CN107502661B - SNP molecular marker combination related to corn stalk rot resistance and application thereof - Google Patents

SNP molecular marker combination related to corn stalk rot resistance and application thereof Download PDF

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CN107502661B
CN107502661B CN201710758666.7A CN201710758666A CN107502661B CN 107502661 B CN107502661 B CN 107502661B CN 201710758666 A CN201710758666 A CN 201710758666A CN 107502661 B CN107502661 B CN 107502661B
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stalk rot
snp
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邹继军
李晓鹏
卢东林
阮祥经
谈存梅
钟敬
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Anhui Longping High Tech Seeds Co ltd
Longping Biotechnology Hainan Co ltd
Yuan Longping High Tech Agriculture Co ltd
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Abstract

The invention relates to molecular biology, and particularly discloses an SNP molecular marker combination related to resistance of corn stalk rot and application thereof. The invention utilizes sequencing data and multi-year-multipoint phenotypic data to carry out correlation analysis to obtain SNP loci related to the corn stalk rot, further develops and develops 11 SNP molecular markers related to the resistance of the corn stalk rot, and improves the detection accuracy by a molecular marker combination detection method, and the accuracy can reach more than 90 percent under the condition of less influence of environmental factors. The invention also identifies the chromosome segment related to the disease resistance of the stem rot, and provides a basis for further gene cloning and utilization. The molecular marker developed by the invention is used for auxiliary breeding, so that the rapid identification of resistance can be realized, and the field test cost is reduced, thereby saving the breeding cost and accelerating the breeding process.

Description

SNP molecular marker combination related to corn stalk rot resistance and application thereof
Technical Field
The invention relates to molecular biology, in particular to SNP molecular markers related to resistance to corn stalk rot.
Background
Corn stalk rot, also called basal stalk rot, is an important fungal soil-borne disease commonly occurring in corn production areas in the world. The disease incidence rate is generally between 10% and 20% according to 18 provinces investigation of Guangxi, Zhejiang, Hubei, Shaanxi, Hebei, Shandong, Liaoning and the like, the disease incidence rate can reach 50% to 60% in severe years, the yield is reduced by about 20%, and the serious disease is even no more than income. The onset period is generally from the filling stage of corn, and the peak period from the late stage of milk ripening to the wax ripening stage. When the disease is developed, the disease usually decays from the root, then the disease spreads upwards, obviously withers leaves until the whole plant withers, the internodes turn light brown, the bracts of the fruit cluster are dry, the ear stem is flexible, the fruit cluster droops and is not easy to separate, the ear stem is soft, the grains are dry and flat, and the threshing is difficult. The stem rot of corn is usually withered and died suddenly in late stage of maturity, and the died plant is green, so it is also called bacterial wilt. The disease symptoms appear from the initial appearance of diseased leaves to the whole plant, the disease onset time is generally about one week, the duration is only 1 to 3 days in short time, and the duration is more than 15 days in long time.
Corn stalk rot is mainly divided into two types, one is mainly caused by obliteration of erwinia chrysanthemi, eubacterales, erwinia, and the other is mainly caused by infection of pythium and fusarium, fungi imperfecti, myceliophthora, tumorous acne, fusarium, pythium algae, oomycetes, pythiales, pythium, and the stalk rot is common in production. At present, the disease resistance identification of the corn stalk rot generally comprises the steps of uniformly inoculating pathogens near roots by a manual inoculation method, investigating secondary disease rate and disease grade at the late stage of corn milk maturity and the mature stage, and determining whether the inoculation is successful according to the disease infection expression of a standard variety.
The identification of the field phenotype of the corn stalk rot mainly comprises two methods of morbidity and disease grade, wherein the morbidity is the percentage of diseased plants in the total number of plants, and the disease grade is the damage degree of individual plants. If grades 1, 3, 5, 7, 9 represent the criteria for very weak, medium, strong, very strong 5 resistance assessments, respectively, then a phenotype of grade 1: the whole plant withers and falls down, the vascular bundle at the base of the stem is broken, and the grains are shriveled; grade 2 phenotype: the leaves of the plants have typical withered symptoms, the stem base parts obviously become soft but not fall, the fruit ears droop, and the seeds are not full; grade 3 phenotype: the leaves of the whole plant have typical withered symptoms, the base part of the stem is discolored and slightly soaked in water, and the ears are basically normal; phenotype of grade 4: withering symptoms appear on the whole plant leaves, the stem base grows normally, and the fruit ears grow normally; grade 5 phenotype: the whole plant grows normally, the leaf of the middle and lower part has the symptoms of withered or yellow withered, the stem base grows normally, and the fruit cluster grows normally.
