SNP (Single Nucleotide Polymorphism) molecular marker closely linked with sweet corn kernel weight major QTL (Quantitative Trait Loci) and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker closely linked with a sweet corn kernel weight major QTL (Quantitative Trait Loci) and application of the SNP molecular marker, and belongs to the technical field of SNP molecular markers. The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, the mutation site is the base of the 242797157th site on the first chromosome of a B73 reference genome (RefGenv4), the base of the site is T, and the sweet corn with the genotype of TT shows the high grain weight character; the basic group of the site is C, and the sweet corn with the genotype of CC shows the character of low grain weight. Based on the SNP molecular marker, a method for identifying the sweet corn grain weight character and a method for screening the sweet corn with the high grain weight character are provided, and the methods are high in accuracy, high in detection speed, easy in result interpretation and low in cost and have important significance in improvement of corn breeding efficiency and large-scale high-yield planting of corn.

Description

Technical Field

[0001] The present invention relates to molecular marker detection technology, and particularly relates to an SNP molecular marker closely linked to a major QTL for sweet corn kernel weight and its application. Background Art

[0002] Maize (Zea mays) belongs to the genus Zea of the tribe Maydeae in the family Poaceae. It has high yield and strong adaptability and is widely planted in temperate and tropical regions. It is an important food crop and also an important feed and industrial raw material. Fresh-eating maize refers to the general term for special maize mainly for eating fresh ears at the milk-ripe stage, including sweet corn, waxy corn and sweet-waxy corn. With the improvement of people's living standards and the change of diet structure, fresh-eating maize has entered the diet structure of the people in our country as a vegetable and is deeply loved by people. This makes how to improve the yield of fresh-eating maize an important research and development direction in this field.

[0003] The maize kernel weight trait refers to the characteristics shown by maize kernels in terms of weight. It is one of the important components of maize yield and also one of the main economic traits of fresh-eating maize. The maize kernel weight trait is usually measured by the thousand-kernel weight or the hundred-kernel weight, that is, the weight of 1000 or 100 maize kernels, and the unit is generally grams, which can reflect the size, plumpness and potential nutritional value of maize kernels, etc. In addition to external factors such as environmental factors and cultivation management measures, the maize kernel weight is mainly determined by genetic genes, that is, there are significant differences in the kernel weight trait among different maize varieties. However, at present, the breeding of fresh-eating maize in our country, especially sweet corn, mainly relies on phenotypic selection, which has high cost, low efficiency and long cycle. At the same time, after sweet corn matures, the kernels shrink, which increases the difficulty of collecting kernel traits. And there are few research reports on the excavation of molecular markers for sweet corn. Therefore, providing SNP molecular markers related to the sweet corn kernel weight trait and using them in the breeding of sweet corn is of great significance in improving the breeding efficiency of sweet corn and the large-scale high-yield planting of sweet corn. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an SNP molecular marker closely linked to a major QTL for sweet corn kernel weight, which promotes the molecular breeding process of fresh-eating maize kernel-related traits.

[0005] The purpose of the present invention also lies in providing a method for identifying the sweet corn kernel weight trait and a method for screening sweet corn with high kernel weight traits. The methods have high accuracy, fast detection speed, easy result interpretation and low cost, and are of great significance in improving the breeding efficiency of maize and the large-scale high-yield planting of maize.

[0006] In order to achieve the above invention purposes, the present invention provides the following technical solutions:

[0007] The present invention provides an SNP molecular marker closely linked to the major QTL of sweet corn kernel weight, and the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1; when the base at the 142nd position from the 5'-end of the nucleotide sequence shown as SEQ ID NO.1 is T, the sweet corn exhibits the trait of high kernel weight; when the base at the 142nd position from the 5'-end of the nucleotide sequence shown as SEQ ID NO.1 is C, the sweet corn exhibits the trait of low kernel weight.

[0008] The present invention also provides the application of the above SNP molecular marker in identifying the sweet corn kernel weight trait.

[0009] The present invention also provides the application of the above SNP molecular marker in screening or breeding sweet corn with the trait of high kernel weight.

