CN115820893A - Application of corn HRM molecular marker in corn genetic breeding - Google Patents

Abstract

The invention discloses an application of a corn HRM molecular marker in corn genetic breeding, and relates to the technical field of biology, wherein the SNP locus of the molecular marker is positioned in c.C1248T, c.G405A or c.A1398G of No. 2 chromosome. The method utilizes the corn whole genome second-generation sequencing technology to scan the corn varieties with the head smut and the head smut resistance, searches SNP sites between the corn varieties, develops the SNP sites into specific molecular markers based on the HRM technology, can realize efficient genotyping of the corn with the head smut and the head smut resistance, and provides reliable and rapid technical tools for gene mining, variety identification, molecular breeding and the like of the corn in the future.

Description

Application of corn HRM molecular marker in corn genetic breeding

Technical Field

The invention relates to the technical field of biology, in particular to application of a corn HRM molecular marker in corn genetic breeding.

Background

Previous researches show that the excellent characteristics of wild rice include disease and insect resistance, stress tolerance and other characteristics (such as cytoplasmic male sterility and high quality), and scientists have utilized these resources to breed a series of excellent rice varieties (lines). However, due to the complex genetic background of wild rice, many good trait genes are linked with bad traits, and the application of the wild rice in the modern rice improvement breeding is seriously hindered.

In the traditional breeding process, breeders mainly rely on long-term practical experience to select phenotypes, and due to the influence of various environmental conditions, the selection efficiency is very low, the accuracy is poor, and the breeding period is long. Particularly in the field distant hybridization breeding practice, due to the complex genetic background of the wild rice, hybrid progeny is separated fiercely, and the difficulty of phenotype selection is larger. By means of advanced molecular biology technology, the breeding efficiency and success rate can be greatly improved by carrying out directional selection from the genome level. Molecular Marker-assisted Selection (MAS) is to combine molecular Marker technology with traditional crop breeding and to effectively reduce linkage drag through Marker Selection, thereby realizing rapid improvement of crops. In the corn crossbreeding process, tracking detection is carried out by using molecular markers, and various excellent characters can be efficiently aggregated by combining phenotype selection, so that linkage drag of unfavorable genes is quickly eliminated. Among them, molecular markers provide powerful detection tools for distinguishing introgressed chromosome segments in hybrid plants, and have been widely applied in analysis of high-generation backcross populations.

The selection of the appropriate marker type is critical to the success of molecular marker assisted selection. Due to the many advantageous features, SNP markers are becoming important marker selection tools in current plant molecular breeding. In plants, SNPs are high in density and distributed in large numbers, with about 1 SNP per 268 bases in maize, which is distributed at a much higher frequency than InDel and SSR. Chen et al selected 51478 site from 1000 ten thousand SNPs of the re-sequencing result of 801 rice varieties and developed it into gene chip, and the frequency of this gene chip can almost reach 1 polymorphic SNP per gene. However, the traditional SNP chip has the defects of high price, poor flexibility and the like, thereby limiting the large-scale application of SNP markers in breeding. With the advent of HRM technology, the above-mentioned difficulties can be solved well. As a new technology for detecting SNP, the method has the advantages of high resolution, simple operation, high efficiency, low cost and the like. In addition, the technology can also detect the variation of InDel and SSR types, so the technology has good application prospect in plant molecular breeding. Carrying out SNP genotyping by adopting a high-resolution melting curve, such as Lu Sha Hua, and the like to obtain an SNP marker closely linked with the Tssd of the peach; functional markers Wx-a/b and BADH2-E7 based on an HRM system are developed by the romanone and the like, and the gene Wx for controlling the amylose content of the rice and the gene fgr formed by the aroma are well genotyped; the HRM is utilized by Zhao-Yuan and the like to analyze the genotypes of a G/A converted SNP marker and an SSR marker and an InDel marker which are difficult to distinguish by non-denaturing polyacrylamide gel electrophoresis, and the HRM has incomparable superiority compared with the traditional gel electrophoresis analysis; marker molecules Pi2-HRMF1/R3 based on the HRM system were developed for functional regions of rice blast resistance gene Pi2 such as Jinming-Na, which can discriminate various functional variations of Pi2 gene and various types of heterozygosity of variations well. A functional molecular marker based on an HRM system is developed aiming at a functional region of a tms5 gene of a two-line sterile line, gold famous marker and the like, and can be used for resource identification and molecular marker-assisted improvement of the tms5 gene, so that technical support is provided for accurate and efficient molecular breeding of the two-line sterile line. Meanwhile, another SNP detection method, competitive Allele-Specific PCR (Kompet-ive Allele Specific PCR), abbreviated as KASP technology, has been developed in recent years. The technology can carry out accurate biallelic genotyping on target SNPs and InDels. Feng et al utilize KASP SNP markers to carry out positioning and polymerization breeding of drought-resistant and low-nitrogen-resistant QTLs of rice and obtain excellent multi-trait multi-site polymerization strains. However, KASP label detection platforms are expensive and generally only affordable by larger biotech service companies or laboratory public platforms.

