A liquid-phase breeding chip for whole genome selection of maize and its application

Abstract

The present invention relates to the fields of bioinformatics and genome breeding, and relates to a liquid-phase breeding chip for whole genome selection of corn and an application thereof. The liquid-phase breeding chip for whole genome selection of corn is used for corn variety breeding. The SNP sites include SNP0001-SNP5851. The chip contains multiple trait-associated sites and important structural variations identified by GWAS, which are distributed in the whole genome. In the breeding stage, the functional sites in the chip can be used to perform preliminary identification on corn inbred lines, thereby improving the efficiency of genome selection.

Description

Technical Field

[0001] The invention relates to the fields of bioinformatics and genome breeding, and in particular to a liquid-phase breeding chip for whole genome selection of corn and its application. Background Art

[0002] Corn is an ideal crop for the application of whole genome selection (GS) breeding technology. The cost of genotyping is still a major obstacle to the promotion and application of whole genome selection technology. Most breeding companies are limited by the long-term cost of genotyping and cannot apply it on a large scale. At the same time, GS has also shown great potential in the field of hybrid breeding. Since the genotypes of hybrids can be inferred based on their parental genotypes instead of re-sequencing, the sequencing cost is greatly reduced. Hybrid breeding is the main strategy to improve the productivity of many crops, especially for rice and corn. Hybrid vigor is an effective strategy to increase their yield, and parent selection is the core of hybrid vigor breeding. At present, the selection of parents mostly relies on the experience of breeders, which has great uncertainty and consumes a lot of time and labor costs. Using GS technology, only the genotypes of the parents and the phenotypes of some of the hybrids can be obtained to predict the phenotypes of all potential hybrids of a given parent. GS undoubtedly provides a better choice for the selection of excellent hybrids. This will increase the chances of selecting truly superior hybrids at the lowest cost, which will not only shorten the corresponding breeding cycle and save time, but also reduce costs and improve the predicted genetic gain to a certain extent.

[0003] With the rapid development of biotechnology, a variety of chip-based genotyping platforms have been constructed in corn based on Illumina and Affymetrix systems. The initial representative chips, such as MaizeSNP50 BeadChip, MaizeSNP600K and Maize6H-60KSNP. Although international research institutions and multinational breeding companies have implemented GS technology in breeding populations, the high cost of genotyping is the main limiting factor for the large-scale breeding application of GS in small and medium-sized enterprises. At present, the developed corn solid phase chips and corn liquid phase chips are mainly high-density and medium-high-density. The cost of crop breeding-specific chips is still high, making it difficult to use them on a large scale in breeding programs. Although the cost of breeding chips has been greatly reduced, the cost of genotyping is still high when applied to large breeding populations, which limits its large-scale application in crop breeding. Therefore, a high-quality and reasonably priced genotyping platform is a prerequisite for GS application. In addition, the current chips cannot effectively integrate structural variations, which may cause the loss of heritability. The accuracy of GS is limited by the variation heritability of the target traits. Currently, most GS studies are based on GS predictions based on genome-wide SNP markers, ignoring many structural variations that are associated with traits but not linked to adjacent SNPs. Summary of the invention

[0004] The present invention provides a SNP molecular marker combination for maize genotyping, a maize whole genome chip (maize 6K chip) and its application. The chip of the present invention is basically evenly distributed on the chromosome, covering functional sites related to yield, plant type, disease and pest resistance, stress resistance and other traits. The product is highly flexible, based on probe hybridization targeted capture sequencing technology, marker sites can be added according to demand, further expanding the application scenarios of the product.

[0005] The technical solution provided by the present invention is as follows:

[0006] The SNP molecular marker combination for maize genotyping consists of 5851 SNPs and 153 structural variation sites. It is characterized in that the SNP sites include SNP0001 to SNP5851, the chromosome information corresponding to the SNP0001 to SNP5851 is derived from the database Ensemb1, its genome assembly corresponds to GCA_000005005.6, the reference genome is Zea_mays.B73 RefGen_v4, and the chromosome sites corresponding to the SNP0001 to SNP5851 are as described in Table 1.