[0010] The present invention also provides a primer pair for detecting the above SNP molecular marker, the sequence of the upstream primer is shown as SEQ ID NO:2, and the sequence of the downstream primer is shown as SEQ ID NO:3.

[0011] The present invention also provides a kit for identifying the sweet corn kernel weight trait, including a DNA extraction kit, a PCR amplification kit and a sequencing kit; the PCR amplification kit includes the above primer pair.

[0012] The present invention also provides the application of the above primer pair or kit in identifying the sweet corn kernel weight trait.

[0013] The present invention also provides the application of the above primer pair or kit in screening or breeding sweet corn with the trait of high kernel weight.

[0014] The present invention also provides a method for identifying the sweet corn kernel weight trait, including the following steps: using the genomic DNA of the sweet corn to be tested as a template, performing PCR amplification with the above primer pair to obtain a PCR product; sequencing the PCR product; when the base at the 142nd position of the PCR product is T and the genotype is TT, it exhibits the trait of high kernel weight; when the base at the 142nd position of the PCR product is C and the genotype is CC, it exhibits the trait of low kernel weight.

[0015] The present invention also provides a method for screening sweet corn with the trait of high kernel weight, the method includes: using the genomic DNA of the sweet corn to be tested as a template, performing PCR amplification with the above primer pair to obtain a PCR product; sequencing the PCR product; selecting and retaining the individuals with the base at the 142nd position of the PCR product being T and the genotype being TT.

[0016] The present invention also provides the application of the above method in sweet corn breeding.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0018] The present invention provides an SNP molecular marker closely linked to the major QTL of sweet corn kernel weight. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1. The mutation site of the SNP molecular marker of the present invention is at the base at position 242797157 on chromosome 1 of the B73 reference genome (RefGen_v4). When the base at the 142nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is T, that is, the base at position 242797157 on chromosome 1 is T, the genotype of sweet corn is TT, showing the trait of high kernel weight; when the base at the 142nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is C, that is, the base at position 242797157 on chromosome 1 is C, the genotype of sweet corn is CC, showing the trait of low kernel weight. The present invention verified that the kernel weight of sweet corn with the genotype TT is significantly higher than that of individuals with the genotype CC (P<0.01).

[0019] Based on the SNP molecular marker, the present invention further provides a method for identifying the trait of sweet corn kernel weight and a method for screening sweet corn with high kernel weight trait. The method has high accuracy, fast detection speed, easy result interpretation, low cost. By only detecting the genotype of a specific locus, the hundred-kernel weight of sweet corn can be predicted. It has the advantages of clear selection target, early detection can be achieved and is not affected by the environment, and is of great significance in improving the breeding efficiency of sweet corn and large-scale high-yield planting of sweet corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the heat map of the haplotype analysis result of the candidate region;

[0021] Figure 2 is the T-test of the hundred-kernel weight of sweet corn with TT genotype and CC genotype. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention provides an SNP molecular marker closely linked to the major QTL of sweet corn kernel weight. The nucleotide sequence of the SNP molecular marker is

[0023]

[0024] (The bold and underlined site is the mutation site, which is the 142nd position of this sequence), as shown in SEQ ID NO.1.

[0025] Through QTL analysis, the present invention found that there is a major QTL on chromosome 1 of the B73 reference genome (RefGen_v4), located between 242,637,476 bp and 243,390,300 bp, with a contribution rate of 10.07% to the 100-kernel weight phenotype of sweet corn. By using candidate region association analysis, the present invention obtained an SNP molecular marker closely linked to the major QTL for sweet corn kernel weight. This locus is S1_242797157, and the base at this locus is T. That is, when the 142nd base from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is T, the genotype of sweet corn is TT, and sweet corn exhibits the trait of high kernel weight; when the base at this locus is C, that is, when the 142nd base from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is C, the genotype of sweet corn is CC, and sweet corn exhibits the trait of low kernel weight. The present invention verified that the kernel weight of sweet corn with the genotype TT is significantly higher than that of individuals with the genotype CC (P < 0.01).

