A SNP molecular marker combination for zea mays whole genome typing, a detection chip and application thereof

Abstract

The application provides a SNP molecular marker combination for whole genome genotyping of explosive corn, a detection chip and application, and relates to the technical field of molecular breeding.The application provides a SNP molecular marker combination for whole genome genotyping of explosive corn, and the genotyping object comprises 6419 SNP sites;the SNP markers in the application uniformly cover the whole corn reference genome, and can provide high-quality molecular markers for map-based cloning of explosive corn specific agronomic trait control genes.Meanwhile, the application can also perform genotyping on different types of corn.The explosive corn germplasm resource is narrower than other types of corn germplasm resources, and the chip of the application can make up for the low diversity of the chip based on the diversity of food corn in explosive corn genotyping.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically a combination of SNP molecular markers, a detection chip, and applications for whole-genome typing of popcorn. Background Technology

[0002] Popcorn (Zea mays L. var. everta Sturt.) is a special type of maize, distinguished from other maize types by its compact ears and small kernels. A key characteristic of its kernels is their composition of hard, horny starch. Under suitable moisture conditions, popcorn kernels expand rapidly during heating. The dramatic increase in internal pressure causes the seed coat to rupture, ultimately forming a butterfly-shaped or spherical puffed structure. This characteristic makes it the sole raw material for popcorn production. Other types of maize, such as flint and dent maize, can also produce less popping deformation after heating, but their popping expansion capacity is much lower than that of popcorn. In the field of breeding, the creation and selection of superior inbred lines are fundamental to the development of high-quality popcorn hybrids. Non-popcorn types of maize have a significant impact on popping characteristics during breeding improvements. The molecular characteristics of the genetic diversity, population structure, and genetic relationships of maize genetic resources developed for specific breeding objectives can help breeders understand how to utilize these resources for further improvement. Simple sequence repeats (SSRs), random amplified polymorphic DNA (RAPD), and single nucleotide polymorphisms (SNPs) have been used for the molecular characterization of popcorn. In popcorn research, SSR markers are more commonly used for diversity analysis of popcorn populations, but their density is relatively low. Currently, SNP markers are widely used in molecular marker-assisted breeding, germplasm fingerprinting, and variety protection of crops due to their genetic stability, large number, uniform distribution, and ease of automation. In medium-density marker detection, liquid chromatography-mass spectrometry (LC-MS) is widely used due to its low cost, high efficiency, flexible application, and wide adaptability. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a combination of SNP molecular markers for whole-genome typing of popcorn. The SNP markers in this invention uniformly cover the entire maize reference genome and can provide high-quality molecular markers for map-based cloning of genes controlling specific agronomic traits in popcorn.

[0004] The present invention also aims to provide a detection chip for genotyping of popcorn, which can perform efficient, rapid and low-cost genotyping of popcorn germplasm resources.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a combination of SNP molecular markers for whole-genome genotyping of pop maize. The combination consists of 6419 SNP loci located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, as shown in the table below:

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[0064] Preferably, the SNP molecular marker combination includes gene sequences within 350 bp before and after the SNP site.

[0065] The present invention also provides a detection chip for popcorn genotyping, including the aforementioned SNP molecular marker combination, which consists of 6419 SNP sites located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0.

[0066] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in popcorn genotyping.

[0067] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the genome-wide association analysis of popcorn.

[0068] This invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the identification of popcorn germplasm resources and kinship.

[0069] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the gene selection breeding of popcorn.

[0070] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the assessment of genetic diversity in popcorn.

[0071] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the construction of a popcorn genetic map.

[0072] The present invention also provides an application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in the identification of the authenticity of popcorn varieties.

[0073] Compared with the prior art, the present invention has the following advantages:

[0074] (1) This invention provides a combination of SNP molecular markers for whole-genome genotyping of popcorn, including 6419 SNP loci. The SNP markers used in this invention are derived from 103 popcorn resequencing data. All markers are located in gene regions, and all marker loci are directly associated with gene fragments. Furthermore, based on existing QTLs related to the popcorn burst shape, meta-QTL analysis was performed to increase the marker density in the meta-QTL region. The SNP markers in this invention uniformly cover the entire maize reference genome, providing high-quality molecular markers for map-based cloning of genes controlling specific agronomic traits in popcorn.

[0075] (2) This invention also develops a detection chip for popcorn genotyping, which can perform efficient, rapid, and low-cost genotyping of popcorn germplasm resources. Popcorn germplasm resources are narrower than other types of maize germplasm resources, and this chip can overcome the shortcomings of existing chips developed based on the diversity of grain maize in popcorn genotyping due to their low diversity. Attached Figure Description

[0076] Figure 1 The distribution of 6419 high-quality SNP loci on the maize genome;

[0077] Figure 2 The figure shows the phylogenetic tree constructed based on the maximum likelihood method for different types of maize germplasm. The background of non-explosive maize germplasm is yellow. Detailed Implementation

[0078] This invention provides a combination of SNP molecular markers for whole-genome typing of pop maize. The combination of SNP molecular markers consists of 6419 SNP sites located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, which can be queried in the MaizeGDB database.

[0079] In this invention, the SNP molecular marker combination preferably includes gene sequences within 350 bp before and after the SNP site; more preferably, it includes gene sequences within 250 to 300 bp before and after the SNP site.