Disclosure of Invention

The invention aims to provide application of a corn HRM molecular marker in corn genetic breeding to solve the problems in the prior art, and the corn HRM molecular marker provided by the invention can be used for accurately identifying corn varieties with head smut resistance.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of a corn HRM molecular marker in corn genetic breeding, wherein SNP loci of the molecular marker are positioned in c.C1248T, c.G405A or c.A1398G of No. 2 chromosome.

Further, the application is to screen corn varieties with head smut resistance.

The invention also provides a method for screening the maize variety with head smut resistance, which is characterized by comprising the following steps:

(1) Obtaining genome DNA of corn to be detected;

(2) Carrying out PCR amplification to obtain a gene fragment containing the SNP locus, and carrying out genotyping to obtain the head smut resistant corn variety; the SNP locus is positioned in c.c1248T, c.G405A or c.A1398G of No. 2 chromosome.

Furthermore, when the SNP locus is positioned at c.C1248T of chromosome 2, the primer pair amplified by the PCR is shown as SEQ ID NO. 1-2.

Further, when the SNP locus is located at c.G405A of chromosome 2, the primer pair amplified by the PCR is shown as SEQ ID NO. 3-4.

Further, when the SNP locus is located at c.A1398G of chromosome 2, the primer pair amplified by the PCR is shown as SEQ ID NO. 5-6.

Further, the reaction procedure of the PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 10s at 72 ℃ and 45 cycles; 1min at 95 ℃; 1min at 40 ℃; 1s at 65 ℃;97 ℃ for 1s.

Further, the reaction system for PCR amplification comprises 10-20 ng of genomic DNA,2 × EasyTaqPCR SuperMix,0.1 μ M forward and reverse primers, 0.5 μ L20 × EvaGreen fluorescent dye, and ddH ₂ Make up to 10. Mu.L of O.

The invention also provides a kit for screening the maize variety with the head smut resistance, which comprises a primer pair shown in SEQ ID NO.1-2, SEQ ID NO.3-4 or SEQ ID NO. 5-6.

The invention discloses the following technical effects:

single Nucleotide Polymorphism (SNP) sites are widely distributed in plants. The SNP marker developed on the basis has become an important molecular tool for crop genetic research at present due to the advantages of high resolution, co-dominance and the like. The invention establishes the SNP molecular marker based on the high-resolution melting curve (HRM) technology, thereby realizing the high-efficiency genotyping of the maize with the head smut and the head smut resistance, and providing a reliable and quick technical tool for the gene mining, the variety identification, the molecular breeding and the like of the maize in the future.