[0026] The sweet corn varieties of the present invention include but are not limited to SHL01, SHL03, SHL10, and SHL11.

[0027] The present invention also provides the application of the above SNP molecular marker in identifying the kernel weight trait of sweet corn, screening sweet corn with high kernel weight traits, or breeding sweet corn with high kernel weight traits. That is, the present invention can predict the 100-kernel weight of sweet corn by detecting the base type at the S1_242797157 locus.

[0028] The present invention also provides a primer pair for detecting the above SNP molecular marker. The upstream primer is F: 5'-AACCACAAGATCCCAAATACCA-3', and the sequence is as shown in SEQ ID NO: 2. The sequence of the downstream primer is 5'-GAGAAGGGTAAAACCGAGCC-3', and the sequence is as shown in SEQ ID NO: 3. Using the genomic DNA of sweet corn to be identified as a template, the primer pair can be used to amplify a sequence containing the above mutation site, including the sequence shown in SEQ ID NO.1, and the amplified fragment length is 330 bp for subsequent sequencing, so as to judge the genotype of sweet corn and the kernel weight trait of sweet corn.

[0029] The present invention also provides a kit for identifying the kernel weight trait of sweet corn, including a DNA extraction kit, a PCR amplification kit, and a sequencing kit; the PCR amplification kit includes the above primer pair. Preferably, the kit further includes a standard positive template, and the standard positive template can be Marker or the nucleotide shown in SEQ ID NO.1. The present invention does not limit the NDA extraction kit and the sequencing kit, and conventional kits in the art can be used.

[0030] The present invention also provides the use of the primer pair or kit in identifying the sweet corn kernel weight trait, screening sweet corn with high kernel weight trait or breeding sweet corn with high kernel weight trait. That is, the present invention can determine the base type of the sweet corn to be tested at the site by detecting the nucleotide sequence containing the S1_242797157 site, so as to achieve the identification of the sweet corn kernel weight trait, and then screen or breed sweet corn with high kernel weight trait.

[0031] The present invention also provides a method for identifying the sweet corn grain weight trait, comprising the following steps: using the sweet corn genomic DNA to be tested as a template, performing PCR amplification using the above primer pair to obtain a PCR product; sequencing the PCR product; the 142nd base of the PCR product is T, the genotype is TT, and it exhibits a high grain weight trait; the 142nd base of the PCR product is C, the genotype is CC, and it exhibits a low grain weight trait.

[0032] The present invention also provides a method for screening sweet corn with high grain weight trait, the method comprising: using the genomic DNA of the sweet corn to be tested as a template, performing PCR amplification using the above primer pair to obtain a PCR product; sequencing the PCR product; and selecting individuals whose base at position 142 of the PCR product is T and whose genotype is TT.

[0033] The PCR amplification system of the present invention is preferably: 25 μL 2×Taq Master Mix, 2 μL left primer (10 μM), 2 μL right primer (10 μM), 2 μL template DNA, ddH 2 O is used to fill in 50 μL. The PCR amplification procedure is preferably: 95°C pre-denaturation for 3 minutes, followed by 35 cycles (95°C for 15 seconds, 60°C for 15 seconds and 72°C for 30 seconds), and finally 72°C thorough extension for 5 minutes to obtain a PCR product. The present invention detects the obtained PCR product by agarose gel electrophoresis, and after the detection is qualified, it is sequenced, preferably by direct sequencing. The present invention analyzes the sequencing results. If the 142nd base from the 5' end of the PCR product has T, its genotype is a TT individual; if the 142nd base from the 5' end of the PCR product is C, its genotype is a CC individual.

[0034] The present invention also provides the application of the above method in sweet corn breeding, wherein the application is preferably molecular polymer breeding of traits.

[0035] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] In the following embodiments, unless otherwise specified, all are conventional methods.

