Cotton 10k functional site breeding chip and application thereof

Abstract

The application belongs to the technical field of molecular biology, and specifically discloses a cotton 10K functional site breeding chip and application thereof. The breeding chip is named "Cotton Chip No. 1", and comprises a 10K functional site target capture probe group designed based on the reference genome of the modern upland cotton Nongda 8 in terms of disease resistance, high quality and high yield. The probe group comprises 11159 SNP sites, and the SNP site information is shown in Table 1 in the specification. The application discloses a cotton 10K functional site breeding chip and application thereof. The breeding chip comprises more functional sites of index functional genetic variations, 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 correlation analysis, so that the breeding efficiency can be significantly improved, and the application is more extensive.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a cotton 10K functional site breeding chip and its application. Background Technology

[0002] In agricultural production, cotton, as an important economic crop, has always had its yield and quality as key focuses of breeding efforts. Traditional cotton breeding primarily relies on phenotypic selection, involving field screening and selection based on the appearance, yield, and quality of cotton plants. However, this method has several significant limitations. Firstly, traditional breeding is time-consuming. Developing a superior cotton variety typically takes 8-10 years or even longer. The breeding process requires multiple generations of self-pollination, hybridization, and selection. Each generation requires waiting for the cotton to grow, flower, and bear fruit before phenotypic identification can be performed. This not only consumes a significant amount of time and manpower but also results in low breeding efficiency, making it difficult to quickly meet market demands for new cotton varieties. Secondly, traditional breeding is easily affected by environmental factors. Cotton phenotype is the result of the interaction between genotype and environment. Differences in field environments, such as soil fertility, climate conditions, and the occurrence of pests and diseases, can all influence cotton phenotype, leading to inaccurate phenotypic identification results. For example, in a certain year, abnormal climate may affect the yield and quality of cotton, making it difficult for breeders to accurately determine whether the superior traits of a plant are determined by genotype or environmental factors, thus increasing the difficulty of selecting superior genotypes and reducing the accuracy of breeding.

[0003] Furthermore, traditional breeding methods are less effective at capturing some recessive beneficial alleles. Many important agronomic traits, such as disease resistance and stress tolerance, may be controlled by recessive genes, which are difficult to observe directly in phenotype. Traditional breeding methods struggle to accurately identify these recessive beneficial alleles in the early stages, leading to these superior genes being easily overlooked during the breeding process and affecting the quality of new varieties.

[0004] With the development of molecular biology techniques, molecular breeding has gradually become an important method in cotton breeding. Among these, gene chip-based molecular breeding technology can detect and analyze the genetic information of cotton at the whole-genome level, providing a more precise tool for cotton breeding. Currently, most cotton gene chips on the market are high-density chips containing a large number of SNP loci. However, these high-density chips are expensive, creating a huge economic burden when testing large-scale breeding populations, limiting their widespread application in practical breeding work. At the same time, existing high-density chips contain a large number of redundant loci. These redundant loci may not be directly related to the target agronomic traits, increasing detection costs, complicating data analysis, and reducing detection efficiency. Moreover, most existing chips are based on ancient reference genome designs, resulting in low matching with the genetic background of modern cotton breeding populations. With the continuous advancement of cotton breeding, the genetic structure of modern breeding populations has changed significantly. Ancient reference genomes cannot accurately reflect the genetic information of modern breeding populations, affecting the accuracy and reliability of chip detection 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] This invention aims to provide a cotton 10K functional locus breeding chip and its application. This breeding chip contains more functional loci that indicate genetic variations, resulting in higher detection accuracy among different cotton varieties. It can be 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 a wider range of practical applications.

[0006] A cotton 10K functional site breeding chip, named "Cotton Core No. 1", includes a set of 10K functional site targeted capture probes designed based on the reference genome of Nongda Cotton No. 8; 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 reference genome link of Nongda Cotton 8 is as follows: 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, tolerance to low phosphorus, tolerance to low nitrogen, and salt tolerance.

[0009] This 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 traits identified include flowering time, boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, tolerance to low phosphorus, tolerance to low nitrogen, 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 the thermodynamic stability algorithm, liquid-phase capture probes were designed to target the GC content and sequence specificity of the target sites, and a target probe set containing 11,159 sites was constructed.