Practice proves that planting disease-resistant varieties is an economic and effective fundamental measure for preventing and treating the disease, and the resistance sources of China are rich, thereby providing guarantee for breeding work and utilization of disease-resistant varieties. The resistance of the same maize inbred line to the stem rot can be kept at a certain resistance level only in different regions and under different natural conditions, the disease resistance of the maize inbred line is stable and reliable, and hybrids prepared by using the maize inbred line as a parent can have better disease resistance in production. Therefore, in conventional breeding, the resistance level of the breeding material to the stem rot can be determined by multi-point identification for many years, so that the time and the cost for variety breeding are greatly increased, and the breeding efficiency can be improved to a great extent by directly screening the breeding material with the stem rot resistance by a molecular marker means.
Corn stalk rot is now becoming one of the most devastating corn diseases in world corn producing areas, especially in colder areas, such as northern corn producing areas in China. There are many relevant studies and reports on how to better control maize stalk rot, and most of the studies indicate that maize stalk rot is a quantitative trait controlled by multiple QTLs (quantitative trait loci) with additive effects. The QTL which is obtained by positioning at present is distributed on other chromosomes except the 8 th chromosome and the 9 th chromosome, but the QTL which is obtained by fine positioning is few, and the reported research results show that the mechanism of the resistance action of the maize stalk rot is a very complex process and relates to biological pathways in various aspects. Considering the serious harmfulness and the very complex resistance mechanism of the corn stalk rot, more QTL or genes must be accurately positioned to explain the resistance mechanism of the corn stalk rot from the genetic point of view, and the resistance of the corn variety to the stalk rot is improved by developing a molecular marker-assisted breeding method, so that an excellent corn variety with high stalk rot resistance is cultivated.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a molecular marker related to resistance to corn stalk rot and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in the first aspect, 317333 high-quality SNP markers obtained by genome sequencing and screening and different maize inbred lines are utilized to perform whole genome association analysis on multi-point stem rot disease-resistant identification results of years in the field, and significant SNP loci related to the resistance to maize stem rot are developed through functional annotation and analysis screening.
Based on the research, the invention provides an SNP molecular marker combination related to the resistance of the corn stalk rot, and the molecular marker combination comprises the following 11 SNP molecular markers:
marking Chromosome of Site of the body Gene Alleles Resistance-associated alleles
S1_207099347
1 207099347 GRMZM2G043353 A/G A
S5_204577972 5 204577972 GRMZM2G317450 C/T C
S6_102712427
6 102712427 GRMZM2G099691 A/G A
S8_132714904
8 132714904 GRMZM2G005350 G/C G
S8_132722742 8 132722742 GRMZM2G005163 A/C A
S8_132792977 8 132792977 GRMZM2G075000 C/T C
S8_132809091
8 132809091 GRMZM2G102674 G/T G
S8_133476574
8 133476574 GRMZM2G141873 A/T A
S8_133615415 8 133615415 GRMZM2G047219 C/T C
S8_134605298 8 134605298 GRMZM2G098039 C/T C
S8_134884224 8 134884224 GRMZM2G175236 T/C T
The above information is based on the maize genome version: b73RefGen _ v3,2013/10/24.
In a second aspect, based on the research results, the invention provides the application of the molecular marker combination in the identification of resistance of maize stalk rot germplasm materials or detection of resistance genes.
And the application of the molecular marker combination in the maize stalk rot resistance marker-assisted breeding.
Specifically, the corn stalk rot resistance marker assisted breeding method comprises the step of detecting the molecular marker combination.
The method comprises the following specific steps:
(1) designing KASP marker according to the physical position of SNP site of the molecular marker and the base sequence near the site;
(2) extracting DNA of a sample to be detected, and carrying out amplification detection by using a designed KASP marker through an SNP parting device to obtain the genotype of an SNP locus;
(3) and selecting a sample with corn stalk rot resistance for breeding work.
The operations involved in the present invention are those conventional in the art unless otherwise specified.
The invention has the beneficial effects that:
the invention utilizes sequencing data and multi-year-multipoint phenotypic data to carry out correlation analysis to obtain the SNP locus related to the corn stalk rot, further develops and develops the SNP molecular marker related to the resistance of the corn stalk rot, and improves the detection accuracy by more than 90 percent through a molecular marker combination detection method.
The invention also provides a candidate genome interval for cloning the candidate disease-resistant gene, identifies a chromosome segment related to the stem rot disease resistance, and provides a basis for further gene cloning and utilization.
The molecular marker developed by the invention is used for auxiliary breeding, so that the rapid identification of resistance can be realized, and the field test cost is reduced, thereby saving the breeding cost and accelerating the breeding process.
Drawings
FIG. 1 is a graph showing the result of dividing the Structure optimal subgroup (K value) in example 2 of the present invention.
FIG. 2 is a graph showing the result of dividing the Structure optimal sub-groups (K values) in example 2 of the present invention.