[0037] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0038] Example 1

[0039] 1. Construction of the genetic map of the sweet corn recombinant inbred lines (RIL) population

[0040] The backbone inbred lines SHL01 and SHL03 of sweet corn were crossed and self-crossed for 6 generations to construct an RIL population with a population size of 232. The RIL population was planted in the Zhuanghang Base of the Shanghai Academy of Agricultural Sciences. At the seedling stage, the leaves of the RIL were sampled for DNA extraction. Genotyping was performed using a liquid-phase chip, and a total of 4199 SNP loci were obtained. Quality control was carried out using the vcftools software, including a missing rate of less than 20%, a heterozygosity rate of less than 20%, and a minor allele frequency of greater than or equal to 0.05. A total of 4199 SNPs were retained. The genetic map was constructed using the ICIMapping software. The total size of the genetic map was 2243.84 cM, and the average genetic distance between SNP markers was 0.53 cM, as shown in Table 1.

[0041] Table 1 Statistical information of the genetic map

[0042] Chromosome Number of SNPs Length (cM) 1 751 419.1995 2 476 341.5352 3 475 203.3578 4 421 179.5186 5 456 208.2899 6 340 146.6081 7 317 155.8949 8 319 294.9602 9 339 158.3638 10 305 136.114 Total 4199 2243.842

[0043] 2. Measurement of the 100-kernel weight of sweet corn kernels

[0044] The RIL population was planted in the Zhuanghang Base of the Shanghai Academy of Agricultural Sciences and the Lingshui Base in Hainan, and a randomized block experimental design was adopted. Each RIL material was planted in 2 rows, with 3 replicates, and normal field management was carried out. After the RIL materials' kernels matured, an automatic seed grader was used to measure the 100-kernel weight. Using the Meta-R software, based on two environments and three replicates, the BLUP value of the 100-kernel weight of the RIL materials was calculated for subsequent QTL identification.

[0045] 3. Obtaining the major QTL for the 100-kernel weight trait of sweet corn

[0046] Using ICI Mapping software, quantitative trait locus (QTL) analysis of 100-kernel weight was performed on the RIL population based on composite interval mapping. A total of 1000 random tests were conducted at a significance level of P<0.05 to determine the logarithm of the odds (LOD) of the likelihood ratio. The 2.5-LOD interval method was used to determine the confidence interval of the QTL. A total of two QTLs were identified. Among them, qHKW1-1 located at 242,637,476 bp to 243,390,300 bp on chromosome 1 had a contribution rate of more than 10%, which was a major QTL, as shown in Table 2. The other, qHKW10-1 located at 136,130,642 bp to 136,965,507 bp on chromosome 10, had a contribution rate of 5.82% and was a minor QTL. The major QTL qHKW1-1 was selected for molecular marker development.

[0047] Table 2 Results of QTL for 100-kernel weight in sweet corn

[0048] QTL Name Chromosome Start Position (bp) End Position (bp) LOD Contribution Rate (%) qHKW1-1 1 242637476 243390300 5.94 10.07 qHKW10-1 10 136130642 136965507 3.45 5.87

[0049] 4. Candidate region association analysis and molecular marker development

[0050] A total of 166 sweet corn inbred lines were selected for candidate region genome-wide association analysis. The sweet corn population was planted in the Zhuanghang Base of Shanghai Academy of Agricultural Sciences and the Lingshui Base in Hainan, and the 100-kernel weight phenotype was measured. The best linear unbiased prediction (BLUP) value was calculated using Meta-R software. Genotype identification was performed by whole-genome resequencing. In the qHKW1-1 region, a total of 2800 single nucleotide polymorphism (SNP) loci were identified. The candidate region association analysis was performed using the general linear model (GLM) of Tassel5 software. A threshold of 1 / 2800 was used, i.e., p<3.4E-4. An SNP locus S1_242797157 significantly associated with the 100-kernel weight of sweet corn was identified, which was located at 242,797,157 bp on chromosome 1 of the B73v4 genome. The p-value of S1_242797157 in the association analysis results was 1.0214E-4, as Figure 1 shown. The S1_242797157 locus contained two allelic genotypes, namely TT and CC, in the association analysis population. Primers for the S1_242797157 locus were designed using primer3 software. The upstream primer was F: 5’-AACCACAAGATCCCAAATACCA-3’, and the sequence of the downstream primer was 5’-GAGAAGGGTAAAACCGAGCC-3’. The amplified sequence was

[0051] The marker locus was located at position 142 of the sequence.