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

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

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

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

[0018] S13. Through genome-wide association analysis, functional loci significantly associated with 13 agronomic traits were screened, and genotype-phenotype relationships were quantified by combining phenotypic data.

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

[0020] T1. DNA Sample Extraction and Quality Control: DNA is extracted using the magnetic bead method, and the concentration and integrity of the DNA sample are measured.

[0021] T2. Targeted capture library construction: DNA fragments are broken down to 150-200 bp, INDEX adapters are ligated, and amplification is performed;

[0022] T3. Target sites are captured by liquid-phase hybridization, non-target fragments are eluted and amplified by PCR to construct a targeted sequencing library;

[0023] T4. Library quality control 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] This invention discloses a cotton 10K functional locus breeding chip and its application. Using the superior modern upland cotton variety Nongda Cotton 8 as a reference genome, this chip offers numerous significant advantages. It integrates 11,159 loci from Chinese cotton breeding populations that are environmentally stable and have significant genetic effects, including 3,981 superior trait loci 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, tolerance to low phosphorus, tolerance to low nitrogen, and salt tolerance. Testing has verified that the chip's sample detection rate and repeatability are both greater than 99%, demonstrating excellent performance. Based on these characteristics, this chip is suitable for various applications such as parent identification, trait selection, and variety evaluation. In 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 trait; and in variety evaluation, it provides a basis for comprehensively assessing the genetic potential of varieties. 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, cultivating higher-quality, higher-yielding, and more resistant cotton varieties, enhancing the adaptability and stability of varieties in different environments, while reducing breeding costs. It provides a powerful tool for whole-genome selection breeding and identification of superior traits in cotton, laying a solid foundation for the sustainable development of the cotton industry.

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

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

[0030] Figure 4 The genotypic concordance rate of the breeding chip sample provided in Example 1;

[0031] Figure 5 Test of key trait sites of the breeding chip provided in Example 1. Detailed Implementation

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

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

[0034] Source of experimental materials:

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

[0036] Example 1

[0037] A cotton 10K functional site 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 germplasm accessions 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 sites are retained.

[0041] S13. Through genome-wide association analysis, functional loci significantly associated with 13 agronomic traits were screened, and genotype-phenotype relationships were quantified by combining phenotypic data.

[0042] S2. Probe Design: Based on the GC content and sequence characteristics of the genome near the target region, probes are designed according to the principle of thermodynamic stability. Factors such as the complexity of the target species' genome, the location of the target site, and the GC content near the target site are comprehensively considered to ensure 100% capture in conventional regions. For highly complex regions, a multi-layered "shingled" probe placement scheme is used to improve the effective coverage of these regions. Specific parameters are as follows: 1) GC content is 30%–80%; the number of sites near a single target site is <10; genome specificity is >50%, and the microarray is scored, with the set of sites having the lowest average score being selected; 2) GWAS-related sites are not considered, and all scores are retained. Liquid-phase capture probes are designed to construct a targeting probe set containing 11,159 SNP sites. SNP site information is shown in Table 1, and the functional site distribution density diagram is shown in... Figure 1This breeding chip 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 functional loci significantly correlated with phenotypes is shown in Table 2.

[0043] S3. The 11,159 SNP loci selected were then used by Huazhi's independently developed liquid-phase probe precise localization sequencing and genotyping technology (Genotyping by Pinpoint Sequencing of liquid). c aptured target, c GPS) was developed into a 10K liquid phase breeding chip for cotton. c GPS is based on an optimized thermodynamic stability algorithm model. It designs specific probes for the target region sequence, and then uses the synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions through liquid phase hybridization. Then, it constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel sites in the target region.