[0007] Table 1 Chromosome loci corresponding to SNP0001 to SNP5851

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[0029] The structural variation sites include SV1 to SV153, and the chromosomal sites corresponding to SV1 to SV153 are shown in Table 2.

[0030] Table 2 Chromosome loci corresponding to SV1~SV153

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[0033] A SNP chip, characterized in that it comprises probes corresponding to the SNP sites, and the nucleotide sequences of the probes corresponding to the SNP sites SNP0001 to SNP5851 correspond to the chromosome positions shown in Table 3.

[0034] Table 3 Chromosome locations corresponding to the nucleotide sequences of the probes corresponding to SNP0001 to SNP5851

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[0070] A SNP chip, characterized in that it comprises probes corresponding to the structural variation sites, and the nucleotide sequences of the probes corresponding to the structural variation sites SV1 to SV153 correspond to the chromosome positions shown in Table 4.

[0071] Table 4 Chromosome locations corresponding to the nucleotide sequences of the probes corresponding to the structural variation sites SV1 to SV153

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[0074] Compson CAGT targeted capture sequencing technology was used to design this targeted capture liquid phase chip containing 5851 SNPs and 153 structural variation sites. The chip integrates the functional SNP sites identified by GWAS analysis, reported functional SNP sites, structural variation and resequencing, and existing commercial chip sites, covering maize agronomic traits, quality traits, pest and disease resistance, and stress resistance. The addition of these sites will promote the progress of breeding for corresponding traits. The maize 6K chip reference genome is Zea_mays.B73_RefGen_v4.

[0075] The application directions of this chip: genomic breeding value estimation, whole genome selection breeding, detection of excellent trait loci, detection of disease resistance traits and molecular marker-assisted breeding.

[0076] The purpose of the present invention is achieved by the following technical solutions:

[0077] The present invention provides application of a corn 6K liquid phase chip in corn variety identification.

[0078] The present invention provides application of the corn 6K liquid phase chip in detecting corn breeding materials.

[0079] The present invention provides application of the corn 6K liquid phase chip in corn molecular marker-assisted breeding.

[0080] The present invention provides application of the corn 6K liquid phase chip in corn whole genome selection breeding.

[0081] The present invention provides an application of the SNP site in preparing a liquid-phase breeding chip for whole-genome selection of corn. The liquid-phase breeding chip for whole-genome selection of corn is used for corn variety selection. The SNP site includes SNP0001 to SNP5851. The chromosome sites corresponding to SNP0001 to SNP5851 are shown in Table 1 of the specification.

[0082] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in improving corn traits.

[0083] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in prediction of corn traits.

[0084] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in predicting corn plant height traits.

[0085] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in predicting corn ear length traits.

[0086] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in prediction of corn ear coarseness traits.

[0087] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in predicting the number of corn ear rows trait.

[0088] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in predicting the trait of kernel number per row of corn.

[0089] The present invention provides application of a liquid-phase breeding chip for whole-genome selection of corn in prediction of corn ear weight traits.

[0090] Furthermore, the prediction methods include GBLUP, PLS, LASSO, RKHS, BayesB, and XGBoost.

[0091] The 6K liquid phase chip of the present invention is used for whole genome prediction and analysis of hybrid phenotypes, which is more accurate, more efficient and less costly, and will accelerate the breeding process of new high-quality corn varieties.

[0092] Beneficial Effects

[0093] The present invention involves a total of 6052 probes, of which 5751 probes are for SNP and 301 probes are for SV. When designing the chip marker sites, SNPs are strictly screened, and the selected sites have good specificity and high capture rate, covering functional sites related to yield, plant type, disease and pest resistance, stress resistance and other traits. The site typing accuracy is greater than 99%, the detection rate is greater than 93%, the product stability is good, and the genotype consistency rate of repeated samples is greater than 99%.

