CN116590466B - KASP primer group related to wheat grain weight and application - Google Patents

KASP primer group related to wheat grain weight and application Download PDF

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CN116590466B
CN116590466B CN202310789870.0A CN202310789870A CN116590466B CN 116590466 B CN116590466 B CN 116590466B CN 202310789870 A CN202310789870 A CN 202310789870A CN 116590466 B CN116590466 B CN 116590466B
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王永刚
杨婧怡
马鸿翔
姜朋
郭佳晖
马海港
高玉姣
戴毅
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Abstract

The application provides a set of KASP primer groups related to QTL locus Qgw-4B affecting thousand kernel weight of wheat and application thereof; qgw-4B is located on the wheat 4B chromosome, and the physical position on the China spring reference genome is 427.49Mb-427.50 Mb; the KASP primer group related to Qgw-4B consists of primers KASP_IAA2880_F1, KASP_IAA2880_F2 and KASP_IAA2880_R, the nucleotide sequences of which are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the KASP primer group provides a molecular marker with simple detection and reliable results for screening high-grain-weight wheat, can finish screening of excellent genotype materials in early generation or in a seedling stage of which seeds are not yet set, is suitable for high-efficiency detection of large-scale breeding lines, shortens a breeding period, can assist in realizing aggregation of excellent thousand-grain weight sites and other excellent characters, and accelerates breeding of new wheat varieties with outstanding yield and excellent comprehensive characters.

Description

KASP primer group related to wheat grain weight and application
Technical Field
The invention relates to the field of wheat genetic breeding, in particular to development and application of a KASP marker related to a wheat grain weight QTL locus Qgw-4B.
Background
Wheat (Triticum aestivum L) is one of the most important grain crops in the world, and improving and stabilizing wheat yield is a major strategic problem related to grain safety and national life, and is also a primary target of wheat genetic research and wheat breeding work. Among the three factors of wheat yield, namely grain weight (generally expressed in thousand grain weight), ear number and ear grain number, grain weight is an important limiting factor affecting high yield of wheat (Mu Xiaoqian, mumei wealth, liu Xincheng. (2010) correlation study of several traits of high yield wheat. Chinese ecological agriculture journal 18 (04): 787-791).
Grain weight is a complex trait under the combined action of multiple genes, mainly an additive effect, belongs to typical quantitative trait inheritance, is greatly influenced by environmental factors (Cao S, xu D, hanif M, et al (2021) Genetic architecture underpinning yield component traits in heat. Theor Appl Genet133 (6): 1811-1823), and in addition, the trait can be measured and analyzed only after wheat grain is mature and harvested, so that genetic improvement of the wheat grain by adopting a conventional breeding method often faces the problems of complex work, long period, low efficiency and the like. With the rapid development of modern molecular biology technology, modern molecular breeding technology has also developed, through the localization analysis of QTL (Quantitative trait loci ), the influence of environment on target traits can be greatly eliminated by combining molecular marker-assisted selection, and the result is accurate, which provides a powerful tool for analyzing the genetic nature of quantitative genetic traits and breeding utilization (MalikN, dwivedi N, singh a K, et al (2016) An integrated genomic strategy delineates candidate mediator genes regulating grain size and weight in price. Sci Rep 6 (1): 23253). To date, genetic mapping studies on wheat grain weight have reported that QTLs controlling wheat grain weight are distributed on multiple chromosomes of wheat (Cao S, xu D, hanif M, et al (2021) Genetic architecture underpinning yield component traits in WHEAT. Theor Appl Genet133 (6): 1811-1823;Li T,Deng G,Su Y,et al (2022) Genetic dissection ofquantitative trait loci for grain size and weight by high-resolution genetic mapping in bread wheat (Triticum aestivum l.). Theorappl Genet 135 (1): 257-271). However, compared to the identification, cloning and breeding utilization of grain weight related QTL/genes (e.g., GW2, GW7, qHKW1, qKW, etc.) in other major food crops such as rice, maize (Chen K,A,Jaremko/>et(2021) Genetic and molecular factors determining grain weight in price. Front Plant Sc 12:605799; fernandez J, messina C, salina A, et al (2022) Kernel weight contribution to yield genetic gain of maize: a global review and US case publications.J Exp Bot73 (11): 3597-3609), the genetic basis of grain weight in wheat remains poorly understood, and molecular markers, QTL/genes, etc., which can be used for breeding, lack. This is mainly due to: wheat is heterohexaploid, has huge genome, complex structure and low genetic positioning accuracy; the used parent genetic background is narrow, the breeding potential is low, and the effect is limited in population assembly and molecular marker assisted selection; mapping population differences and molecular marker differences lead to different localization results and often confidence intervals are too large, QTLs are too high or false positive QTLs are present, decreasing the practical efficiency of molecular assisted selection (Ding P, mo Z, tang H, et al (2022) A major and stable QTL for wheat spikelet number per spike validated in different genetic backgroups.j. Intergagar 21 (6): 1551-1562;Saini D,SrivastavaP,Pal N,et al. (2022) Meta-QTLs, ortho-Meta-QTLs and candidate genes for grain yield and associated traits in wheat (Triticum aestivum l.). Theorappl Genet 135 (3): 1049-1081). Therefore, based on backbone strain, the grain weight related QTL positioning and linkage marker combined with the high-quality genetic map is easier to be applied to breeding, and has great significance for improving the wheat yield.
