Major QTL, SNP molecular markers, KASP detection primer sets and their applications for controlling maize kernel dehydration rate

Abstract

The present invention provides a major QTL controlling the dehydration rate of corn kernels, wherein the major QTL is qAUDDC2.2 and is located in the 212.83Mb to 213.91Mb interval on corn chromosome 2. The present invention also provides SNP molecular markers tightly linked to the major QTL controlling the dehydration rate of corn kernels, wherein the SNP molecular markers are chr2_212831691 and chr2_213913274. The present invention also provides a KASP primer set for detecting the SNP molecular markers, wherein the KASP primer set is named KASP-qAUDDC2.2-2_213891067, and the KASP primer set comprises a forward competitive primer 1, a forward competitive primer 2, and a reverse universal primer. The main effect QTL, SNP molecular marker and KASP primer set for detecting SNP molecular marker that control the dehydration rate of corn kernels in the present invention are applied to the genetic improvement of corn kernel dehydration rate and its breeding, thereby obtaining corn varieties with different moisture content or dehydration rates for the selection of machine-harvested corn varieties, thereby reducing breeding production costs.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetic technology, and in particular relates to a major effect QTL, a SNP molecular marker, a KASP detection primer set and an application thereof for controlling the dehydration rate of corn kernels. Background Art

[0002] Maize kernel desiccation rate is a typical quantitative trait that is easily affected by environmental conditions, making its phenotypic investigation and identification complex. Traditional breeding methods for kernel desiccation-specific germplasm selection suffer from long breeding cycles and low efficiency. Molecular marker-assisted selection (MAM) offers the advantages of rapid selection, high sensitivity, strong specificity, and high accuracy and reliability. Furthermore, it is unaffected by factors such as the organism's growth and development stage and external environmental conditions, potentially accelerating the screening of kernel desiccation-specific germplasm.

[0003] Currently, there are limited QTL loci and developed molecular markers for maize kernel dehydration rate. Therefore, providing a QTL loci and developed molecular markers that can control maize kernel dehydration rate is an urgent problem to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a major effect QTL, SNP molecular marker, KASP detection primer set and application for controlling the dehydration rate of corn kernels. The major effect QTL site in the present invention has the function of controlling the moisture content and dehydration rate traits of corn kernels, and this major effect QTL has never been reported in previous public materials.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: a major effect QTL controlling the dehydration rate of corn kernels, the major effect QTL is qAUDDC2.2, and the major effect QTL is located in the 212.83Mb to 213.91Mb interval on the second chromosome of corn.

[0006] The present invention also provides SNP molecular markers tightly linked to the above-mentioned major effect QTL controlling the dehydration rate of corn kernels. The SNP molecular markers are chr2_212831691 and chr2_213913274. The nucleotide sequence of chr2_212831691 is shown in SEQ ID NO.4, and the nucleotide sequence of chr2_213913274 is shown in SEQ ID NO.5. The 101st base in the nucleotide sequences of chr2_212831691 and chr2_213913274 is a polymorphic site. The polymorphic site of chr2_212831691 is A / G, and the polymorphic site of chr2_213913274 is T / C.

[0007] The present invention also provides a KASP primer set for detecting the above-mentioned SNP molecular marker. The KASP primer set is named KASP-qAUDDC2.2-2_213891067. The KASP primer set includes a forward competitive primer 1, a forward competitive primer 2, and a reverse universal primer. The nucleotide sequence is 5'-3'. The forward competitive primer 1 is KASP-2_213891067-1, shown in SEQ ID NO. 1; the forward competitive primer 2 is KASP-2_213891067-2, shown in SEQ ID NO. 2; and the reverse universal primer is KASP-2_213891067-3, shown in SEQ ID NO. 3.

[0008] The application of the above-mentioned major effect QTL controlling the dehydration rate of corn kernels in genetic improvement and molecular breeding of corn kernel dehydration rate.

[0009] The application of the above-mentioned SNP molecular markers tightly linked to the major effect QTL controlling the dehydration rate of corn kernels in genetic improvement and molecular breeding of corn kernel dehydration rate.