[0094] The chip of the present invention has the characteristics of low cost and good effect, and can be applied to corn genome selection breeding, providing powerful technical support and guiding basis for corn hybrid breeding.

[0095] The chip contains multiple trait association sites identified by GWAS, distributed throughout the genome. In the breeding stage, the functional sites in the chip can be used to conduct preliminary identification of corn inbred lines, improve the efficiency of genomic selection, accelerate breeding progress, and formulate reasonable selection strategies. This product has good practicality, basically covers the published important trait-related markers, has high flexibility, and based on probe hybridization targeted capture sequencing technology, marker sites can be added according to needs to further expand the application scenarios of the product.

[0096] The present invention provides a breeding chip containing multiple functional sites of corn and its application, providing a germplasm resource genotype identification tool for corn molecular breeding, providing technical guidance for whole genome prediction of corn hybrid breeding, and accelerating the breeding process of new excellent corn varieties. Therefore, this study screened the variant sites and excellent alleles with important application value for genetic improvement, combined with the known corn functional gene information and GWAS analysis, and developed a low-density liquid phase chip for whole genome selection breeding.

[0097] The product is cost-effective and based on domestic design, detection and analysis processes, it greatly reduces typing costs; it uses automated detection processes to improve detection throughput and stability. The application of 6K liquid phase chips will greatly improve the efficiency of genotyping, shorten detection time and detection costs, provide efficient molecular detection tools for breeding applications of corn whole genome selection, and accelerate the selection and breeding of new excellent corn varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Distribution map of chip loci on chromosomes;

[0099] Figure 2 The predictive power of different traits under different models for 6K and 40K chips. DETAILED DESCRIPTION

[0100] The technical solution provided by the present invention is described in detail below in conjunction with the examples, but is not limited to the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The various experimental materials, reagents and carriers used in the following examples can all be obtained through commercial channels.

[0101] Marker development process:

[0102] (1) Obtaining the initial dataset

[0103] The chip sites are composed of four modules, namely, self-researched functional sites, reported functional sites, structural variation sites, and resequencing and existing commercial chip sites.

[0104] Functional loci studied by ourselves: obtained through analysis of two maize inbred line populations, GWAS and candidate gene analysis were performed on 24 traits using 216,095 genotype data from 346 maize inbred lines of our research group, and a total of 696 loci were identified, of which 73 candidate genes involved have been proven by research; GWAS analysis was performed on 9 traits and the general combining ability of these 9 traits using 108,541 genotype data from 266 maize inbred lines of our research group, and a total of 169 loci were identified.

[0105] Reported functional sites: Through literature search, this chip selected sites related to maize agronomy, quality, disease resistance, insect resistance, cold tolerance and salt-alkali tolerance.

[0106] Structural variation sites: 6746 PAV (Presence / absence Variation) data from pan-genome data of 13 backbone maize inbred lines (A632, Chang 7-2, Huangzao-4, Mo17, OH43, PH207, S37, Ye 178, Ye 478, Zheng 58, Dan 340, Jing 724 and Jing 92), and 180 PAVs detected in 5 materials or more were retained.

[0107] Resequencing and existing commercial chip sites: 17,851,196 SNPs were generated by resequencing of 350 maize inbred lines, with a sequencing depth greater than 10X. After data quality control, 504 SNPs were evenly selected based on the principle of uniform distribution of sites in the whole genome. 43,606 SNPs were generated by chip genotyping of 417 maize inbred lines in our research group. After data quality control, 403 SNPs were evenly selected based on the principle of uniform distribution of sites in the whole genome. Finally, these 907 SNPs were used as the background sites of the chip.

[0108] Microarray site screening

[0109] According to the custom chip design principles of Beijing Compson Biotechnology Co., Ltd., by evaluating and designing SNP sites, 103 successfully designed adjacent sites and all failed design sites were deleted, and a total of 5851 sites were selected. According to the 1X probe design strategy, a total of 5751 probes were used, with a coverage rate of 100%. For SVs data, probes were designed within 120bp on both sides of the missing sequence, and a total of 153 SVs data were retained, with a probe number of 301 and a coverage rate of 100%. Finally, the 6K corn chip retained 5851 SNPs and 153 SVs.