The wheat varieties Yangmai 158 and Ningmai 9 are high-yield and high-quality wheat varieties developed by the agricultural scientific institute in the lower river region of Jiangsu province and the agricultural academy of Jiangsu province respectively, are prominent in important traits such as grain weight, spike number and the like, and have been mainly produced in wheat regions in the middle and lower Yangtze river (the definition of the wheat regions is shown in Chengjiu and the like 2012) (Chengjie, guo Wenshan, wang Longjun and the like 2012 in south China, jiangsu scientific technology publishing society), and the varieties developed by taking the wheat varieties as backbone parents are nearly hundreds, such as Yangmai 23, yangmai 4, ning Mai 14 and the like, are mainly cultivated in large-area wheat regions in the middle and lower Yangmai, however, genetic analysis of important yield traits such as Guan Yangmai, ningmai 9 and the like are reported, and the molecular markers which are applicable to high-throughput accurate detection of excellent mutation are particularly deficient.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) data is widely used for development and application of molecular markers due to its characteristics of high density, genetic stability, easy automated analysis, etc. The competitive allele-specific PCR (kompetitive allele-specific PCR, KASP) technology established based on SNP can realize accurate judgment of single-site bi-alleles, has the advantages of high throughput, strong operability and the like, is suitable for the requirement of high-efficiency detection of a large number of samples in breeding work, and has great application potential in the fields of crop trait improvement and the like (Yang Qingqing, tang Guqi, zhang Changquan and the like (2022) application and hope of KASP marking technology in main crops, and biotechnology report 38 (04): 58-71).
Disclosure of Invention
In order to solve the problems in the technology, the application designs a KASP primer group and application thereof based on the first discovered QTL locus Qgw-4B of wheat grain weight. The QTL locus Qgw-4B can obviously increase the grain weight of wheat, and the KASP mark related to the QTL locus has high typing precision, so that the QTL locus Qgw-4B has high application value for high-yield molecular breeding of wheat.
Specifically, the technical scheme provided by the application is as follows:
first, the present application provides a set of KASP primer group related to wheat grain weight, which consists of forward competitive primer KASP_IAAA2880_F1 with nucleotide sequence shown in SEQ ID NO.1, forward competitive primer KASP_IAA2880_F2 with nucleotide sequence shown in SEQ ID NO.2 and reverse universal primer KASP_IAA2880_R with nucleotide sequence shown in SEQ ID NO. 3.
The KASP primer group is developed based on wheat grain weight QTL locus Qgw-4B (self-named by the applicant), wherein the QTL locus is on a 4B chromosome of wheat, the physical position of the QTL locus on a China spring reference genome IWSSC RefSeq v1.0 is 427.49Mb-427.50Mb, and SNP molecular markers on two sides of the QTL locus are IAAV2880 and GENE-2847_1060.
The 5' end of the KASP_IAAV2880_F1 is connected with a FAM fluorescent probe label (the nucleotide sequence of which is shown as SEQ ID NO. 4); the 5' end of KASP_IAAV2880_F2 is connected with HEX fluorescent probe label (the nucleotide sequence is shown as SEQ ID NO. 5).
