A set of KASP primers related to wheat grain weight, their applications, and reagent kits.

Abstract

This invention discloses a set of KASP primers related to wheat grain weight and their applications. The primer set includes primers with nucleotide sequences as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.6. This KASP primer set can be used to detect loci on wheat chromosome 5D that are associated with thousand-grain weight. Ktkw-5D, Allele C / T typing was performed at this locus. Wheat carrying allele C had a significantly higher thousand-grain weight than wheat carrying allele T, thus enabling early screening of wheat grain weight and accelerating the breeding process.

Description

Technical Field

[0001] This application relates to the field of wheat breeding, and in particular to a KASP marker related to wheat grain weight, its application, and a kit. Background Technology

[0002] High yield is an important goal in wheat breeding. Grain weight is one of the components of yield, mainly controlled by additive effects, and has the highest heritability among yield factors, reaching 59-80% (Xiao Shihe, He Zhonghu. Improvement of wheat yield potential and quality. In: Zhuang Qiaosheng. Improvement of Chinese wheat varieties and pedigree analysis. Beijing: China Agriculture Press, 2003. pp 497-542). Wheat grain weight is a quantitative trait, exhibiting continuous variation and regulated by multiple genes with minor effects (Zhang LY, Liu DC, Guo XL, Yang W L, Sun JZ, Wang DW, Zhang A. Genomic Distribution of Quantitative Trait Loci for Yield and Yield-Related Traits in Common Wheat. Journal of Integrative Plant Biology, 2010, 52: 996-1007). Numerous studies have been reported on the genetic mapping of wheat grain weight. Mason et al. investigated wheat yield traits under different sowing dates and detected two thousand-grain weight QTLs on chromosomes 2D and 5A (Mason R E, Hays DB, Mondal S, Ibrahim AMH, Basnet B R. QTL for Yield, Yield Components and Canopy Temperature Depression in Wheat under Late Sown Field Conditions. Euphaitica, 2013, 194:243-259). Liu Lihua et al. conducted multi-environment yield trait identification on 248 winter wheat varieties bred in northern China, and identified 24 loci significantly associated with thousand-grain weight through association analysis (Liu Lihua, Liu Yangna, Zhou Yue, Li Hongbo, Zhang Mingming, Qu Pingping, Zhao Changping, Pang Binshuang. Genome-wide association analysis of wheat yield-related traits based on efficient SNP chip. Journal of Triticeae Crops, 2023, https: / / link.cnki.net / urlid / 61.1359.S.20230914.0914.004). Due to differences in mapping populations and molecular markers, the localization results varied considerably.

[0003] With the development of molecular biology, molecular markers have evolved from RFLP, RAPD and SSR to SNP markers in recent years. SNPs are characterized by genetic stability, large numbers, and wide distribution, and are suitable for high-throughput detection. Genotype chips developed based on them, such as 9k, 50k, 90k, and 660k chips, aggregate tens of thousands of SNP markers, greatly improving the accuracy of genetic mapping (Cavanagh CR, Shiaoman C, Shichen W, Bevan Emma H, Stuart S, Seifollah K, Kerrie F, Cyrille S, Brown-Guedira GL, Alina A. Genome-Wide Comparative Diversity Uncovers Multiple Targets of Selection for Improvement in Hexaploid Wheat Landraces and Cultivars. Proceedings of the National Academy of Sciences of the United States, 2013, 110: 8057-8062; Wang S, Wong D, Forrest K, Allen A, Chao S, Huang BE, Maccaferri M, Salvi S, Milner SG, Cattivelli L. Characterization). ofPolyploid Wheat Genomic Diversity Using a High-Density 90,000SingleNucleotide Polymorphism Array.Plant Biotechnology Journal,2014,12:787-796;CuiF,Zhang N,Fan X,Zhang W,Zhao C,Yang L,Pan R,Chen M,Han J,Zhao X.Utilization of a Wheat660k Snp Array-Derived High-Density Genetic Map for High-ResolutionMapping of a Major QTL for Kernel Number. Scientific Reports, 2017,7:3788).Based on the differences in SNP sites, KASP (Kompetitive Allele Specific PCR) markers with specific matching primer terminal bases can be developed. This can accurately identify biallelic genes at SNP sites and has high throughput, making it suitable for molecular marker detection of large numbers of samples. It is compatible with breeding selection and has broad application prospects in breeding (Semagn K, Babu R, Hearne S, Olsen M. Single Nucleotide Polymorphism Genotyping Using KompetitiveAllele Specific Pcr (Kasp): Overview of the Technology and Its Application in Crop Improvement. Molecular Breeding, 2014, 33: 1-14).