[0052] Example 2

[0053] A method for identifying the kernel weight trait of sweet corn, comprising the following steps:

[0054] Extract the genomic DNA of 160 sweet corn inbred lines (from the Zhuanghang Base of the Shanghai Academy of Agricultural Sciences and the Lingshui Base in Hainan) using a genomic extraction kit, and use the extracted sweet corn genomic DNA as a template to perform PCR amplification on the marker sequence using primer pairs. The PCR amplification system is: 25 μL of 2× Taq Master Mix, 2 μL of left primer (10 μM), 2 μL of right primer (10 μM), 2 μL of template DNA, and ddH 2 O is supplemented to 50 μL. The PCR amplification program is: pre-denaturation at 95 °C for 3 mins, followed by 35 cycles (95 °C for 15 sec, 60 °C for 15 sec, and 72 °C for 30 sec), and finally a thorough extension at 72 °C for 5 mins to obtain the PCR product; sequence the PCR product.

[0055] The base at the 142nd position of the PCR products of 68 sweet corn DNAs is T, and the genotype is TT. The base at the 142nd position of the PCR products of 92 sweet corn DNAs is C, and the genotype is CC. The average 100-kernel weight of 68 sweet corns with the genotype TT was counted as 14.57 g, showing a high kernel weight trait; the average 100-kernel weight of 92 sweet corns with the genotype CC was counted as 13.19 g, showing a low kernel weight trait. Perform a T-test on the 100-kernel weights of the sweet corn inbred lines with the TT genotype and the CC genotype. The P-value is 0.0012, indicating that there is a highly significant difference in the 100-kernel weights between the sweet corn inbred lines with the TT genotype and the sweet corn inbred lines with the CC genotype. The results are as Figure 2 shown.

[0056] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A SNP molecular marker tightly linked to the major QTL of sweet corn kernel weight, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1; when the base at the 142nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is T, the sweet corn exhibits a high grain weight trait; when the base at the 142nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 is C, the sweet corn exhibits a low grain weight trait.

2. Use of the SNP molecular marker described in claim 1 in identifying the trait of sweet corn kernel weight.

3. Use of the SNP molecular marker according to claim 1 in screening or breeding sweet corn with high grain weight trait.

4. A primer pair for detecting the SNP molecular marker according to claim 1, characterized in that: The sequence of the upstream primer is shown in SEQ ID NO.2, and the sequence of the downstream primer is shown in SEQ ID NO.

3.

5. A kit for identifying the weight trait of sweet corn kernels, characterized in that: It comprises a DNA extraction kit, a PCR amplification kit and a sequencing kit; the PCR amplification kit comprises the primer pair according to claim 4.

6. Use of the primer pair according to claim 4 or the kit according to claim 5 in identifying the trait of sweet corn kernel weight.

7. Use of the primer pair according to claim 4 or the kit according to claim 5 in screening or breeding sweet corn with high grain weight trait.

8. A method for identifying the weight trait of sweet corn kernels, characterized in that: The following steps are involved: Using the sweet corn genomic DNA to be tested as a template, PCR amplification is performed using the primer pair described in claim 4 to obtain a PCR product; The PCR products were sequenced; the 142nd base of the PCR product was T, the genotype was TT, and it showed a high grain weight trait; the 142nd base of the PCR product was C, the genotype was CC, and it showed a low grain weight trait.

9. A method for screening sweet corn with high grain weight trait, characterized in that: The method comprises: using the sweet corn genomic DNA to be tested as a template, performing PCR amplification using the primer pair of claim 4 to obtain a PCR product; sequencing the PCR product; and selecting an individual whose base at position 142 of the PCR product is T and whose genotype is TT.

10. Use of the method according to any one of claims 8 or 9 in sweet corn breeding.

Images