A molecular marker of a gene related to corn flowering period and application thereof

Abstract

The present application relates to a kind of molecular marker and application related to corn flowering period gene.The present application is mined to the SNP site significantly related to flowering period by the method of whole genome association analysis, and further develops molecular marker ME1.The molecular amplification product 645 bases are G in early flowering material, and the amplification product 645 bases are T in late flowering multi-leaf material.The marker can significantly affect corn flowering period, but has no significant effect on kernel size.Therefore, the present application provides a new technical means for screening high-quality high-yield early-maturing corn materials.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular marker of a gene related to the flowering period of corn and application thereof, and belongs to the field of corn breeding and molecular biology. BACKGROUND

[0002] Maize is an annual grass, and the time from sowing to maturity varies greatly with geographical distribution, from 35 days to 120 days. Compared with temperate maize, maize in tropical and subtropical regions has rich genetic variation, and has excellent traits such as high plant type, strong stress resistance and high yield. Flowering time, as an important trait, has a great influence on the maturity period, seed setting rate, yield and environmental adaptability of maize (Colasanti J, et al. (2006) The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants [J]. BMC Genomics 7: 158-162; Tallury S, Goodman M. (1999) Experimental evaluation of the potential of tropical germplasm for temperate maize improvement [J]. Theoretical and Applied Genetics 98: 54-61; Buckler ES, et al. (2009) The genetic architecture of maize flowering time. Science 325: 714-718; Swarts K, et al. (2017) Genomic estimation of complex traits reveals ancient maize adaptation to temperate North America. Science 357: 512-515.). Flowering time is one of the important target traits in maize breeding, and is closely related to the regional distribution and seasonal adaptation of maize, directly or indirectly affecting yield, stress resistance and disease resistance and other agronomic traits. If tropical maize is directly planted in temperate regions without artificial improvement, it will lead to significant delay in flowering time or even no flowering and seed setting. The flowering time of maize adapted to the regional climate can make the most of local light and temperature resources, avoid stress risks, and thus improve maize yield.Therefore, it is of great significance to study the flowering regulation mechanism of maize for breeding excellent varieties and germplasm dissemination worldwide (Oyervides M, et al. (1985) Evaluation of improved maize populations in Mexico and the US Corn Belt 1 [J]. Crop Science 25: 115-120; Jung C, Muller AE. (2009) Flowering time control and applications in plant breeding [J]. Trends in Plant Science 14: 563-566; Sun H, et al. (2020) dlf1 promotes floral transition by directly activating ZmMADS4 and ZmMADS67 in the maize shoot apex [J]. New Phytologist 228: 1386-1400.).

[0003] With the development of molecular marker technology, QTL (quantitative trait locus, QTL) positioning combined with phenotype trait data and DNA markers can effectively detect chromosome segments and quantity effects linked to target traits. In recent years, the emerging genome-wide association study (GWAS) combined with deeper sequencing depth and wider genome coverage can accurately locate single nucleotide polymorphism sites in the whole genome, which greatly helps to further understand the genetic regulation mechanism of corn flowering date. At present, the second and third generation sequencing technology is becoming mature, and the association analysis through the whole genome level has higher positioning accuracy and has been widely used in the research of important agronomic traits of plants (Myles S, et al. (2009) Association mapping: critical considerations shift from genotyping to experimental design [J]. The Plant Cell 21: 2194-2202; Patrick F, Byrne. (2005) Quantitative trait locus (QTL) analysis [J]. Journal of Natural Resources and Life Sciences Education 34: 121-125.). SUMMARY

[0004] One of the purposes of the present application is to provide a molecular marker of a gene related to corn flowering date.

[0005] Another purpose of the present application is to provide the application of the molecular marker of the gene related to corn flowering date.

[0006] The primer pair for amplifying the molecular marker ME1 of the gene related to corn flowering date, wherein the sequence of the forward primer of ME1 is SEQ ID NO. 1, and the sequence of the reverse primer is SEQ ID NO. 2.

[0007] The molecular marker of the gene related to corn flowering date, characterized in that the related gene is located on the first chromosome of corn, the molecular marker linked to the gene is ME1, the molecular marker is obtained by amplifying the primer pair, and the length of the amplified fragment is 901 bp.