Secondly, the application of the KASP primer group in detecting the grain weight of wheat is provided, namely, genome DNA of the wheat to be detected is used as a template, the KASP primer is utilized for fluorescence quantitative PCR amplification, fluorescent signal scanning is carried out on an amplified product, and genotyping is carried out on the wheat material to be detected according to the fluorescent signal, wherein the method comprises the following steps: if the forward primer KASP_IAAV2880_F1 of the KASP primer has a fluorescent probe signal (fluorescence is polymerized on a sample which is close to a Y axis and shows blue color) and does not contain the forward primer KASP_IAAV2880_F2, the wheat material to be tested contains a wheat grain weight QTL locus Qgw-4B and is of an A genotype (Yangma 158 genotype); if the forward primer KASP_IAAV2880_F2 of the KASP primer has a fluorescent probe signal (fluorescence is polymerized near the X axis and shows red color) and does not contain the fluorescent probe signal of the forward primer KASP_IAAV2880_F1, the wheat material to be tested does not contain a wheat grain weight QTL locus Qgw-4B and is marked as a B genotype (Ningmai 9 genotype); the grain weight of the genotype a wheat strain is considered to be significantly higher than that of the genotype B wheat strain. The wheat variety is preferably wheat in the middle and downstream wheat regions in Yangtze river.
The PCR amplification reaction system described above was 10. Mu.L, and it included: genomic DNA at a concentration of 120ng/ul 2.5. Mu.L, 2 XSKASP Master Mix (LGC) 5. Mu.L, KASP Assay Mix 0.14. Mu.L, add ddH 2 O was filled to 10. Mu.L;
the KASPASSAy Mix preparation method comprises the following steps: each 100. Mu.L KASPASSAY Mix contains 12. Mu.L of each of two forward competitive primers at a concentration of 100. Mu.M, 30. Mu.L of the reverse universal primer at a concentration of 100. Mu.M, and 46. Mu.L of ddH 2 O was made up to 100. Mu.L.
The PCR amplification reaction procedure was as follows: 1) Thermally activating at 94 ℃ for 15min; 2) Denaturation at 94 ℃ for 20s, annealing and extension at 61-55 ℃ for 1min, 10 times of circulation, and 0.6 ℃ of annealing and extension temperature reduction in each circulation; 3) Denaturation at 94℃for 20s, annealing at 55℃for 1min,28 cycles. The amplified product is incubated at 30 ℃ and fluorescent signals are collected.
The object of the present invention also includes the use of the provided KASP primers, comprising any one of the following: 1) Application in detecting wheat grain weight related QTL locus Qgw-4B and gene localization; 2) The method is applied to breeding and creating wheat resources with different grain weights; 3) The method is applied to popularization of polymerization breeding between the QTL locus Qgw-4B related to grain weight and other excellent character loci of wheat.
The high-generation recombination selfing population is constructed by using Yangmai 158 and Ningmai 9, a QTL locus Qgw-4B for controlling the grain weight of wheat is positioned for the first time, qgw-4B is a novel QTL which is positioned on a 4B chromosome and is used for controlling the grain weight of wheat and has the physical position of 427.49Mb-427.50Mb, the QTL can remarkably increase the grain weight of the wheat, not only can provide novel functional loci and gene resources for high-yield breeding of the wheat, but also can further combine a molecular marker to assist in breeding, promote the molecular improvement of grain weight characters, improve the breeding efficiency and have strong practicability, and have high utilization value for the high-yield breeding of the wheat.
The application further develops KASP molecular markers which can be used for accurately and efficiently detecting the grain weight QTL Qgw-4B based on the sites, the molecular markers are closely linked with Qgw-4B, and the method has the advantages of stable amplification, good accuracy and convenience and rapidness in detection, and very meets the accurate detection requirement of the wheat breeding on the genotypes of large-scale genetic strains, and can be used for accurately and efficiently detecting the grain weight. The molecular marker primer combination can promote the application of the grain weight QTL Qgw-4B in high-yield wheat breeding, realize early identification and auxiliary selection of wheat taking yield as a breeding target, break the character identification difficulty caused by growth stage limitation and improve the breeding efficiency; and the polymerization of excellent grain weight sites and other excellent characters can be assisted, and the breeding of new wheat varieties with outstanding yield and excellent comprehensive characters can be quickened.
Drawings
FIG. 1 is a schematic diagram of Qgw-4B genetic mapping.
