Major QTL, SNP molecular marker and KASP detection primer group for controlling dehydration rate of corn kernels and application of major QTL, SNP molecular marker and KASP detection primer group

Abstract

The invention provides a major QTL (Quantitative Trait Loci) for controlling the dehydration rate of corn kernels, the major QTL is qAUDDC2.2, and the major QTL is located in the interval of 212.83 Mb to 213.91 Mb on the second chromosome of corn; the invention also provides an SNP (Single Nucleotide Polymorphism) molecular marker which is closely linked with the major QTL for controlling the dehydration rate of the corn kernels, and the SNP molecular marker is chr2212831691 and chr2213913274; the invention further provides a KASP primer group for detecting the SNP molecular marker, the KASP primer group is named as KASP-qAUDDC2.2-2213891067, and the KASP primer group comprises a forward competitive primer 1, a forward competitive primer 2 and a reverse universal primer. The major QTL for controlling the corn kernel dehydration rate, the SNP molecular marker and the KASP primer group for detecting the SNP molecular marker are applied to genetic improvement of the corn kernel dehydration rate and breeding of the corn kernel dehydration rate, so that corn varieties with different water contents or dehydration rates are obtained and used for breeding of mechanically harvested corn varieties, and the breeding production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics, 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] The dehydration rate of corn kernels is a typical quantitative trait that is easily affected by environmental conditions, and its phenotypic investigation and identification process is complicated. The selection of germplasm resources for kernel dehydration rate through traditional breeding methods has problems such as long breeding cycle and low efficiency. Molecular marker-assisted selection technology has the advantages of fast breeding speed, high sensitivity, strong specificity, accuracy and reliability, and is not affected by factors such as the growth and development stage of the organism and external environmental conditions, which can accelerate the screening process of kernel dehydration germplasm resources.

[0003] At present, there are limited QTL loci and developed molecular markers for the dehydration rate of corn kernels. Therefore, it is an urgent problem to provide a QTL loci and developed molecular markers that can control the dehydration rate of corn kernels. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a major effect QTL, SNP molecular marker, KASP detection primer set and application for controlling the dehydration rate of corn kernels in view of the deficiencies of the above-mentioned prior art. 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 the 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 QTL that controls the dehydration rate of corn kernels, wherein the major QTL is qAUDDC2.2, and the major QTL is located in the interval of 212.83Mb to 213.91Mb 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, wherein the SNP molecular markers are chr2_212831691 and chr2_213913274, the nucleotide sequence of the chr2_212831691 is shown in SEQ ID NO.4, the nucleotide sequence of the chr2_213913274 is shown in SEQ ID NO.5, the 101st base in the nucleotide sequences of the chr2_212831691 and the chr2_213913274 are both polymorphic sites, the polymorphic site of the chr2_212831691 is A / G, and the polymorphic site of the 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 comprises 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, as shown in SEQ ID NO.1; the forward competitive primer 2 is KASP-2_213891067-2, as shown in SEQ ID NO.2; the reverse universal primer is KASP-2_213891067-3, as 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 SNP molecular markers tightly linked to the major QTL controlling the dehydration rate of corn kernels in genetic improvement of corn kernel dehydration rate and molecular breeding.

[0010] Application of the above 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 effect QTL in the present invention can significantly improve the dehydration rate of corn kernels, which can not only provide new functional sites and gene resources for corn kernel mechanical harvesting breeding, but also can be further combined with molecular marker-assisted selection to realize the transfer and breeding utilization of corn genetic enhancement sites / genes to future generations, promote the molecular improvement of kernel dehydration rate, and has a high utilization value for corn 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 an application method thereof, which can be used to accurately, efficiently and intuitively detect the grain dehydration rate site qAUDDC2.2. The 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 is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It 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 grain dehydration rates of corn inbred lines carrying genotypes a and b respectively. DETAILED DESCRIPTION

[0019] Example 1

[0020] In this embodiment, a major QTL for controlling the dehydration rate of corn kernels is provided, wherein the major QTL is qAUDDC2.2, and the major QTL is located in the interval of 212.83Mb to 213.91Mb on the second chromosome of corn;

[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 embodiment, the method for obtaining the main effect QTL and marker interval controlling the dehydration rate of corn kernels is as follows:

[0023] S1. The maize inbred line PB80 with a slow grain dehydration rate was used as the female parent and the inbred line PHJ65 with a fast grain dehydration rate was used 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 the single-seed transmission method.

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

[0025] S3. In 2020, the high-generation recombinant inbred line genetic segregation populations obtained in S1 were planted in Xinxiang (E113°97′, N35°05′), Anyang (E114°38′, N36°10′), and Zhoukou (E115°07′, N33°41′), respectively. Single-row sowing was adopted, with a row length of 3.00 m, a 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 days, 45 days, 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 pierced into the kernels, and recorded the moisture content of the kernels; selected 5 plants with the same growth from each family for measurement, and repeated measurements were performed on each ear for 3 times, and the average value was taken as the phenotypic value of the plant; calculated the area under the moisture content curve (AUDDC) as an indicator of the dehydration rate of the kernels; (AUDDC: moisture content variation index; evaluation method reference: Yang J, Carena Mand Uphaus J. Area under the dry down curve (AUDDC): amethod to evaluate rate of dry down in maize [J]. Crop Sci., 2010, 50 (6): 2347-2354.);

[0026] S4. Combining genotype and phenotype data, using 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), using ICIM-ADD, and using LOD value 2.5 as the significance threshold; finally, a stable QTL locus qAUDDC2.2 detected in all three environments was located; qAUDDC2.2 is located on the second chromosome of maize, and the SNP molecular markers on both sides are chr2_212831691 and chr2_213913274, and the physical position on the reference genome B73 (V4) is between 212.83Mb and 213.91Mb ( Figure 1 shown).