[0010] Application of the above-mentioned KASP primer set in molecular detection of the major effect QTL and breeding selection.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] 1. The major QTL in the present invention can significantly increase the dehydration rate of maize kernels, which not only provides new functional loci and gene resources for maize kernel mechanical harvesting breeding, but can also be further combined with molecular marker-assisted selection to achieve the transfer of maize genetic enhancement loci / genes to future generations and breeding utilization, promoting molecular improvement of kernel dehydration rate, and has high utilization value for maize kernel mechanical harvesting breeding.

[0013] 2. Based on the SNP molecular markers tightly linked to the major effect QTL, the present invention has developed a KASP molecular marker primer set (KASP primer set) and its application method for accurately, efficiently, and intuitively detecting the grain dehydration rate locus qAUDDC2.2. This KASP molecular marker primer set can achieve accurate typing of dominant allele variation, has the advantages of stable amplification, simple detection, and intuitive results, and can be used for accurate detection of genotypes of large-scale genetic lines and auxiliary prediction and evaluation of phenotypes in corn breeding.

[0014] 3. The KASP primer set provided by the present invention can promote the application of the maize kernel dehydration rate enhancement QTL locus qAUDDC2.2 in machine-harvested maize breeding, realize early identification and auxiliary selection of maize with kernel dehydration rate as the breeding target, and improve breeding efficiency.

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the location map of the main effect QTL of the present invention.

[0017] Figure 2 It is fluorescence signal detection and genotyping clustering.

[0018] Figure 3 This is a comparison chart of the dehydration rates of the kernels of maize inbred lines carrying genotypes a and b respectively. DETAILED DESCRIPTION

[0019] Example 1

[0020] This embodiment provides a major QTL for controlling the dehydration rate of maize kernels. The major QTL is qAUDDC2.2, and the major QTL is located in the interval 212.83Mb to 213.91Mb on maize chromosome 2.

[0021] Also provided are SNP molecular markers tightly linked to the major effect QTL controlling the dehydration rate of corn kernels. The SNP molecular markers are chr2_212831691 and chr2_213913274. The nucleotide sequence of chr2_212831691 is shown in SEQ ID NO.4, and the nucleotide sequence of chr2_213913274 is shown in SEQ ID NO.5. The 101st base in the nucleotide sequences of chr2_212831691 and chr2_213913274 is a polymorphic site. The polymorphic site of chr2_212831691 is A / G, and SEQ ID NO.4 shows that the 101st base is G. The polymorphic site of chr2_213913274 is T / C, and SEQ ID NO.5 shows that the 101st base is C.

[0022] In this example, the method for obtaining the main effect QTL and marker interval controlling the dehydration rate of corn kernels is as follows:

[0023] S1. A maize inbred line PB80 with a slow kernel dehydration rate was hybridized with an inbred line PHJ65 with a fast kernel dehydration rate as the male parent to obtain the F1 generation. The F1 generation was self-fertilized to obtain the F2 generation. A high-generation recombinant inbred line genetic segregation population containing 310 lines was obtained through single-seed descent.

[0024] S2. A whole-genome scan was performed on the genetic segregating population of 310 high-generation recombinant inbred lines constructed from PB80×PHJ65 in S1 using high-throughput simplified genome sequencing technology. A bin map was constructed using the maximum likelihood recombination inference (MPR) method. The map covers all 10 maize chromosomes, with a total length of 1237.36 cM.

[0025] In 2020, the high-generation recombinant inbred line genetic segregation population obtained in S1 was planted in Xinxiang (E113°97′, N35°05′), Anyang (E114°38′, N36°10′), and Zhoukou (E115°07′, N33°41′). Single-row sowing was done manually with a row length of 3.00 m, an intra-row spacing of 0.60 m, and a row spacing of 0.25 m. The moisture content of corn kernels was continuously measured at 35, 45, and 55 days after pollination using a portable HB-300 moisture meter (Kett Electric). Laboratory) penetrated the bracts in the middle of the ear and into the kernels, and recorded the moisture content of the kernels. Five plants with consistent growth were selected from each family for measurement. Each ear was measured three times, and the average value was taken as the phenotypic value of the plant. The area under the moisture content curve (AUDDC) was calculated as an indicator of the kernel dehydration rate (AUDDC: moisture content variation index; evaluation method reference: Yang J, Carena Mand Uphaus J. Area under the dry down curve (AUDDC): a method to evaluate the rate of dry down in maize [J]. Crop Sci., 2010, 50(6): 2347-2354.).