[0044] The 11,159 SNP loci screened in this embodiment 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 superior 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 cotton 10K functional locus breeding chips includes the following steps:

[0046] T1. DNA Sample Extraction and Quality Control: DNA was extracted from the samples using the magnetic bead method, and the DNA samples underwent quality control. Quality control included determining DNA concentration using a Qubit quantitative PCR instrument and checking DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0047] T2, Targeted capture library construction:

[0048] T21. Take 200 ng of qualified genomic DNA, digest the DNA sample with a fragmentation enzyme, repair the enzyme ends and add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0049] T22. Using T4 ligase, the adapter fragments were ligated to both ends of the DNA. The ligation products were purified using fragment sorting magnetic beads. The concentration of the purified products was detected by a Qubit fluorescence quantitative instrument, and the fragment size was detected by agarose gel electrophoresis.

[0050] T23. Perform PCR amplification on the purified ligation products, and use magnetic beads to screen the amplified products for fragments. The concentration of the screened products is detected using a Qubit real-time fluorescence instrument, and the fragment size is detected by agarose gel electrophoresis.

[0051] T24. Place the qualified library, blocking reagent, and 10K liquid phase breeding chip probe on a PCR instrument for hybridization reaction. Incubate at 55℃ for a minimum of 16 hours and a maximum of 24 hours.

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

[0053] T4. Library Quality Control and Sequencing: PE150 sequencing was performed using the DNBSEQ-T7 platform. The raw data after high-throughput sequencing underwent quality control filtering and other processing. FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality clean reads. BWA software was used to align the clean reads with a reference genome and perform positional sorting to obtain the sorted BAM file. GATK software was used to analyze the sequencing results for variant sites, obtaining genotyping results for the target loci. The genotyping process flow is as follows: Figure 2 .

[0054] Table 1 SNP locus information

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[0090] Table 1 shows the chromosome numbering information, base position information, reference genotype information, and variant genotype information for the 11,159 loci described in the cotton 10K chip.

[0091] 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 the Nongda Cotton 8.

[0092] Table 2. Statistics on the number of functional loci and phenotypic significantly correlated loci.

[0093]

[0094] Table 2 shows the number of some functional sites in the cotton 10K chip, including 148 sites related to disease resistance, 62 sites related to cotton boll weight, 795 sites related to fiber length, 16 sites related to fiber fineness, 58 sites related to fiber strength, 29 sites related to insect resistance, 9 sites related to cotton lint percentage, 5 sites related to low nitrogen tolerance, and 14 sites related to cotton seed finger.

[0095] The effectiveness of the breeding chip provided in Example 1 above was verified:

[0096] 1. Evaluate the genotyping effect of the breeding chip provided in Example 1 above:

[0097] Eighty-two samples were selected for production testing and verification of the cotton 10K chip system. Thirteen samples were randomly selected from these eighty-two for a technical replication, named the original sample number -re, resulting in a total of 95 sample data. The results are shown below. Figure 3 And Table 3.

[0098] Table 3 Detection rate of breeding chip samples

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[0102] From Table 3 and Figure 3 The locus detection rates of the 95 samples ranged from 98.22% to 99.83%, with an average detection rate of 99.26%; the heterozygosity ranged from 1.36% to 20.72%, with an average heterozygosity of 5.42%. The locus detection rates of the reference genome samples ranged from 99.74% to 99.83%, with an average detection rate of 99.79%.

[0103] The test involved 13 technical repetitions, and the results are as follows. Figure 4 And Table 4.

[0104] Table 4 Genotype Detection Concordance Rate of Breeding Chips

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[0106] As shown in Table 4, the genotypic consistency rate of the duplicate samples with sample names 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 locus is missing, that locus is not included in the consistency statistics), with an average consistency rate of 99.08%. The average genotypic consistency rate of the duplicate samples of reference genome samples 1 and 2 was 99.78%. The results show that this breeding chip has extremely high detection accuracy for different cotton varieties, with a detection rate of over 99%, and good stability. The genotyping results are accurate and reliable, and it can be fully used for genotyping detection of different cotton samples.

[0107] 2. Evaluate the application effect of the breeding chip provided in Example 1 in the identification of breeding traits:

[0108] This breeding chip was used to identify 13 key agronomic traits of cotton, including flowering date, boll weight, lint percentage, daughter index, fiber length, fiber strength, micronaire value, disease resistance, insect resistance, herbicide resistance, tolerance to low phosphorus, tolerance to low nitrogen, and salt tolerance. The daughter index identification results for three example samples are shown below. Figure 5 .