[0004] Thousand-grain weight has a very important impact on wheat yield (Morgounov A, Zykin V, Belan I, Roseeva L, Zelenskiy Y, Gomez Becerra HF, Budak H, Bekes F (2010) Genetic gains for grain yield in high latitude spring wheat grown in Western Siberia in 1900–2008. Field Crops Research 117:101-112; Song JM, Dai S, Li HS, Cheng DG, Liu AF, Cao XY, Liu JJ, Zhao ZD (2013) Evolution of Agronomic and Quality Traits of Wheat Cultivars Released in Shandong Province Recently. Scientia Agricultura Sinica 46:1114-1126; Underdahl JL, Mercoum M, Ransom JK, Schatz BG (2008) Agronomic Traits Improvement and Associations in Hard Red Spring Wheat Cultivars Released in North Dakota from 1968 to 2006 (Crop Science 48:158-166). Identifying the genetic control loci for thousand-grain weight in local wheat varieties in my country has important guiding significance for further targeted selection of wheat yield. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a set of KASP primers associated with the grain weight control site Ktkw-5D on the wheat 5D chromosome. This set can rapidly perform grain weight-related genotyping on wheat materials from the middle and lower reaches of the Yangtze River, thereby accelerating the breeding process.

[0006] Specifically, this application first provides a set of KASP primers related to wheat grain weight, including primer AX-109454027-F1 with nucleotide sequence as shown in SEQ ID NO.7, primer AX-109454027-F1 with nucleotide sequence as shown in SEQ ID NO.8, and universal primer AX-109454027-R with nucleotide sequence as shown in SEQ ID NO.6.

[0007] Secondly, this application also provides the application of the above-mentioned KASP primer set in the detection of wheat grain weight. The specific steps are as follows: using wheat sample DNA as a template, a PCR detection system is prepared using the above-mentioned KASP primer set, and the sample wheat is subjected to fluorescent PCR detection. If red fluorescence is produced, it corresponds to the dominant allelic variant C; if blue fluorescence is produced, it corresponds to the non-dominant allelic variant T. The thousand-grain weight of wheat carrying the dominant allelic variant gene C is significantly higher than that of wheat carrying the non-dominant allelic variant gene T. The above-mentioned wheat is preferably from the wheat-growing region of the middle and lower reaches of the Yangtze River.

[0008] Furthermore, the above PCR detection system includes: 2.5 μL of 2×KASP Master Mix, 0.07 μL of KASP Assay Mix, and 2.43 μL of template DNA at a concentration of 20 ng / μL;

[0009] The preparation method for each 100 μL KASP Assay Mix is ​​as follows: 12 μL (100 μM) of primer AX-109454027-F1 with nucleotide sequence as shown in SEQ ID NO.7, 12 μL (100 μM) of primer AX-109454027-F2 with nucleotide sequence as shown in SEQ ID NO.8, 30 μL (100 μM) of primer AX-109454027-R with nucleotide sequence as shown in SEQ ID NO.6, and 46 μL of ultrapure water.

[0010] The PCR reaction program was as follows: Step 1: 94℃, 15 min; Step 2: 94℃, 20 s, 61~55℃, 1 min, decreasing by 0.6℃ for each cycle, for a total of 10 cycles; Step 3: 94℃, 20 s, 55℃, 1 min, for a total of 26 cycles.

[0011] Third, the present invention also provides a kit for detecting wheat grain weight, comprising: 2×KASPMaster Mix 2.5μL and KASP Assay Mix 0.07μL; wherein, the KASP Assay Mix is ​​prepared as follows per 100μL: primer AX-109454027-F112μL (100μM) with nucleotide sequence as shown in SEQ ID NO.7, primer AX-109454027-F212μL (100μM) with nucleotide sequence as shown in SEQ ID NO.8, primer AX-109454027-R 30μL (100μM) with nucleotide sequence as shown in SEQ ID NO.6, and 46μL of ultrapure water.

[0012] In this application, the definition of the term "wheat region in the middle and lower reaches of the Yangtze River" is as disclosed in the literature "Cheng Shunhe, Guo Wenshan, Wang Longjun. Wheat in Southern China. Nanjing: Jiangsu Science and Technology Press, 2012.pp 23-26".

[0013] This application, through the determination and association analysis of the thousand-grain weight of 103 wheat varieties in the middle and lower reaches of the Yangtze River, detected a thousand-grain weight control locus on chromosome 5B, and further converted this SNP locus into a KASP marker, and verified that this locus has a significant impact on the thousand-grain weight of wheat, and can be applied to breeding selection. Attached Figure Description

[0014] Figure 1 The Ktkw-5D map of the site;

[0015] Figure 2 The results of KASP sequence fluorescence detection are shown in Table 1 of the examples. Detailed Implementation

[0016] All wheat varieties mentioned in the examples are preserved in the applicant's laboratory.