[0080] This invention also provides a detection chip for pop maize genotyping, including the aforementioned SNP molecular marker combination, which consists of 6419 SNP sites located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0. This invention uses GenoBaits technology based on liquid-phase probe capture to design the detection chip. This invention extracts 7,519,610 SNP sites and 300 bp sequences before and after each site from the maize B73V5 reference genome to evaluate the suitability of probe design. The preferred evaluation criteria are: GC content of 30%–70% and homology ≤5 on the reference genome. It is estimated that 5,249,049 sites were successfully designed. Through Meta-QTL analysis, 73 QTLs associated with popping traits were summarized into 43 Meta-QTLs. Based on the Meta-QTL regions, 3458 sites within these regions were preferably retained. Other region selection probes are distributed evenly at 2961 sites on the chromosome, for a total of 6419 sites. These sites cover the ten chromosomes of the maize genome almost evenly.

[0081] This invention also provides the application of the aforementioned SNP molecular marker combination or the aforementioned detection chip in popcorn genotyping, popcorn genome-wide association analysis, popcorn germplasm resource and kinship identification, popcorn gene selection breeding, popcorn genetic diversity assessment, popcorn genetic map construction, or popcorn variety authenticity identification.

[0082] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0083] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0084] Example 1: Bursting Maize Germplasm Resequencing

[0085] One hundred and three representative popcorn germplasm resources were selected, including ten Latin American popcorn varieties, eight Chinese popcorn varieties, and 85 popcorn breeding inbred lines, and planted at the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences. After emergence, DNA was extracted from leaf tissues of the plants using the CTAB method. The samples were sequenced using Illumina sequencing technology, with B73 (version V5, https: / / download.maizegdb.org / Zm-B73-REFERENCE-NAM-5.0 / Zm-B73-REFERE NCE-NAM-5.0.fa.gz) as the reference genome. Alignment was performed using BWA, Picard, and Freebayes. Repetitive sequence markers and SNP variant sites were identified, resulting in a total of 7,519,610 SNP sites (Table 1).

[0086] Table 1. Variation identification results from popcorn resequencing data.

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[0088] Example 2: SNP Tag Selection and Chip Development Method

[0089] This embodiment uses GenoBaits technology based on liquid-phase probe capture for chip design. 7,519,610 SNP loci and 300 bp sequences before and after each locus were extracted from the maize B73V5 reference genome to evaluate the suitability of probe design. The evaluation criteria were: GC content between 30% and 70% and homology ≤ 5 on the reference genome. An estimate showed 5,249,049 successfully designed loci. Meta-QTL analysis revealed 73 burst trait-related QTLs, which were summarized into 43 Meta-QTLs. Based on the Meta-QTL regions, 3458 loci within these regions were preferentially retained. Another 2961 loci, evenly distributed across the chromosomes, were selected from other regions, totaling 6419 loci. These loci are essentially evenly distributed across the ten chromosomes of the maize genome. Figure 1 ).

[0090] Example 3: Application of SNP Genotyping Detection Chip in Popcorn

[0091] In this embodiment, 6 popcorn germplasm samples and 10 other types of maize germplasm samples (other types of maize germplasm include tropical and temperate grain maize) were selected. The popcorn SNP genotyping detection chip developed in Example 2 was used for genotyping. The steps are as follows:

[0092] (1) Sample preparation and DNA extraction solution: In the laboratory, 16 test maize inbred lines were germinated until they reached the three-leaf stage. The leaves were then sampled and quick-frozen in liquid nitrogen. The CTAB method was then used to extract DNA from the leaves.

[0093] (2) Sequence capture and sequencing: The sample DNA is randomly broken into fragments of 300-500 bp. The probes of the chip are used to capture the DNA fragments. Based on the captured DNA fragments, a sequencing library is constructed, and finally sequencing is performed on the Illumina platform.

[0094] (3) Target site genotype identification: The sequence obtained from sequencing was aligned to the B73 reference genome using BWA software, and repetitive sequences were removed using Picard. The HaplotayeCaller module of GATK was used for SNP identification.

[0095] A total of 6419 SNP loci were obtained using the above methods. The MAF of these SNP loci ranged from 0.05 to 0.50. The results indicate that the popcorn SNP genotyping chip can not only be used for genotyping of fresh corn germplasm but also for distinguishing other types of corn. Phylogenetic trees were constructed for these 16 corn germplasm accessions using the maximum likelihood method, based on... Figure 2 The results showed that popcorn and other types of maize germplasm could be distinguished.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of SNP molecular markers for whole-genome typing of popcorn, characterized in that, The SNP molecular marker combination includes gene sequences of 350 bp before and after the SNP site. The SNP site consists of 6419 SNP sites located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, as shown in the table below:

2. A detection chip for genotyping of popcorn, characterized in that, Includes probes for detecting the SNP sites as described in claim 1.

3. The application of the detection chip according to claim 2 in popcorn genotyping.

4. The application of the detection chip according to claim 2 in the genome-wide association analysis of popcorn.

5. The application of the detection chip according to claim 2 in the identification of popcorn germplasm resources and kinship.

6. The application of the detection chip according to claim 2 in gene selection breeding of popcorn.

7. The application of the detection chip according to claim 2 in the assessment of genetic diversity in popcorn.

8. The application of the detection chip according to claim 2 in the construction of the genetic map of popcorn.

9. The application of the detection chip according to claim 2 in the identification of the authenticity of popcorn varieties.

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