FIG. 2 is a graph showing amplification results of different primers within the Qgw-4B interval.
Wherein the values of the axes represent the intensity of the allele fluorescent signal; the black dots represent negative control (NTC) (i.e., ddH 2 0) Blue and red dots respectively represent different genotypes; x represents an unknown genotype, i.e. the primer did not achieve a typing effect.
FIG. 3 is a graph showing the genotype test results of a test material using the KASP molecular marker combination developed in the present invention.
Wherein a is a detection result in the parent high-generation backcross population, and b is a detection result in the wheat high-generation strain material; the values of the axis represent the intensity of the allele fluorescence signal, and the black dots represent the negative control (NTC) (i.e., ddH) 2 O), blue and red dots represent different genotypes, respectively.
FIG. 4 is a graph showing the results of comparison of particle weights of two different genotypes detected by KASP molecular markers in 2 test populations.
Wherein: a is a result graph of the parent high-generation backcross population, and b is a result graph of the wheat high-generation strain material; GW represents thousand kernel weight.
Detailed Description
The following examples are given in detail to make the objects, technical contents and advantages of the present invention more apparent.
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless specifically stated otherwise. In the examples, various processes and methods not described in detail are conventional methods well known in the art.
Reagents, instruments, etc. used in the examples described below are commercially available unless otherwise specified.
The wheat materials used in the invention are all germplasm resource materials saved by Jiangsu province grade germplasm resource library (crops) -university of Yangzhou agricultural college, and can be obtained and used by the technical staff and researchers in the field.
Example 1 acquisition of wheat grain weight QTL locus QTkw-4B and marker Interval
(1) In the embodiment, wheat variety Ningmai 9 is taken as a female parent, yangmai 158 is taken as a male parent, F1 generation is obtained by hybridization, F2 generation is obtained by F1 generation selfing, and a high-generation Recombination Inbred Line (RIL) population containing 282 strains is obtained by a single seed transmission method; meanwhile, the Yangmai 158 is taken as a recurrent parent, and the BC1F7 population containing 480 families is constructed through backcrossing with F1 and continuous selfing.
(2) The RIL population comprising 282 lines constructed in nigella sativa 158 was genomically scanned using the illuminea 90k gene chip to construct a linkage genetic map covering 21 chromosomes of wheat with full length 3022cM (see disclosure in literature "Jiang P, zhang X, wu L, et al (2020) A novel QTL on chromosome 5AL of Yangmai 158increases resistance to Fusarium headblight in Wheat.Plant Pathol 69:249-258").
(3) RIL populations were grown in series 2 years on Yangzhou university test bases (119.40E, 32.34N), single row sown, row length 1.2m, row spacing 0.3m,3 replicates in 2020-2021 and 2021-2022. The test base soil conditions were uniform and, prior to mature harvest, gave complete uniformity of field management by the methods disclosed in reference to Ma Hongxiang et al (2021) (Ma Hongxiang, gu Kejun, chen Huaigu (2021) & lt 100 & gt, chinese agricultural press & ltd & gt, key practical technology for wheat industry). After the seeds are ripe, 5 plants with uniform growth vigor are randomly harvested from each plant line, the seeds are naturally aired after threshing, 1,000 seeds are randomly selected from the seeds to measure the weight, the thousand-seed weight represents the weight of the seeds, and the average value is taken for data analysis. The same method is adopted for measurement: 1) 213 wheat (applicant named itself BC, as shown in table 2) thousand kernel weight randomly selected from BC1F7 population; 2) 156 wheat (the applicant has self-denominated Va as shown in table 5) thousand grain weight randomly selected from wheat high-generation (F7, F8) breeding strain materials obtained by hybridization of applicant by utilizing wheat regions in the middle and downstream of Yangtze river in a large area, wherein the wheat regions comprise Huamai No.5, zhengmai 9023, huai wheat 28, anhui wheat 54, xiangmai 25, yangmai 11, annong 92484, zhenmai No. 9, yangtze wheat No.3, sumai 188, duchei 6010, ruihua 306, nannong 9918 and the like.