[0017] Example 1: SNP site detection

[0018] 1. Materials and Methods

[0019] One hundred and thirty-three wheat varieties approved in the wheat-growing region of the middle and lower reaches of the Yangtze River between 1972 and 2016 (see reference "Jiang, P., P. Zhang, L. Wu, Y. He, C. Li, H. Ma, et al. 2021. Linkage and association mapping and Kompetitive allele-specific PCR marker development for improving grain protein content in wheat. Theoretical and Applied Genetics 134:3563-3575") were used as materials. These materials were planted in single-row plots at the Jiangsu Academy of Agricultural Sciences' experimental base and Liuhe base for two consecutive growing seasons (2017-2018 and 2018-2019), with 60 seeds per row, a row length of 1.6 m, and a row spacing of 0.25 m. The plants were replicated twice and managed using conventional methods. After grain maturity, the thousand-grain weight was determined using a Wanshen grain quality tester (SC-A1, Hangzhou, Zhejiang). In addition, the thousand-grain weight of 261 wheat high-generation lines harvested from the Liuhe base during the 2019–2020 growing season (Table 4) was measured for subsequent verification. These 261 wheat lines were all bred from some commonly used parents in the wheat-growing areas of the middle and lower reaches of the Yangtze River, including the Ningmai series (such as Ningmai 13 and Ningmai 14), the Yangmai series (Yangmai 9, Yangmai 23, and Yangmai 20), and the Zhenmai series (such as Zhenmai 13).

[0020] Genomic DNA was extracted using the CTAB method (Porebski S, Bailey L, Baum B (1997) Modification of CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15:8-15). Genotypes were obtained using an Affymetrix 50K gene chip (Beijing Bio-Tech Co., Ltd., Beijing). Quality control of the genotype data was performed using TASSEL V5.2.13 software, deleting marker sites with a minimum gene frequency ≤5% and a deletion rate exceeding 10%.

[0021] Association analysis was conducted using the GAPIT software package in R (Lipka A, Tian F, Wang Q, Peiffer J, Li M, Bradbury P, Gore M, Buckler E, Zhang Z (2012) GAPIT: Genome Association and Prediction Integrated Tool. Bioinformatics 28:2397-2399). The BLINK (Bayesian-information and linkage-disequilibrium iteratively nested keyway) method was selected (Huang M, Liu X, Yao Z, Summers R, Zhang Z (2018) BLINK: A package for the next level of genome-wide association studies with both individuals and markers in the millions. GigaScience 8). Principal components were used as covariates to reduce the false positive rate, and the screening threshold was set to 1×10⁻⁶. -3 .

[0022] KASP molecular markers were developed by designing PCR amplification primers based on SNP sites and flanking sequences. For each marker, two SNP-specific primers (F1 / F2) and one universal primer (R) were designed. The F1 primer was tailed with a specific sequence GAAGGTGACCAAGTTCATGCT that binds to FAM fluorescence, and the F2 primer was tailed with a specific sequence GAAGGTCGGAGTCAACGGATT that binds to HEX fluorescence. KASP primers were designed using Polymarker (http: / / www.polymarker.info / ) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0023] The total KASP reaction volume was 5 μL, containing 2.5 μL of 2×KASP Master Mix, 0.07 μL of KASP Assay Mix (primer working solution), and a concentration of 20 ng / μL. -1 2.43 μL of template DNA.

[0024] Each 100 μL Assay Mix contains: 12 μL (100 μM) each of SNP-specific primers (F1 / F2), 30 μL of universal primer (R) (100 μM), and 46 μL of ultrapure water.

[0025] The KASP reaction procedure is as follows: Step 1: 94℃, 15min; Step 2: 94℃, 20s, 61~55℃, 1min, decreasing by 0.6℃ in each cycle, for a total of 10 cycles; Step 3: 94℃, 20s, 55℃, 1min, for a total of 26 cycles.

[0026] PCR reactions were performed in a water bath PCR instrument (LGC Hydrocycler model). 16 The PCR results were analyzed using a KASP fluorescence analyzer (LGC model PHERAstar plus).

[0027] 2. Results and Analysis

[0028] 2.1 Association analysis detected a thousand-grain weight control locus on chromosome 5D of wheat varieties such as Yangmai 158, Ningmai 23, Yangfumai 4, and Zhenmai 9, located within the physical interval of 549-557 Mb. Figure 1 The applicant named it Ktkw-5D.

[0029] 2.2 To better utilize the Ktkw-5D site for breeding, this embodiment developed a KASP marker suitable for high-throughput genotyping based on its region marker sequence. First, two SNP markers with low homology were selected from its marker region for primer design (Table 1). After amplification and verification, the results are as follows... Figure 2 As shown, Figure 2 In the figures, A and B represent the amplification results of primers AX-110894043 and AX-110894043, respectively. Red corresponds to the dominant allelic variant C, blue to the non-dominant allelic variant T, and black represents the blank control. It is evident that AX-110894043 failed to genotype successfully, while AX-109454027 showed good amplification results, suggesting potential for further development and breeding applications.

