. The results showed that the breeding chip had extremely high detection accuracy for different cotton varieties, with detection rates higher than 99%, good stability, and accurate and reliable typing results, making it fully applicable for genotyping detection of different cotton samples.

[0112] 2. Evaluate the application effect of the breeding chip provided in Example 1 above in the identification of breeding traits:

[0113] The breeding chip was used to identify 13 key agronomic traits of cotton, including flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, low phosphorus tolerance, low nitrogen tolerance, and salt tolerance. The seed index trait identification results of three sample samples are as follows: Figure 5 .

[0114] Depend on Figure 5 The microarray successfully detected 14 association loci for the zygote-related traits, and the different germplasms showed significant genetic differences: the superior germplasm No. 6 carried 14 (100%) zygote-dominant alleles, BZ2H contained 13 (7.1%), and BZ14 retained only one (1.3%).

[0115] The prediction of the whole genome selection model and field phenotypic verification showed that the fiber length trait showed a high genetic correlation with the molecular marker detection results. At the same time, the chip also performed well in the identification of other important trait loci such as insect resistance and disease resistance. Based on the excellent molecular marker precision identification function of this breeding chip, the following can be achieved in the early stages of breeding parent selection: (1) prediction of the genetic potential of target traits; (2) assessment of the abundance of dominant alleles; and (3) molecular design for the coordinated improvement of multiple traits. This molecular marker-driven pre-screening strategy effectively shortens the traditional phenotypic screening cycle and provides a reliable genotype selection basis for the construction of excellent trait aggregates.

[0116] 3. Evaluate the application effect of the breeding chip provided in Example 1 above in whole genome selection:

[0117] The Bayesian model (BRR / BL / BayesA / BayesB / BayesC) based on this breeding chip demonstrated the robustness of whole-genome selection technology in 10-fold cross-validation, especially in modeling important cotton traits. The model cross-validation indicators are shown in Table 6.

[0118] Table 6 Whole genome selection model constructed based on breeding chip and 10-fold cross validation accuracy results

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[0121] As shown in Table 6, the chip's prediction correlation for the LP trait (BayesC_CV_COR = 0.717) was significantly superior to that for other traits (e.g., LU CV_COR = 0.630-0.635), and its error control was stable (CV_RMSE = 2.985), validating the chip's ability to resolve complex traits. For high-yield traits, the prediction accuracy of BW (BL_CV_COR = 0.496, CV_MAE ≈ 0.290) and the prediction results for the high-quality trait FL (BayesB_CV_COR = 0.705, CV_RMSE = 1.094) both reached high levels, demonstrating the advantages of multi-trait collaborative prediction. Cross-model analysis showed that "Cotton Core No. 1" maintained algorithm compatibility across 13 traits (covering high yield, quality, and stress resistance), and the prediction results were highly consistent across different models (CV_COR difference < 0.03), highlighting the adaptability of its marker system to the multi-model framework. By integrating high-density SNP markers across the genome (covering key sites in known QTL regions), the chip provides a molecular basis for difficult-to-fix traits such as disease resistance and insect resistance. Its standardized design can effectively support the joint analysis of multi-environmental data. The error fluctuation of 10-fold cross-validation based on 10,000 iterations is less than 15%, indicating that the algorithm stability meets the requirements for breeding applications. As the most comprehensive cotton breeding chip covering traits, "Cotton Core No. 1" significantly improves the practicality of whole-genome selection in micro-effect multi-gene controlled traits by optimizing marker density and genetic effect modeling, providing an efficient tool for large-scale aggregation breeding of complex traits.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A cotton 10K functional site breeding chip, characterized in that: The breeding chip is named "Cotton Core No. 1" and includes a 10K functional site targeted capture probe group designed based on the reference genome of Agricultural University Cotton No. 8; the probe group contains 11,159 SNP sites, of which 3,981 are functional sites associated with 13 agronomic traits and 7,178 are background sites reflecting genetic diversity; the SNP site information is shown in Table 1 of the specification.

2. The cotton 10K functional site breeding chip according to claim 1, characterized in that: The Nongda cotton 8 reference genome link is https: / / www.cottongen.org / data / download / genome_tetraploid / AD1.

3. The cotton 10K functional site breeding chip according to claim 1, characterized in that: The 13 agronomic traits include flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, low phosphorus tolerance, low nitrogen tolerance, and salt tolerance.

4. Use of the cotton 10K functional site breeding chip according to any one of claims 1 to 3 in genotyping of upland cotton populations, identification of agronomic traits, genome-wide association analysis, and genome-wide selection.

5. The cotton 10K functional site breeding chip according to claim 1, characterized in that: The breeding chip is obtained by the following method: S1, functional site screening; S2. Probe Design: Based on a thermodynamic stability algorithm, liquid-phase capture probes were designed based on the GC content and sequence specificity of the target sites, and a targeted probe set containing 11,159 sites was constructed. S3. The breeding chip is developed using liquid phase probe precise positioning sequencing typing technology.

6. The cotton 10K functional site breeding chip according to claim 5, characterized in that: In S1, the functional site screening includes the following steps: S11. Perform high-depth resequencing and variant detection on the cotton core collection to obtain SNP variant data. The filtering parameters are: sequencing depth ≥3X for each variant site, missing data rate ≤20%, and only retaining the minimum allele frequency ≥5%; S12. Based on population linkage disequilibrium, heterozygosity polymorphism, non-SNP hotspot regions and non-high copy regions, 40,000 candidate loci were retained; S13. Through genome-wide association analysis, functional loci significantly associated with 13 agronomic traits were screened, and the genotype-phenotype relationship was quantified in combination with phenotypic data.

7. The genotyping method for cotton 10K functional site breeding chip according to claim 1, characterized in that: The steps include: T1. DNA sample extraction and quality control: DNA was extracted using the magnetic bead method and the concentration and integrity of the DNA sample were determined; T2, Targeted Capture Library Construction: Fragment the DNA into 150-200 bp, ligate with INDEX adapters, and amplify; T3, capture the target site by liquid phase hybridization, elute the non-target fragments and perform PCR amplification to construct a targeted sequencing library; T4. Library quality inspection and sequencing: PE150 sequencing was performed using the DNBSEQ-T7 platform, and the data were compared and analyzed to obtain genotyping results.

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