[0110] Example 1: Summary of Corn 6K Targeted Capture Chip Test

[0111] The corn chip was used to perform genotyping on 18 (including 3 replicates) corn inbred line materials. After the raw data was downloaded, the data was quality controlled. The clean data was aligned to the reference genome using the bwa software, and the alignment rate and repetition rate of each sample data were counted to prepare for subsequent variant detection. Then the data was aligned with the reference genome. The following is the statistics of the alignment results. 18 samples were sequenced this time, and the alignment results are shown in Table 5. The average best alignment rate was 94.6%, and the average dup ratio was 4.3%. The alignment results were normal and contaminant-free, and can be used for subsequent variant detection and related analysis.

[0112] Table 5 Comparison results statistics

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[0114] According to the comparison results of Clean Reads in the reference genome, the HaplotypeCaller module in the software GATK was used to generate SNP and Indel variant site files, and the target sites were extracted, and then filtered by 10X, totaling 5786 sites. Then the uniformity of the sites was tested. Generally, 10%X mean depth coverage rate (%) reached more than 97%, with better uniformity, and the uniformity of the product was 97.52%. Next, the detection rate of all test samples was statistically analyzed. As shown in Table 6, when the coverage depth of the target site was greater than 5X, the average site detection rate was 93.77%. Then, the detection of SNP sites on the chip of three repeated samples was statistically analyzed, that is, the number of sites and typing consistency of the analysis were analyzed. The results of Table 7 show that the typing consistency rate of the three repeated samples was more than 99%, and the verification results showed that the corn 6K liquid phase chip product has a high degree of reliability and accuracy.

[0115] Table 6 Sample detection rate

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[0118] Table 7 Consistency of typing of repeated samples

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[0120] Embodiment 2:

[0121] There are 280 SNPs with known superior alleles in the functional loci of the chip. The distribution of these 280 SNPs in leaf angle, number of tassel branches, flowering days, silking days, silking-flowering interval days, ear height, ear height / plant height, corn lethal necrosis, corn rough dwarf disease, corn gray leaf spot, corn borer and salt tolerance traits is shown in Table 8.

[0122] Table 8 Excellent allele type loci

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[0124] The chip was used to perform genotyping on 316 maize inbred lines, and the distribution of the number of excellent allele loci in some important inbred line materials was studied. The results are shown in Table 9. The following are the actual characteristics of some materials: Zong 31 has a semi-compact plant type, relatively harmonious male and female, and is resistant to lodging. Drought resistance and moderate waterlogging resistance. Resistant to large and small leaf spot, head smut, smut, ear rot and stem rot; Zheng 58 is tolerant to fertilizer and water, has strong drought tolerance, compact plant type, strong lodging resistance, and is resistant to large and small leaf spot and smut. Ji 853 inbred line has made significant breakthroughs in important traits such as yield, quality, and resistance; Dan 340 is resistant to multiple diseases, resistant to lodging, has high combining ability, and has a moderate growth period. It is one of the important parents of several large-ear excellent maize hybrids with the largest breeding and promotion area in my country; P138 has a compact plant type and harmonious male and female. The root system is well developed, with strong resistance to lodging, drought, waterlogging, disease and wide adaptability; K12 has a compact plant type and good coordination between male and female. It has strong drought tolerance, lodging resistance, resistance to large and small spot diseases, brown spot disease, and mild tumor smut. Among them, Zong 31 has a relatively compact plant type, and its loci for controlling the reduction of leaf angles are relatively few, while Zheng 58 and P138 are both relatively compact, and their loci for controlling the reduction of leaf angles are relatively more. In addition, K138 and K12 have good coordination between male and female, and their loci for controlling the reduction of silking and flowering intervals are relatively more. Ji 853, Dan 340 and P138 have strong disease resistance, and have more loci for rough dwarf disease, gray leaf spot disease, etc. In some materials, the judgment based on these loci is in line with the actual situation of these materials in some aspects, indicating that the chip can provide a preliminary reference for the identification of inbred corn.