FIG. 3 is a diagram illustrating the Structure partition result in embodiment 2 of the present invention.
Fig. 4 is a graph of the cluster analysis result in embodiment 2 of the present invention.
FIG. 5 is a graph showing the results of analysis of linkage disequilibrium distance per chromosome (LD distance) of the related population in example 2 of the present invention.
FIG. 6 is a graph showing the results of Principal Component Analysis (PCA) in example 2 of the present invention.
Fig. 7 is a model prediction chart (QQ chart) of the correlation analysis result in embodiment 2 of the present invention.
Fig. 8 is a manhattan chart (Manhaten scattergram) of the correlation analysis result in example 2 of the present invention.
FIG. 9 shows the differences in the genotypes of the SNP sites in the extreme susceptor material according to example 3 of the present invention.
FIG. 10 is a diagram showing the results of correlation analysis of candidate genes in example 4 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 SNP marker development pathway associated with disease resistance of Stem rot
1) Sorting and analyzing phenotype data;
2) sorting and screening genotype data;
3) performing correlation analysis by combining the phenotype data and the genotype data;
4) the detailed analysis and functional annotation are carried out on the result of the correlation analysis to obtain the stability
5) Developing SNP markers according to the SNP sites, and verifying the functionality of the markers.
Example 2 correlation of analytical results
1) Results of population Structure analysis
Firstly, the Structure of the associated population is predicted and divided by Structure software (figures 1-3), clustering analysis and linkage disequilibrium analysis are carried out by the Tassel software (figures 4-5), PCA analysis is carried out by the Tassel software and the R software (figure 6), and the result shows that the population is suitable for being used as the population for association analysis.
2) Correlation analysis result chart
Firstly, the results of multi-point phenotype identification for years are subjected to variance analysis and normal distribution analysis, the results show that phenotype data are in normal distribution, the inheritance rate is close to 90%, and the phenotype data are proved to be slightly influenced by environmental factors. Then, the Tassel 5.0 software is used for screening genotype data, the Tassel software is used for association analysis by adopting a composite linear model to obtain an expected model QQ diagram and a series of significant SNP sites (figures 7 and 8), websites such as MaizeGDB and the like and related software are used for screening analysis and functional annotation of candidate genes on association analysis results to finally obtain candidate SNP sites (Table 1).
TABLE 1 significant SNP sites obtained by multi-point association analysis over years
Figure BDA0001392752920000061
Example 3 genotype differences in the extremely resistant Material at SNP sites
Extreme maize inbred line materials which show high resistance or high sense in multiple environments are selected, genotype differences of the extreme maize inbred line materials at target SNP sites are compared, as can be seen from figure 9, candidate SNP sites show obvious differences in anti-sense materials, meanwhile, as can be seen from table 9, genotypes of extreme disease-resistant materials are almost completely consistent, genotypes of extreme disease-sensitive materials have some differences, many disease-sensitive materials carry some disease-resistant sites, and according to the results of all genotypes and phenotypes of multiple points in multiple years, 145 excellent maize inbred line materials carry more disease-resistant sites, especially extreme disease-resistant materials, so that the situation that many individual disease-resistant materials simultaneously carry more disease-resistant sites can also occur, for example, the disease-sensitive material PHN37 only carries one disease-sensitive site, but the situation is just a few examples. Meanwhile, all materials with more susceptible sites are shown as more susceptible materials or extremely susceptible materials under different environments, and the evidence is combined to show that the sites are most likely to be susceptible sites, namely, the susceptible sites are used for judging whether the materials are susceptible or not, the accuracy of identifying the disease resistance is higher, and the accuracy of identifying the susceptible materials in the identified materials is close to 100%. The resistance of the corn stalk rot is simultaneously controlled by a plurality of quantitative character sites, and the high disease resistance of a corn inbred line cannot be guaranteed by only identifying individual disease-resistant sites by 100 percent, so that the obtained SNP sites are required to be fully utilized, and the accuracy and the authenticity of the phenotype prediction are guaranteed by a way of jointly utilizing a plurality of SNP markers.
According to the results of 5 environment and different physiological race phenotype identifications, 15 materials with stronger resistance and 15 materials with weaker resistance (about 10% of the total number of materials at each station) are respectively taken, the percentage of the total number of sites of the number station, which contains disease-resistant sites, of the material with stronger resistance is obtained to be between 93.3% and 97.0%, and the percentage of the total number of sites, which contains disease-resistant sites, of the material with stronger resistance is obtained to be between 46.1% and 58.8%. If the influence caused by the environment and other factors is eliminated, the difference is definitely larger, for example, the extremely susceptible material identified by any environment is necessarily the extremely susceptible material in other environments if the extremely susceptible material carries a large number of susceptible sites, and the identification result of the extremely susceptible material in other environments is probably a more resistant material if the extremely susceptible material carries a large number of resistant sites, on the other hand, the SNP sites are very likely to be the susceptible sites, namely, the material is highly likely to be the susceptible material if the material carries more susceptible sites, and the conclusion is consistent with the identification results of all materials in all environments.