[0027] Example 2

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

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

[0030] In order to better utilize the main effect QTL locus qAUDDC2.2 of corn kernel dehydration rate obtained by genetic mapping in Example 1 for breeding, based on the composition of SNP molecular markers closely linked to the interval, KASP molecular markers suitable for high-throughput typing were further developed. The specific operation is as follows:

[0031] S1. As shown in Table 1, the SNP molecular markers are chr2_212831691 and chr2_213913274 respectively; first, the sequence information of 150bp upstream and downstream of the above two SNP molecular markers is retrieved respectively, and multiple sets of amplification primers are designed according to the base composition of the target SNP site using KrakenTM software (LGC Biosearch Technologies, Hoddesdon, UK). Each set of amplification primers includes 3 primers, two forward specific primers and one reverse universal primer. The two forward primers correspond to two fluorescent signals. After PCR reaction, the fluorescence values ​​of the two fluorescent signals are finally detected to determine the sample typing. The KASP primer set sequence involved is shown in Table 1. Each set of primers consists of 3 sequences, namely: forward competitive primer 1: FAM label sequence + amplification primer sequence; forward competitive primer 2: HEX label 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. In order 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) Randomly select 50 materials from the genetic segregation population of the high-generation recombinant inbred line constructed by the above PB80×PHJ65, take young leaves together with the two parents, extract genomic DNA from them using the CTAB method, and then dilute the DNA to a concentration of about 50 ng / ul with sterile ultrapure water.

[0037] 2) KASP primer set reaction test

[0038] The 2μL PCR amplification reaction system was tested using the Douglas Scientific Array Tape platform: 0.75μL 2×KASP Master mix from LGC, 0.03μL KASP Assay Mix, 50ng template DNA, and ddH2O to 2μL. The KASP Assay Mix contained forward primers, forward competitive primer 1, forward competitive primer 2, and reverse universal primers, and the primers with a concentration of 100μM were mixed in a volume ratio of 2:2:5;

[0039] 2) The obtained KASP primer set was used for PCR amplification procedure: 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 and 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: The fluorescence data were read using the 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 ​​aggregated close to the Y axis) were bound to the FAM fluorescent group and were of the PB80 genotype; samples with blue fluorescence signals in the genotyping cluster diagram (fluorescence signal values ​​aggregated close to the X axis) were bound to the HEX fluorescent group and were of the PHJ65 genotype; samples aggregated close to the origin and displayed black were blank controls without DNA added;

[0041] like Figure 2 As shown in the typing results, the KASP molecular marker primer set developed by the present invention has high amplification efficiency, clear typing, and accurate detection of SNP sites, which is completely consistent with the gene chip detection results of the corresponding genotype parents and each strain, indicating that the KASP molecular marker has been successfully developed. The inventor named the 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 corn kernel dehydration rate enhancement site qAUDDC2.2-2. Its dominant allele variation comes from PHJ65, and the non-dominant allele variation comes from PB80.

[0042] Example 3

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

[0044] In order to verify 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 verification materials. Genomic DNA was extracted and fluorescent quantitative PCR amplification and genotyping were performed. DNA extraction, KASP reaction system, amplification procedure and genotyping method were consistent with the method described in Example 2. The results of fluorescent signal detection and genotyping clustering are shown in Figure 2. Figure 2 as shown in .

[0045] SPSS19.0 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 the grain dehydration rate (AUDDC) 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 corn inbred line materials with the genotype of grain dehydration rate (genotype a) and (genotype b). The results are shown in Table 3: The grain dehydration rate of the corn inbred line materials with genotype a was significantly higher (p<0.001) than that of the corn inbred line materials with genotype b, indicating that the corn 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 the dominant allele variation. 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 of large-scale genetic generations and breeding lines in the breeding process, meets the breeding needs of early screening of target traits and evaluation of grain mechanical 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 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 change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A major QTL controlling the dehydration rate of maize kernels, characterized in that: The major QTL is qAUDDC2.2, and the major QTL is located in the interval of 212.83Mb to 213.91Mb on the second chromosome of maize.

2. A SNP molecular marker tightly linked to the major QTL controlling the dehydration rate of corn kernels according to claim 1, characterized in that: The SNP molecular markers are chr2_212831691 and chr2_213913274, the nucleotide sequence of chr2_212831691 is shown in SEQ ID NO.4, 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 are both polymorphic sites, the polymorphic site of chr2_212831691 is A / G, and the polymorphic site of chr2_213913274 is T / C.

3. The KASP primer set for detecting the SNP molecular marker according to claim 2, characterized in that: The KASP primer set is named KASP-qAUDDC2.2-2_213891067, and the KASP primer set includes a forward competitive primer 1, a forward competitive primer 2 and a reverse universal primer, and the nucleotide sequence is 5'-3', the forward competitive primer 1 is KASP-2_213891067-1, as shown in SEQ ID NO.1; the forward competitive primer 2 is KASP-2_213891067-2, as shown in SEQ ID NO.2; the reverse universal primer is KASP-2_213891067-3, as shown in SEQ ID NO.

3.

4. Use of a major effect QTL controlling the dehydration rate of corn kernels as claimed in claim 1 in genetic improvement and molecular breeding of corn kernel dehydration rate.

5. Use of a SNP molecular marker tightly linked to a major QTL controlling the dehydration rate of corn kernels as claimed in claim 2 in genetic improvement of the dehydration rate of corn kernels and molecular breeding.

6. Use of the KASP primer set as claimed in claim 3 in molecular detection of the major effect QTL and breeding selection.

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