SNP (Single Nucleotide Polymorphism) molecular marker combination for pop corn whole genome typing, detection chip and application

Abstract

The invention provides an SNP molecular marker combination for pop corn whole genome typing, a detection chip and application, and relates to the technical field of molecular breeding. The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker combination for genotyping of the whole genome of the pop corn, a genotyping object comprises 6419 SNP loci, the SNP markers uniformly cover the whole corn reference genome, and a high-quality molecular marker can be provided for map-based cloning of a pop corn specific agronomic trait control gene. Meanwhile, different types of corn can also be subjected to genotyping. Burst corn germplasm resources are narrower than other types of corn germplasm resources, and the chip can overcome the defect that an existing chip developed based on grain corn diversity is low in diversity in pop corn genotyping.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and specifically relates to a SNP molecular marker combination, a detection chip and an application of whole genome typing of popcorn. Background Art

[0002] Popcorn (Zea mays L. var. everta Sturt.) is a special type of corn that is distinguished from other types of corn by its compact cobs and smaller kernels. The notable feature of its kernels is that they are composed of hard keratinous starch. Under appropriate moisture content, popcorn kernels can expand rapidly during heating. The sharp increase in internal pressure causes the seed coat to rupture, eventually forming a butterfly-shaped or spherical puffed structure, which makes it the only raw material for producing popcorn. Other types of corn, such as flint and dent corn, can also produce smaller swelling deformation after heating, but their bursting and swelling capacity is much lower than that of popcorn. In the field of breeding, the creation and selection of excellent inbred lines is the basis for the preparation of high-quality popcorn hybrids. Non-popcorn types have a greater impact on the swelling characteristics in breeding improvement. Molecular characterization of genetic diversity, population structure and genetic relationships of corn genetic resources developed for specific breeding goals can help breeders understand how to use these resources for further improvement. Simple sequence repeat (SSR), randomly amplified polymorphic DNA (RAPD) and single nucleotide polymorphism (SNP) markers have been used for molecular characterization of popcorn. In popcorn research, SSR markers are more commonly used for diversity analysis of popcorn populations, but the density of SSR markers is low. Currently, SNP markers are widely used in the fields of molecular marker-assisted breeding, germplasm fingerprinting and variety protection of crops due to their genetic stability, large number, uniform distribution and easy automation. In medium-density marker detection, liquid microarrays are widely used due to their low cost, high efficiency, flexible application and wide adaptability. Summary of the invention

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

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a SNP molecular marker combination for whole genome typing of popcorn, wherein the SNP molecular marker combination consists of 6419 SNP sites located on the corn B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, and the SNP sites are shown in the following table:

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[0065] Preferably, the SNP molecular marker combination includes gene sequences within 350 bp before and after the SNP site.

[0066] The present invention also provides a detection chip for popcorn genotyping, comprising the SNP molecular marker combination, wherein the SNP molecular marker combination is composed of 6419 SNP sites located on the corn B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0.

[0067] The present invention also provides an application of the SNP molecular marker combination or the detection chip in popcorn genotyping.

[0068] The present invention also provides an application of the SNP molecular marker combination or the detection chip in whole genome association analysis of popcorn.

[0069] The present invention also provides an application of the SNP molecular marker combination or the detection chip in identification of popcorn germplasm resources and kinship.

[0070] The present invention also provides an application of the SNP molecular marker combination or the detection chip in gene selection breeding of popcorn.

[0071] The present invention also provides an application of the SNP molecular marker combination or the detection chip in the evaluation of genetic diversity of popcorn.

[0072] The present invention also provides an application of the SNP molecular marker combination or the detection chip in the construction of a popcorn genetic map.

[0073] The present invention also provides an application of the SNP molecular marker combination or the detection chip in authenticity identification of popcorn varieties.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] (1) The present invention provides a combination of SNP molecular markers for whole-genome genotyping of popcorn, and the genotyping objects include 6419 SNP sites. The SNP markers used in the present invention are derived from 103 popcorn resequencing data, all markers are located in the gene region, and all marker sites are directly related to the gene fragments. At the same time, based on the currently available QTL related to the puff shape of popcorn, the marker density is increased in the Meta-QTL region by performing Meta-QTL analysis. The SNP markers in the present invention evenly cover the entire corn reference genome, and can provide high-quality molecular markers for the map-based cloning of genes controlling specific agronomic traits of popcorn.

[0076] (2) The present 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 corn germplasm resources. This chip can make up for the disadvantage of low diversity in popcorn genotyping of existing chips developed based on grain corn diversity. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The distribution of 6419 high-quality SNP loci on the maize genome;

[0078] Figure 2 This is an evolutionary tree constructed for different types of corn germplasm based on the maximum likelihood method. The background of non-popcorn germplasm in the figure is yellow. DETAILED DESCRIPTION

[0079] The present invention provides a SNP molecular marker combination for whole genome typing of popcorn, wherein the SNP molecular marker combination consists of 6419 SNP sites located on a corn B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, and the corn reference genome version can be queried in the MaizeGDB database.

[0080] In the present 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.