Example 4 functional annotation and analysis of candidate genes
11 candidate genes MaizeGDB (http:// www.maizegdb.org /) corresponding to the maize genome are obtained according to the 11 candidate sites obtained by analysis, the candidate genes are subjected to preliminary functional annotation through related websites, article reports and the like, the candidate genes comprise homologous genes of which the expression sites correspond to arabidopsis thaliana and rice, the functions of the candidate genes in maize can be predicted through related research of the homologous genes, and summary analysis is carried out according to different relativity (figure 10). Firstly, according to the expression condition of the genes in corn, we show that 7 genes are expressed in the root, stem base, phloem and other parts of the corn which are possibly related to the resistance of the corn stalk rot, and in addition, two genes GRMZM2G005350 and GRMZM2G102674 are consistent with a transcript analysis result of fusarium verticillium which is one of the corn stalk rot pathogenic bacteria. According to the functional analysis of homologous genes, four genes are involved in the process of plant stress-resistant response, wherein an expression product bHLH binding protein of a homologous gene AT5G08130.2 of GRMZM2G317450 in Arabidopsis is involved in the regulation process of a plant cold-resistant mechanism, a homologous gene AT3G18690.1 of GRMZM2G099691 in Arabidopsis is involved in the regulation process of the plant stress-resistant mechanism, an expression product WRKY transcription factor of the GRMZM2G099691 is involved in the processes of plant disease resistance, injury, aging-related genes, salicylic acid induction and the like, a homologous gene AT3G55270.1 of GRMZM2G005350 in Arabidopsis is also involved in the synthesis process of salicylic acid to regulate the plant stress-resistant process, the research of the homologous gene AT2G37660.1 of GRMZM2G047219 in Arabidopsis directly shows that the plant stress-resistant response to bacteria is generated, one of the pathogenic bacteria of maize stalk rot is called Erwinia chrysanthemi maize pathogenic bacteria, and the research of gene function of the maize variety predicts that the several sites can play a certain role in the regulation of maize stalk rot resistance, laying a foundation for the research of the genetic mechanism of the corn stalk rot and the development of molecular markers.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (4)

1. The application of SNP molecular marker combination related to corn stalk rot resistance in identifying resistance of germplasm materials of corn stalk rot disease, wherein the molecular marker combination comprises the following 11 SNP molecular markers:
marking Chromosome of Site of the body Gene Alleles Resistance-associated alleles S1_207099347 1 207099347 GRMZM2G043353 A/G A S5_204577972 5 204577972 GRMZM2G317450 C/T C S6_102712427 6 102712427 GRMZM2G099691 A/G A S8_132714904 8 132714904 GRMZM2G005350 G/C G S8_132722742 8 132722742 GRMZM2G005163 A/C A S8_132792977 8 132792977 GRMZM2G075000 C/T C S8_132809091 8 132809091 GRMZM2G102674 G/T G S8_133476574 8 133476574 GRMZM2G141873 A/T A S8_133615415 8 133615415 GRMZM2G047219 C/T C S8_134605298 8 134605298 GRMZM2G098039 C/T C S8_134884224 8 134884224 GRMZM2G175236 T/C T
The above information is based on the maize genomic version of the database MaizeGDB (http:// www.maizegdb.org /): b73RefGen _ v3,2013/10/24.
2. The use of a SNP molecular marker combination related to resistance to maize stalk rot in the auxiliary breeding of maize stalk rot resistance markers, wherein the molecular marker combination is as defined in claim 1.
3. A method for assisting in breeding corn stalk rot resistance markers, which comprises the step of detecting a combination of SNP molecular markers associated with corn stalk rot resistance, wherein the combination of molecular markers is as set forth in claim 1.
4. A method according to claim 3, characterized by the steps of:
(1) designing KASP markers according to the physical positions of the SNP sites of the molecular marker combination and the base sequences near the sites;
(2) extracting DNA of a sample to be detected, and carrying out amplification detection by using a designed KASP marker through an SNP parting device to obtain the genotype of an SNP locus;
(3) and selecting a sample with corn stalk rot resistance for breeding work.
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CN108893550B (en) * 2018-07-10 2021-08-10 北京市农林科学院 Development and application of SNP molecular marker related to resistance of corn stalk rot
CN109234431B (en) * 2018-09-27 2022-11-01 北京大北农生物技术有限公司 Molecular marker of corn stalk rot resistance QTL and application thereof
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