[0008] A molecular marker related to corn flowering period, the molecular marker is a SNP site obtained by PCR amplification using a primer pair and taking corn genome as a template, and there are three SNP sites, wherein the first and third SNP sites are synonymous mutations through amino acid analysis, so the second SNP site is analyzed, and the genotype of the late flowering material is T at the 645th site of the amplification product, and the genotype of the early flowering material is G. The sequence of the primer pair is as follows:

[0009] SEQ ID NO. 1: 5' CCAGCCCTCATTCCTTGCA-3',

[0010] SEQ ID NO. 2: 5' GGCAGGCTGAAGTCCAGATT-3'.

[0011] The primer pair for obtaining the molecular marker related to corn flowering period is also within the protection scope of the present application, and the sequence of the primer pair is as follows:

[0012] SEQ ID NO. 1: 5' CCAGCCCTCATTCCTTGCA-3',

[0013] SEQ ID NO. 2: 5' GGCAGGCTGAAGTCCAGATT-3'.

[0014] The present application mines the SNP site significantly related to flowering period through the method combining association analysis and linkage analysis, and further develops a molecular marker. The marker can significantly affect the flowering period of corn. Therefore, the present application provides a new technical means for screening high-quality, high-yield and early-maturing corn materials. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The whole genome association analysis is positioned to the significant site 1:53369716,

[0016] wherein A is the anthesis phenotype analysis, B is the anthesis phenotype whole genome association analysis,

[0017] Figure 2 Haplotype analysis of ZmEIL3 polymorphism site,

[0018] Figure 3 Analysis of the effect of ZmEIL3 on the anthesis and ear weight phenotypes.

[0019] Figure 4 Analysis of the effect of ZmEIL3 on the anthesis.

[0020] Wherein Hap 1, 2 are two haplotypes, a, b, c represent difference, multiple comparisons are carried out by using minimum significant difference method, if there is significant difference between two groups at p<0.05 level, different lowercase letters are marked. If there is same letter mark, it indicates that there is no significant difference between two groups. DETAILED DESCRIPTION

[0021] The advantages and features of the present application will become more apparent with the description of the specific embodiments. The embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0022] A molecular marker of a corn flowering period related gene, the related gene is located on the first chromosome of corn, and flowering period traits of 369 corn inbred materials are investigated for three years in Gansu. The present application integrates the advantages of association analysis and linkage analysis, determines reliable QTL confidence interval by using linkage analysis in multiple environments, determines candidate genes and functional sites by using extensive variation germplasm resources through association analysis, and further develops a molecular marker.

[0023] The molecular marker linked to the gene is ME1, and the primer pair for amplifying the molecular marker of the corn flowering period related gene, wherein the sequence of the forward primer of ME1 is SEQ ID NO. 1, and the sequence of the reverse primer is SEQ ID NO. 2. The molecular marker amplification product in the late flowering material of flowering period is T, indicating that the material has the existence of late flowering allele, showing late flowering phenotype; the amplification product in the early flowering material is G, indicating that the material has early flowering allele, showing early flowering phenotype.

[0024] Specifically, the molecular marker provided by the present application is obtained by the following method:

[0025] Genome-wide association study: 369 maize inbred lines from domestic and abroad, tropical / subtropical and temperate, are all conventional materials without transgenic components. Using second-generation sequencing technology, whole-genome sequencing of leaf tissue at the trumpet stage was performed, generating 6034.2 Gb of raw data. After filtering by sequencing depth > 2, minimum allele frequency > 0.05, and deletion rate < 0.25, in addition to the further filtering criteria for SNP markers used in genome-wide association study (GWAS) as follows: 1. deleting the long sequence fragments (Scaffolds) assembled from overlapping groups generated during splicing; 2. removing SNP sites with a missing rate greater than 20%; 3. retaining SNP sites with a minor allele frequency (MAF) less than 5%. A total of 3997283 high-quality SNP sites were identified in 369 different maize germplasm resources. Association analysis using a mixed linear model (MLM) that considers population structure and kinship found that 17 significant sites were associated, with three sites (3:53900672, 6:83651448, and 7:117480970) having higher significance. Combining B73_RefGen_V4.36 reference genome information, only one of the 17 sites was located in the gene region, with a physical location of 1:53369716, falling on the Zm00001d028974 (ZmEIL3) gene on chromosome 1. Figure 1 B) in the middle.