[0026] S4. Combining genotypic and phenotypic data, we used the software (Meng L, Li H, Zhang L, et al. (2015) QTLIciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. Crop J 3: 269-283) and the ICIM-ADD method, with an LOD value of 2.5 as the significance threshold. We finally located a stable QTL locus, qAUDDC2.2, which was previously detected in all three environments. qAUDDC2.2 is located on maize chromosome 2, and the SNP molecular markers on both sides of it are chr2_212831691 and chr2_213913274, with a physical position of 212.83Mb to 213.91Mb on the reference genome B73 (V4). Figure 1 shown).

[0027] Example 2

[0028] This example provides a KASP primer set for detecting the SNP molecular marker described in Example 1. The KASP primer set is named KASP-qAUDDC2.2-2_213891067. The KASP primer set comprises a forward competitive primer 1, a forward competitive primer 2, and a reverse universal primer, with a 5'-3' nucleotide sequence. The forward competitive primer 1 is KASP-2_213891067-1, shown in SEQ ID NO. 1; the forward competitive primer 2 is KASP-2_213891067-2, shown in SEQ ID NO. 2; and the reverse universal primer is KASP-2_213891067-3, shown in SEQ ID NO. 3.

[0029] Development, screening and validation of KASP molecular markers

[0030] To better utilize the major QTL locus qAUDDC2.2 for maize kernel dehydration rate identified in Example 1 for breeding, we further developed KASP molecular markers suitable for high-throughput typing based on the tightly linked SNP molecular marker composition within the region. The specific procedures are as follows:

[0031] S1. As shown in Table 1, the SNP markers are chr2_212831691 and chr2_213913274. First, the sequence information of the 150 bp upstream and downstream of the two SNP markers was retrieved. Based on the base composition of the target SNP site, multiple sets of amplification primers were designed using Kraken™ software (LGC Biosearch Technologies, Hoddesdon, UK). Each set of amplification primers included three primers: two forward-specific primers and one reverse universal primer. The two forward primers corresponded to two fluorescent signals. After PCR reaction, the fluorescence values ​​of the two fluorescent signals were finally detected to determine the sample typing status. The KASP primer set sequences involved are shown in Table 1. Each primer set consists of three sequences: forward competitive primer 1: FAM tag sequence + amplification primer sequence; forward competitive primer 2: HEX tag sequence + amplification primer sequence; reverse universal primer 3: amplification primer sequence.

[0032] Table 1 KASP primer set design

[0033]

[0034] Note: In the SNP composition (a / b), a represents the PB80 variant type and b represents the PHJ65 variant type.

[0035] S2. To further test the accuracy and resolution of the developed KASP primer set in identifying the target SNP molecular marker and detecting the grain dehydration rate site, and to prove the successful development of the KASP marker, it is necessary to verify the typing effect of the KASP marker. The specific method is as follows:

[0036] 1) Fifty accessions were randomly selected from the genetic segregation population of the high-generation recombinant inbred lines constructed from PB80 × PHJ65. Young leaves of both parents were taken and genomic DNA was extracted from them using the CTAB method. The DNA was then uniformly diluted with sterile ultrapure water to a concentration of approximately 50 ng / ul.

[0037] 2) KASP primer set reaction test

[0038] Detection was performed using the Douglas Scientific Array Tape platform. A 2μL PCR amplification reaction system consisted of 0.75μL of LGC's 2× KASP Master mix, 0.03μL of KASP Assay Mix, and 50ng of template DNA, made up to 2μL with ddH2O. The KASP Assay Mix contained forward primers (forward competitive primer 1, forward competitive primer 2), and a reverse universal primer, all mixed at a 100μM concentration in a 2:2:5 volume ratio.