The method utilizes the corn whole genome second-generation sequencing technology to scan the corn varieties with the head smut and the head smut resistance, searches SNP sites between the corn varieties and the head smut resistance, and develops the SNP sites into the specific molecular markers based on the HRM technology. Then, the molecular markers are used for carrying out molecular detection on the parent head smut-sensitive corn variety, the head smut-resistant corn variety and the BC1 backcross population of the two varieties so as to verify the effectiveness of the two varieties. Corn resequencing found a total of 4198 SNP sites between disease and disease, which were more evenly distributed across 10 chromosomes. 3 SNP loci are selected on the No. 2 chromosome of the corn and are developed into a specific molecular marker based on the HRM technology. The markers are used for detecting and analyzing BC2 and BC3 groups of the maize variety with the head smut and the head smut resistance, and the markers can accurately distinguish homozygous genotypes and heterozygous genotypes of parents. And detecting the insertion of the head smut resistant corn fragment in the marker interval ZY 1-ZY 1-4 of chromosome 2 of the backcross progeny. The corn re-sequencing genome can be well applied to the development of corn SNP markers. The SNP specific marker developed by the invention can accurately and efficiently carry out genotyping on the maize variety with the head smut and the head smut resistance, and provides an efficient molecular detection means for the molecular genetic research of the head smut resistance maize in the future, and is beneficial to gene mining and breeding application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the distribution of different SNP sites of each chromosome of maize whole genome re-sequencing detection of head smut resistant maize (YLZ) and maize variety (Z207) with head smut sensitivity;

FIG. 2 is a HRM assay of primer c.C1248T on chromosome 2 bin2.09, against head smut maize (YLZ), head smut maize (Z207) cultivars; the blue curve represents the high-resolution melting peak of the head smut resistant corn variety, the red curve represents the high-resolution melting peak of the head smut sensitive corn variety, and the gray curve represents the high-resolution melting peak of the YLZ/Z207F1 progeny heterozygous individual; the upper part of the figure is YLZ and the lower part is Z207.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

1 materials and methods

Material

The 2 parents in this experiment are respectively the head smut-susceptible (female parent) and head smut-resistant (male parent) maize variety. And (2) hybridizing the YLZ serving as a male parent with Z07 to obtain F1, backcrossing the F1 serving as the male parent with the Z07 to obtain BC1F1, planting BC1F1 materials, randomly selecting 20 single plants from the materials, hybridizing the single plants with the Z07 to obtain BC 2F 1, continuously backcrossing the single plants for 1 generation by adopting the same method to obtain BC3F 1, planting BC3F 1 plants, and mixing the plants in a mature period to obtain BC3F2 selfed seeds. 6 and 24 individuals are randomly selected from the BC3F 1 population and the BC3F2 population respectively for sampling, and SNP molecular marker analysis is carried out.

1.1 maize Whole genome resequencing SNP analysis

The genome DNAs of the head smut-sensitive and head smut-resistant maize varieties were extracted using an Omega DNA extraction Kit (SP Plant DNA Kit, cat # D5511), and the specific steps were referred to the protocol.

The DNA for whole genome re-sequencing SNP analysis needs to meet the following requirements:

(1) The DNA concentration is 50-100 ng/mu L;

(2) The ratio of the absorbance OD 260/280 of the DNA is 1.8-2.0, and a clear band is formed during agarose detection.

The above DNA samples were sent to a Nordheim source and sequenced using an illuminaHiSeq-based method. Then, SAMTOOLS (mpileup-m 2-F0.002-d 1000) is used for carrying out SNP and InDel detection to obtain the SNP and InDel variation information of 7 corn varieties. Carrying out functional annotation on the detection results of the SNP and the InDel by using ANNOVAR, and dividing the detection results into a coding region SNP/InDel, an intron region SNP/InDel, a shearing site SNP/InDel, a gene upstream SNP/InDel, a gene downstream SNP/InDel and an intergenic region SNP/InDel according to the positions of the SNP/InDel on a genome; according to the functional mutation caused by SNP/InDel in the coding region, the SNP/InDel is divided into non-synonymous variation SNP, frame shift mutation InDel, non-frame shift mutation InDel, variation SNP/InDel for making the gene obtain the stop codon and variation SNP/InDel for making the gene lose the stop codon.