[0081] The present invention also provides a detection chip for genotyping of popcorn, including the SNP molecular marker combination, which is composed of 6419 SNP sites located on the reference genome version of corn B73V5 Zm-B73-REFERENCE-NAM-5.0. The present invention selects the GenoBaits technology based on liquid probe capture to design the detection chip. The present invention extracts 7519610 SNP sites and 300bp sequences before and after the sites from the corn B73V5 reference genome to evaluate whether the probe design is suitable. The evaluation criteria are preferably: GC content of 30% to 70% and homology number on the reference genome ≤5. After estimation, the number of sites successfully designed is 5249049. The present invention performs Meta-QTL analysis, and 73 QTLs related to the popping trait are summarized into 43 Meta-QTLs. Based on the Meta-QTL region, 3458 sites in this interval are preferably retained. The other regions selected probes at 2961 sites evenly distributed on the chromosomes, a total of 6419 sites. These sites are basically evenly covered on the ten chromosomes of the maize genome.

[0082] The present invention also provides an application of the SNP molecular marker combination or the detection chip in popcorn genotyping, popcorn whole genome 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.

[0083] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0084] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0085] Example 1 Popcorn Germplasm Resequencing

[0086] 103 representative popcorn germplasm resources, including 10 Latin American popcorn local varieties, 8 Chinese popcorn local varieties, and 85 popcorn breeding inbred lines, were selected and planted at the Zhuanghang Experimental Station of Shanghai Academy of Agricultural Sciences. After emergence, DNA from leaf tissues of plants was extracted using the CTAB method. The samples were sequenced using Illumina sequencing technology, and B73 (version number: V5, https: / / download.maizegdb.org / Zm-B73-REFERENCE-NAM-5.0 / Zm-B73-REFERE NCE-NAM-5.0.fa.gz) was used as the reference genome. BWA, Picard, and Freebayes were used for alignment, identification of repeat sequence markers and SNP variant sites, and a total of 7,519,610 SNP sites were obtained (Table 1).

[0087] Table 1 Results of variant identification in popcorn resequencing data

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[0089] Example 2 SNP marker selection and chip development method

[0090] This embodiment uses the GenoBaits technology based on liquid phase probe capture for chip design. 7,519,610 SNP sites and 300bp sequences before and after the sites extracted from the corn B73V5 reference genome are used to evaluate whether the probe design is suitable. The evaluation criteria are: GC content between 30% and 70% and the number of homologs on the reference genome ≤5. After estimation, 5,249,049 sites were successfully designed. By performing Meta-QTL analysis, 73 QTLs related to bursting traits were summarized into 43 Meta-QTLs. Based on the Meta-QTL region, 3458 sites in this interval were preferentially retained. Other regions selected 2961 sites where probes were evenly distributed on chromosomes, for a total of 6419 sites. These sites are basically evenly covered on the ten chromosomes of the corn genome ( Figure 1 ).

[0091] Example 3 Application of Popcorn SNP Typing Detection Chip

[0092] In this example, 6 popcorn germplasms and 10 other types of corn germplasms (other types of corn germplasms include tropical and temperate grain corn) were selected, and the popcorn SNP typing detection chip developed in Example 2 was used for genotyping, and the steps were as follows:

[0093] (1) Sample preparation and DNA extraction solution: The 16 test maize inbred lines were germinated in the laboratory. When the germinated maize had three leaves and one heart, the leaves were sampled and quick-frozen in liquid nitrogen. The CTAB method was then used to extract DNA from the leaves.

[0094] (2) Sequence capture and sequencing: The sample DNA is randomly broken into fragments of 300 to 500 bp. The DNA fragments are captured using the probes on the chip. A sequencing library is constructed based on the captured DNA fragments, and finally sequencing is performed based on the Illumina platform.

[0095] (3) Target site genotype identification: The sequence obtained by sequencing was aligned to the B73 reference genome using BWA software, and Picard was used to remove duplicate sequences. The HaplotypeCaller module of GATK was used for SNP identification.

[0096] A total of 6419 SNP loci were obtained through the above method. The MAF of these SNP loci was between 0.05 and 0.50. The results showed that the popcorn SNP typing detection chip can not only be used for genotyping of fresh corn germplasm, but also for distinguishing other types of corn. The maximum likelihood method was used to construct an evolutionary tree for these 16 corn germplasms. Figure 2 The results showed that popcorn and other types of corn germplasm could be distinguished.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SNP molecular marker combination for whole genome typing of popcorn, characterized in that: The SNP molecular marker combination consists of 6419 SNP sites located on the maize B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.0, and the SNP sites are shown in the following table:

2. The SNP molecular marker combination according to claim 1, characterized in that: The SNP molecular marker combination includes gene sequences within 350 bp before and after the SNP site.

3. A detection chip for popcorn genotyping, characterized in that: It comprises the SNP molecular marker combination as described in claim 1 or 2, wherein the SNP molecular marker combination consists of 6419 SNP sites located on the corn B73V5 reference genome version Zm-B73-REFERENCE-NAM-5.

0.

4. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in popcorn genotyping.

5. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in genome-wide association analysis of popcorn.

6. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in identification of popcorn germplasm resources and kinship.

7. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in gene selection breeding of popcorn.

8. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in the evaluation of genetic diversity of popcorn.

9. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in constructing a genetic map of popcorn.

10. Use of the SNP molecular marker combination according to claim 1 or 2 or the detection chip according to claim 3 in authenticity identification of popcorn varieties.

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