[0039] 2) PCR amplification using the obtained KASP primer set: activation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 65°C for 60 s, 10 cycles, with the annealing / extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing / extension at 55°C for 60 s, 26 cycles;

[0040] 3) Fluorescence Signal Scanning and Genotyping: Fluorescence data were read using a microplate reader, Pherastar (LGC Biosearch Technologies, Hoddesdon, UK), and genotyping clustering was performed. Specifically, samples with red fluorescence signals in the genotyping cluster diagram (fluorescence signal values ​​clustered close to the Y-axis) were bound to the FAM fluorescent group and had the PB80 genotype; samples with blue fluorescence signals in the genotyping cluster diagram (fluorescence signal values ​​clustered close to the X-axis) were bound to the HEX fluorescent group and had the PHJ65 genotype; samples clustered close to the origin and displayed black were blank controls without DNA added;

[0041] like Figure 2 The typing results show that the KASP molecular marker primer set developed by the present invention has high amplification efficiency, clear typing, and accurate SNP detection. The marker is fully consistent with the gene chip detection results of the corresponding genotyped parents and strains, indicating the successful development of the KASP molecular marker. The inventors named this KASP primer set KASP-qAUDDC2.2-2_213891067. The KASP primer set sequence includes three sequences: KASP-2_213891067-1, KASP-2_213891067-2, and KASP-2_213891067-3. It can be used for molecular detection and breeding selection of the maize kernel dehydration rate enhancing locus qAUDDC2.2-2. The dominant allele variant is derived from PHJ65, and the non-dominant allele variant is derived from PB80.

[0042] Example 3

[0043] This example provides the application of the above-mentioned KASP primer set in molecular detection of the major effect QTL and breeding selection.

[0044] To validate the effectiveness of the KASP primer set KASP-qAUDDC2.2-2_213891067 obtained in Example 2, 227 samples were randomly selected from different types of maize inbred lines as molecular marker validation materials. Genomic DNA was extracted and fluorescence quantitative PCR amplification and genotyping were performed. DNA extraction, KASP reaction system, amplification procedure, and genotyping methods were consistent with those described in Example 2. The fluorescence signal detection and genotyping clustering results are shown in Figure 2. Figure 2 As shown in .

[0045] SPSS 19.0 was used for statistical analysis. Comparing the phenotypic data of the grain dehydration rate of the a and b genotypes in Table 2 and the maize inbred line materials carrying these two genotypes, the results showed that the grain dehydration rate (AUDDC) of the maize inbred line materials carrying the a genotype was significantly higher than that of the maize inbred line materials carrying the b genotype (e.g. Figure 3 The independent sample t-test was further used to compare the phenotypic differences between the maize inbred line materials with different genotypes of grain dehydration rate (genotype a) and (genotype b). The results are shown in Table 3: The grain dehydration rate of the maize inbred line materials with genotype a was significantly higher (p < 0.001) than that of the maize inbred line materials with genotype b, indicating that the maize inbred line materials with genotype b have a faster dehydration rate.

[0046] In summary, the KASP primer set KASP-qAUDDC 2.2-2_213891067, which is tightly linked to qAUDDC2.2, can accurately type dominant alleles. The implementation method is simple and fast, the results are accurate and intuitive, and the analysis throughput is high. It is suitable for the efficient detection of excellent genotypes in large-scale genetic generations and breeding lines during the breeding process, meets the breeding needs of early screening of target traits and evaluation of grain harvesting potential, and improves breeding efficiency.

[0047] Table 2 Genotype detection and grain dehydration rate phenotype investigation of 227 different types of maize inbred lines

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[0057] Table 3 Test results

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[0059] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Application of a KASP primer set for detecting SNP molecular markers in corn kernel dehydration rate breeding selection, characterized in that: The KASP primer set is named KASP-qAUDDC2.2-2_213891067. The KASP primer set includes a forward competitive primer 1, a forward competitive primer 2, and a reverse universal primer, with a nucleotide sequence of 5'-3'. The forward competitive primer 1 is KASP-2_213891067-1, shown in SEQ ID NO.1; the forward competitive primer 2 is KASP-2_213891067-2, shown in SEQ ID NO.2; and the reverse universal primer is KASP-2_213891067-3, shown in SEQ ID NO.3.

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