1.2 primer design and information

Previous studies found that primers with an amplified fragment size of 68bp were best suited for HRM typing analysis. Therefore, the size of the PCR amplified target fragment of the HRM detection system in the research is determined to be within 200 bp. According to the sequence comparison result of 4198 polymorphic SNP sites of a corn re-sequencing genome between a head smut-sensitive corn variety and a head smut-resistant corn variety, 100bp sequences at both ends of all SNP sites are downloaded from the national center for biological information of America NCBI (https:// www.ncbi.nlm.nih.gov /), and then HRM Primer design is carried out through a Primer design tool Primer Premier 6. The primer sequence was synthesized by Biotechnology engineering (Shanghai) GmbH.

1.3 PCR reaction System and HRM analysis

The PCR reaction used a 10. Mu.L system containing 10-20 ng of genomic DNA,2 × EasyTaqPCR SuperMix (Transgen, beijing), 0.1. Mu.M forward and reverse primers (Table 1), and 0.5. Mu.L of 20 × Eva Green fluorescent dye (Biotium, USA), plus ddH ₂ O was made up to 10 μ L and the product was run on a Roche white 96-well PCR plate. The PCR amplification procedure was as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 10s at 72 ℃ and 45 cycles; 1min at 95 ℃; 1min at 40 ℃; 1s at 65 ℃;97 ℃ for 1s. After the PCR is finished, a High Resolution Melting curve (High Resolution Melting) is directly collected and analyzed, and the genotypes of a heterozygote and a biparental strain are distinguished according to the change form of the Melting curve.

2 results and analysis

2.1 genome Assembly, annotation and SNP detection analysis

Obtaining Raw data by using base containment (Illumina pipeline CASAVA v1.8.2), and filtering reads containing Illumina library construction joints, reads containing more than 10% unknown bases (N bases) in single-ended sequencing and reads containing more than 50% low-quality bases (sequencing quality value is less than or equal to 5) in single end through a quality control program to obtain Clean data. Clean data was aligned to the reference genome B73-RefGen-v 4-genomic using BWA software (parameters: mem-t 4-k 32-M). SNP detection is carried out by utilizing SAMTOOLS (mpileup-m 2-F0.002-d 1000), and SNP variation information of 7 corn varieties is obtained. Functional annotation was performed on the detection results of SNPs using ANNOVAR.

Through analyzing the differential sites detected between the two parents, 4198 SNP sites in total are found to present polymorphism, namely 1 polymorphic site is found in the average 100kb in the corn genome; wherein, the polymorphic sites of the chromosome 1 are the most and up to 540, the polymorphic sites of the chromosome 10 are the least and 231, and the distribution of the SNP sites on each chromosome is relatively uniform (figure 1), which can meet the follow-up molecular marker tracking requirements of introgression lines; meanwhile, the sequencing result also shows that the genetic difference between the head smut resistant corn variety and the head smut-sensitive corn variety is large, the genetic relationship is far, and the genetic basis of the head smut-sensitive corn variety can be effectively improved by constructing the head smut-resistant corn introgression line.

2.2 Development and validation of SNP markers

According to the distribution condition of SNP loci between the smut resistant corn and the smut susceptible corn, 132 pairs of HRM typing primers are designed relatively uniformly on 10 chromosomes of the corn at the average physical distance of about 10Mb, specifically, the marker distribution of each chromosome is shown in figure 1, the length of an amplification product is 60-150 bp, the mutation types detected by the markers are all mutation of A/T → G/C, and the related information of part of the primers is shown in table 1.