[0030] Table 1

[0031]

[0032] Table 2

[0033]

[0034]

[0035] Table 2 shows the comparison between the genotyping results of the KASP primer set AX-109454027 and the genotype chip. It can be seen that the two results are completely consistent, which indicates that the KASP primer set AX-109454027 can be applied to the breeding selection of wheat thousand-grain weight in the wheat-growing areas of the middle and lower reaches of the Yangtze River based on the SNP site Ktkw-5D. Its dominant allelic variant is C, and its non-dominant allelic variant is T.

[0036] Example 2: KASP Primer Set Validation

[0037] In this embodiment, the successfully developed KASP marker was used to genotype 261 high-generation wheat lines and statistical analysis was performed in combination with phenotypic data (Table 3). It was found that the marker had a significant effect on the selection of thousand-grain weight. The thousand-grain weight of the material carrying the C dominant allele was significantly higher than that of the material carrying the T non-dominant allele. The specific genotype and phenotypic values ​​are shown in Table 4.

[0038] The KASP primer set used in this step is AX-109454027, including primers AX-109454027-F1, AX-109454027-F2, and the universal primer AX-109454027-R.

[0039] AX-109454027-F1:GAAGGTGACCAAGTTCATGCTTTCAGCACAAGTAAACCTGATCA(SEQ IDNO.7)

[0040] AX-109454027-F2:GAAGGTCGGAGTCAACGGATTTTCAGCACAAGTAAACCTGATCG(SEQ IDNO.8)

[0041] AX-109454027-R:CGATTCAAGATGGCGTGCAG(SEQ ID NO.6)

[0042] The total KASP reaction volume was 5 μL, containing 2.5 μL of 2×KASP Master Mix, 0.07 μL of KASP Assay Mix, and a concentration of 20 ng / μL. -1 2.43 μL of template DNA.

[0043] The preparation method for each 100 μL Assay Mix is ​​as follows: primer AX-109454027-F1 12 μL (100 μM), primer AAX-109454027-F2 12 μL (100 μM), primer AX-109454027-R 30 μL (100 μM), and 46 μL of ultrapure water.

[0044] KASP reaction procedure: Step 1: 94℃, 15min; Step 2: 94℃, 20s, 61~55℃, 1min, decreasing by 0.6℃ per cycle, for a total of 10 cycles; Step 3: 94℃, 20s, 55℃, 1min, for a total of 26 cycles.

[0045] PCR reactions were performed in a water bath PCR instrument (LGC Hydrocycler model). 16 The PCR results were analyzed using a KASP fluorescence analyzer (LGC model PHERAstar plus).

[0046] Table 3

[0047]

[0048] Note: The numbers in parentheses represent the quantity of material carrying the corresponding allelic variation.

[0049] Table 4

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] As shown in Table 4, the AX-109454027 primer set can effectively genotype the wheat Ktkw-5D locus. The T / C alleles play a significant role in the selection of thousand-grain weight. Wheat carrying the dominant allele C has a significantly higher thousand-grain weight than wheat carrying the non-dominant allele T. This allows for the screening of wheat genotypes at the seed stage, accelerating the breeding process.

Claims

1. The application of a set of KASP primers related to wheat grain weight in the detection of wheat grain weight, characterized in that, Using wheat DNA as a template, a PCR detection system was prepared using KASP primers and the wheat samples were subjected to fluorescent PCR detection. If red fluorescence was produced, it contained C allelic variants; if blue fluorescence was produced, it contained T allelic variants. The thousand-grain weight of wheat carrying C allelic variants was significantly higher than that of wheat carrying T allelic variants. The KASP primer set consists of primer AX-109454027-F1 with nucleotide sequence as shown in SEQ ID NO.7, primer AX-109454027-F2 with nucleotide sequence as shown in SEQ ID NO.8, and primer AX-109454027-R with nucleotide sequence as shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that, The PCR detection system includes: 2.5 μL of 2×KASP MasterMix, 0.07 μL of KASP Assay Mix, and 2.43 μL of template DNA at a concentration of 20 ng / μL; The KASP Assay Mix is ​​prepared as follows: 12 μL of primer AX-109454027-F1 at a concentration of 100 μM, 12 μL of primer AX-109454027-F2 at a concentration of 100 μM, 30 μL of primer AX-109454027-R at a concentration of 100 μM, and 46 μL of ultrapure water; The PCR reaction program was as follows: Step 1: 94℃, 15 min; Step 2: 94℃, 20 s, 61~55℃, 1 min, decreasing by 0.6℃ for each cycle, for a total of 10 cycles; Step 3: 94℃, 20 s, 55℃, 1 min, for a total of 26 cycles.

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