[0125] Table 9 Distribution of superior allele loci in important inbred line materials

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[0127] Embodiment 3:

[0128] Application of corn chip in GS. In order to illustrate the effectiveness of the locus set obtained by the invention, a 40K commercial corn chip was used as a control. The experimental materials came from 316 inbred lines from 5 heterotic groups and 723 hybrids of these inbred lines. Six traits of hybrids, ear length (EL), ear diameter (ED), ear row number (ERN), row kernel number (KNR), ear weight (EW) and plant height (PH), were investigated. Genotype data were obtained by genotyping of 40K corn chip and 6K corn chip. Six models, GBLUP, PLS, LASSO, RKHS, BayesB and XGBoost, were used. The prediction performance of 6K chip and 40K chip was evaluated by 5-fold cross validation, that is, the population was randomly divided into 5 parts, 4 of which were used as training groups, and the remaining 1 was used as a test group. Each time, 4 samples were used to build a model to predict the other 1, and each sample was used as a test group, so that after 5 predictions, all individuals can be predicted. Predictive power is defined as the absolute coefficient between the predicted value and the observed value. To reduce the variation caused by sample allocation, 20 replicates of 5-fold cross validation were performed in each case, and the average predictive power of the 20 replicates was used.

[0129] Six models were used to estimate the genomic estimated breeding values ​​(GEBV) of six hybrid traits, namely, ear length, ear diameter, number of ear rows, number of grains per row, ear weight, and plant height. The accuracy of GEBV estimation was calculated using a 5-fold cross-validation method. Finally, the GEBV prediction accuracy was compared with that of the 40K chip. The calculation results are shown in the figure. Figure 2 As shown, in the four traits of ear length, ear weight, number of grains per row and plant height, the average prediction accuracy of the 6K chip through the six methods of GBLUP, PLS, LASSO, RKHS, BayesB and XGBoost is slightly higher than that of the 40K chip, which is increased by 4.61%, 3.68%, 16.99% and 0.87% respectively. In the two traits of ear thickness and ear row number, the average prediction accuracy of the 6K chip through the six methods of GBLUP, PLS, LASSO, RKHS, BayesB and XGBoost is slightly lower than that of the 40K chip, which is reduced by 1.62% and 3.11% respectively. In summary, although the SNP density of the corn 6K chip provided by the present invention is greatly reduced compared with the corn 40K chip, the prediction results of the six traits of ear length, ear thickness, ear row number, row grain number, ear weight and plant height show that the GEBV estimation accuracy of the corn 6K chip is not inferior to that of the 40K chip. This shows that the corn 6K chip can meet the analysis needs of genome selection and ensure the smooth implementation of corn genome selection breeding.

Claims

1. A liquid phase chip for maize genotyping, characterized in that: The liquid phase chip includes probes corresponding to the SNP site and the SV site. The nucleotide sequences of the probes corresponding to the SNP sites correspond to the chromosome positions shown in Table 1 below: Table 1 The chromosomal positions corresponding to the nucleotide sequences of the probes corresponding to the SV sites are shown in Table 2 below: Table 2 The liquid phase chip reference genome is Zea_mays.B73_RefGen_v4.

2. The use of the liquid phase chip for corn genotyping in corn trait improvement as claimed in claim 1, characterized in that: The corn traits include corn plant height, ear length, ear diameter, number of ear rows, number of kernels per row and ear weight.

3. The use of the liquid phase chip for corn genotyping in corn trait prediction according to claim 1, characterized in that: The corn traits include corn plant height, ear length, ear diameter, number of ear rows, number of kernels per row and ear weight, and the prediction methods include GBLUP, PLS, LASSO, RKHS, BayesB and XGBoost.

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