TABLE 1 information on 3 pairs of primers on chromosome 2

The genomic DNAs of the head smut-resistant maize and the head smut-sensitive maize were amplified using 3 pairs of primers (Table 1) c.C1248T-c.A1398G, respectively, and the amplification products were subjected to genotyping analysis using HRM genotyping instrument LC 96. Meanwhile, in order to verify the stability and reliability of the designed primers c.c1248T-c.A1398G, 2 repeats are set for the template used for amplification in FIG. 2, and the amplified product is sent to Shenzhen Hua DageneCo for sequencing. HRM typing results showed that the amplification products of all primers exhibited different melting curves between the two parents (FIG. 2). Sequencing results show that the SNP sites of the two parents are completely consistent with the result of HRM typing. In order to evaluate the typing effect of the SNP markers on F1 plants, 2F 1 single plants hybridized by the head smut resistant corn and the head smut-sensitive corn varieties are selected, HRM typing analysis is carried out on the 3 pairs of primers (table 1) above, and the amplification products of all the primers on the F1 plants show different melting curves from those of two parents, namely the designed primers can well distinguish heterozygotes from homozygotes. HRM typing and sequencing of the anti-head smut and head smut-sensitive maize varieties and HRM typing of F1 of both parents with other primers also showed similar results to FIG. 2. The results well prove that the SNP molecular marker analysis method based on the HRM technology can carry out accurate genotyping on the amphiphilic parents and corresponding backcross progeny plants thereof.

2.3 Breeding applications of maize-specific molecular markers

6 plants of the BC1F1 population of introgression lines of Z24 recurrent parent maize were randomly sampled and then HRM typed with the SNP markers in Table 1. The results show that the plants BC1F 1-1, BC1F 1-3, BC1F 1-4 and BC1F1-5 are in a heterozygous state at the marked sites of c.C1248T, c.G405A and c.A1398G, and the Z207 banding pattern of the parent is shown at the marked sites of ZY1-5, while the plants BC1F 1-2 and BC1F 1-6 are in the Z207 banding pattern which is homozygous at all 5 marked sites. The results of the 6 plants tested with the remaining 10 markers showed that they were all homozygous bands of the Z207 parent. The seeds of BC1F 1-1 are harvested, planted in the field for selfing to obtain a BC 2F 2 segregation population, 24 plants are randomly selected from the population for listing and sampling, and HRM typing analysis is carried out on the plants by adopting 3 marks of c.C1248T-c.A1398G. The results show that 24 plants are randomly separated from c.c1248T to c.a1398G, and are presented as Z207 homozygous genotypes at the ZY1-5 marker locus. These results show that SNP molecular markers based on HRM technology can well detect corn genome fragment introgression, thereby providing a technical tool for the discovery of wild excellent genes.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. The application of the HRM molecular marker in corn genetic breeding is characterized in that the SNP locus of the molecular marker is positioned in c.C1248T, c.G405A or c.A1398G of No. 2 chromosome.

2. The use of claim 1, wherein said use is in screening maize varieties for head smut resistance.

3. A method for screening a maize variety resistant to head smut, comprising the steps of:

(1) Obtaining genome DNA of corn to be detected;

(2) Carrying out PCR amplification to obtain a gene fragment containing the SNP locus, and carrying out genotyping to obtain the head smut resistant corn variety; the SNP locus is positioned in c.c1248T, c.G405A or c.A1398G of No. 2 chromosome.

4. The method of claim 3, wherein when the SNP site is located at c.c1248T of chromosome 2, the primer pair for PCR amplification is shown as SEQ ID NO. 1-2.

5. The method as claimed in claim 3, wherein when the SNP site is located at c.G405A of chromosome 2, the primer pair for PCR amplification is shown as SEQ ID NO. 3-4.

6. The method of claim 3, wherein when the SNP site is located at c.A1398G of chromosome 2, the primer pair for PCR amplification is as set forth in SEQ ID nos. 5 to 6.

7. The method of claim 3, wherein the reaction procedure of the PCR amplification is: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 10s at 72 ℃ and 45 cycles; 1min at 95 ℃; 1min at 40 ℃; 1s at 65 ℃;97 ℃ for 1s.

8. The method of claim 3, wherein the reaction system for PCR amplification comprises 10-20 ng of genomic DNA,2 XEasyTaqPCR SuperMix, 0.1. Mu.M forward and reverse primers, 0.5. Mu.L of 20 XEvaGreen fluorescent dye, plus ddH ₂ O make up to 10. Mu.L.

9. A kit for screening a maize variety resistant to head smut is characterized by comprising a primer pair shown as SEQ ID NO.1-2, SEQ ID NO.3-4 or SEQ ID NO. 5-6.

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