[0109] Depend on Figure 5 It can be seen that the chip successfully detected 14 associated loci in the analysis of sub-finger related traits. Furthermore, different germplasm resources showed significant genetic differences: superior germplasm No. 6 carried 14 (100%) dominant sub-finger alleles, BZ2H contained 13 (7.1%), while BZ14 retained only 1 (1.3%).

[0110] Genome-wide selection model prediction and field phenotypic validation showed a high genetic correlation between fiber length and molecular marker detection results. The chip also demonstrated excellent performance in identifying other important trait loci such as insect resistance and disease resistance. Based on the excellent molecular marker-driven identification function of this breeding chip, the following can be achieved in the early stages of parental selection: (1) prediction of the genetic potential of target traits; (2) assessment of the abundance of dominant alleles; and (3) molecular design for synergistic improvement of multiple traits. This molecular marker-driven pre-screening strategy effectively shortens the traditional phenotypic screening cycle and provides a reliable basis for genotypic selection to construct aggregates of superior traits.

[0111] 3. Evaluate the application effect of the breeding chip provided in Example 1 in genome-wide selection:

[0112] The Bayesian model (BRR / BL / BayesA / BayesB / BayesC) based on this breeding chip demonstrated the robustness of genome-wide selection technology in 10-fold cross-validation, especially in modeling important cotton traits. The model cross-validation metrics are shown in Table 5.

[0113] Table 5. Accuracy results of the genome-wide selection model constructed based on breeding chips and 10-fold cross-validation.

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[0116] Table 5 shows that the chip's prediction relevance for the LP trait (BayesC _CV_COR=0.717) is significantly better than other traits (such as LU CV_COR=0.630-0.635), and its error control is stable (CV_RMSE=2.985), verifying the chip's ability to resolve complex traits. Regarding the high-yield trait, the prediction accuracy of BW (BL_CV_COR=0.496, CV_MAE≈0.290) and the prediction results for the quality trait FL (BayesB_CV_COR=0.705, CV_RMSE=1.094) both reach high levels, demonstrating the advantages of multi-trait collaborative prediction. Cross-model analysis shows that "Cotton Core No. 1" maintains algorithm compatibility across 13 traits (covering high yield, quality, and stress resistance), and the prediction results between different models are highly consistent (CV_COR difference <0.03), highlighting the adaptability of its labeling system to multi-model frameworks. This chip integrates high-density SNP markers across the entire genome (covering key sites in known QTL regions), providing a molecular basis for difficult-to-fix traits such as disease resistance and insect resistance. Its standardized design effectively supports the joint analysis of multi-environment data. Based on 10,000 iterations of 10-fold cross-validation, the error fluctuation is less than 15%, indicating that the algorithm's stability meets the requirements for breeding applications. As the cotton-specific breeding chip with the most comprehensive trait coverage, "Cotton Core No. 1" significantly improves the practicality of genome-wide selection in low-impact multi-gene controlled traits by optimizing marker density and genetic effect modeling, providing an efficient tool for large-scale complex trait aggregation breeding.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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, named "Cotton Core No. 1," includes a set of 10K functional site-targeting capture probes designed based on the Nongda Cotton 8 reference genome. The probe set contains 11,159 SNP sites. The SNP site information is shown in the table below. The version number of the reference genome sequence of Nongda Mian 8 is: Gossypium hirsutum (AD1) 'NDM8'genome HEAU_v1.

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

3. The application of the cotton 10K functional site breeding chip according to claim 1 in genotyping, agronomic trait identification, genome-wide association analysis and genome-wide selection of upland cotton populations.

4. The genotyping method for cotton 10K functional locus breeding chips as described in claim 1, characterized in that, Includes the following steps: T1. DNA Sample Extraction and Quality Control: DNA is extracted using the magnetic bead method, and the concentration and integrity of the DNA sample are measured. T2. Targeted capture library construction: DNA fragments are broken down to 150-200 bp, INDEX adapters are ligated, and amplification is performed; T3. Target sites are captured by liquid-phase hybridization, non-target fragments are eluted and amplified by PCR to construct a targeted sequencing library; T4. Library quality control 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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