[0026] Haplotype analysis: Selecting approximately 3Kb upstream and downstream of the target gene, and combining the BLUP data of 2018, 2019, 2020, and 3-year phenotypes, candidate segment association analysis was performed, resulting in 4 significant SNPs sites regulating the shedding stage phenotype, all located in the exon region of the target gene. The 3 single nucleotide polymorphism variation sites have a certain degree of linkage disequilibrium, and the relatively independent (R 2 <0.8) polymorphic sites were selected for haplotype division. To ensure the reliability of the statistical results, when the haplotype frequency was less than 0.02, it was ignored. Among the 369 inbred lines, 326 were HapⅠ(ZmEIL3 C / G / G ) haplotype, 26 were HapⅡ(ZmEIL3 A / T / T ) haplotype, and the frequencies of the other haplotypes were low and had no significant effect on the phenotype, so they were not considered. The shedding stage and spinning stage of HapⅠ haplotype materials were significantly earlier than those of HapⅡ haplotype materials, so HapⅠ(ZmEIL3 C / G / G ) was defined as an early-flowering haplotype, and HapⅡ(ZmEIL3 A / T / T) is a late-flowering haplotype Figure 2 )。

[0027] Molecular marker development: using Primer Premier 5 to design a primer pair for 1:53369716 based on the reference sequence of corn inbred line B73, the length of the amplified fragment is 901bp, and the specific primer pair sequence is as follows:

[0028] Upstream primer: 5' CCAGCCCTCATTCCTTGCA-3'

[0029] Downstream primer: 5'GGCAGGCTGAAGTCCAGATT-3'

[0030] Example 1 Determination of molecular marker

[0031] A molecular marker associated with a corn flowering period-related gene, the related gene is located on the first chromosome of corn, and the genotypes of 200 single plants in the F2 population constructed by early-flowering line B73 and late-flowering line C319 are identified, wherein 48 plants are early-flowering haplotypes and 45 plants are late-flowering haplotypes. The flowering period phenotype is counted, and the phenotype trait separation in the population is observed, and the results show that there is a significant difference between the anthesis period of the homozygous late-flowering haplotype and the anthesis period of the early-flowering haplotype. In addition, the ear weight of the two haplotype materials is investigated and counted respectively, and it is found that the material with early-flowering haplotype has higher ear weight than the material with late-flowering haplotype Figure 3 )。

[0032] Example 2 Screening of high-yield and early-maturing germplasm resources of corn

[0033] The molecular marker ME1 of the present application is used to screen 369 corn germplasms for early-flowering germplasm. The 369 germplasms are planted in the field in Jiuquan, Gansu, and the DNA is extracted from the leaves at the seedling stage, and the ME1 primer pair is used for extension sequencing, and it is found that 1:53369716 is a T genotype material of 26 parts, and a G genotype material of 326 parts. The flowering period is counted when the plants shed pollen and spin silk. It is found through counting that the 26 materials with genotype T have a shedding period of 88.9 days, and the materials with genotype G have a shedding period of 86.6 days, so the materials with genotype T all show a late-flowering phenotype, and the materials with genotype G show an early-flowering phenotype Figure 4 ) The marker can be used for screening of early-flowering corn germplasm.

Claims

1. The application of primer pairs for amplifying the molecular marker ME1, a gene related to maize flowering time, in the screening or identification of germplasm resources with early or late flowering time traits, characterized in that: The SNP site obtained by PCR amplification using a primer pair and taking the corn genome as a template is at the 645th position of the amplification product, the genotype of the late flowering material is T, and the genotype of the early flowering material is G, and the sequences of the primer pair are as follows: SEQ ID NO. 1: 5' CCAGCCCTCATTCCTTGCA-3', SEQ ID NO. 2: 5'GGCAGGCTGAAGTCCAGATT-3'.

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