(4) The software QTL Icimapping v4 is used for carrying out grain weight trait QTL positioning (Meng L, li H, zhang L, et al (2015) QTL Icimapping: integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental potentials. Crop J3:269-283) in combination with phenotype data, and a complete interval mapping method (ICIM-ADD) is adopted, wherein a LOD value of 2.5 is used as a significance threshold. Finally, a stable QTL derived from increased grain weight of Yangmai 158 and detected in various environments was located, which applicant has named itself Qgw-4B, whose genetic orientation is shown in fig. 1. Qgw-4B is located on the wheat 4B chromosome, the SNP molecular markers on both sides are IAAV2880 and GENE-2847_1060, and the physical position on the China spring reference genome IWSSC RefSeq v1.0 is 427.49Mb-427.50 Mb.
EXAMPLE 2 development, screening and validation of KASP molecular markers
(1) SNP detection is carried out on Ningmai No. 9 and Yangmai 158 by utilizing an exome capturing sequencing technology, and SNP in the QTL interval is searched and screened by taking China spring IWSSC RefSeq v1.0 as a reference genome. As shown in Table 1, a total of 6 SNPs were obtained, namely 4B_427494423 (IAAV 2880), 4B_427495111, 4B_427502540, 4B_427505127, 4B_42750635, and 4B_427506435. Respectively calling 150bp sequence information on the upstream and downstream of each SNP, designing a plurality of groups of amplification primers by using Primer Premier5 (http:// www.premierbiosoft.com/primideign /), evaluating the designed Primer mass by using DNAMAN, carrying out Primer specificity detection by using Ensembl Plants database, and converting the screened Primer sequences into KASP primers (Yang Qingqing, tang Guqi, zhang Changquan and the like) (2022) application and hope of KASP marking technology in main crops, wherein the related KASP Primer sequences are shown in table 1, and each pair of primers consists of 3 sequences, namely: forward competitive primer F1: FAM tag sequence + amplification primer sequence; forward competitive primer F2: HEX tag sequence + amplification primer sequence; reverse universal primer: amplifying the primer sequence; wherein the nucleotide sequences of the FAM tag sequence and the HEX tag sequence are as follows:
FAM tag sequence (SEQ ID No. 4): 5'-GAAGGTGACCAAGTTCATGCT-3' (FAM fluorescent group)
HEX tag sequence (SEQ ID NO. 5): 5'-GAAGGTCGGAGTCAACGGATT-3' (HEX fluorescent group)
TABLE 1KASP primer design
(2) From RIL population, 50 parts of material were randomly selected, young leaves were taken along with Ningmai No. 9 and Yangmai 158, reference was made to Stein et al (2001) (Stein N, herren G, and Keller B (2001) Anew DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant seed 120:354-356), genomic DNA was isolated therefrom by CTAB method, and the extracted DNA was uniformly diluted to about 120ng/ul with sterilized ultrapure water.
The reaction system, amplification procedure and genotyping method were as follows:
KASP reaction system (10 μl): comprises 2.5. Mu.L of sample DNA (120 ng/ul), 2 XSP Master Mix 5. Mu.L (LGC Genomics, hoddeston, UK), 0.14. Mu.L KASPassay Mix, add ddH 2 O was made up to 10. Mu.L.
The KASPASSAy Mix preparation method comprises the following steps: each 100. Mu.L KASPASSAY Mix contains 12. Mu.L of each of two forward competitive primers at a concentration of 100. Mu.M, 30. Mu.L of the reverse universal primer at a concentration of 100. Mu.M, and 46. Mu.L of ddH 2 O was made up to 100. Mu.L.
KASP reaction procedure: thermally activating at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ and extension for 1min, 10 times of circulation, and 0.6 ℃ drop in annealing extension temperature each time of circulation; denaturation at 94℃for 20s, annealing at 55℃and extension for 1min,28 cycles. The amplified product is incubated at 30 ℃ and fluorescent signals are collected.
Genotyping: fluorescent signals were scanned and genotyping was performed using a fluorescent quantitative PCR instrument Applied Biosystems ABI Viia7 Real Time PCR System (Thermo Scientific, USA), specifically: the fluorescence type of the sample polymerized near the Y axis and showing blue is FAM fluorescent group type and Yangmai 158 genotype type; the fluorescence type of the sample polymerized near the X axis and showing red is HEX fluorescent group type and Ningmai No. 9 genotype type; samples that were polymerized near the origin and displayed black were blank.
Fluorescent quantitative PCR amplification was performed using the KASP primer set obtained in example 2 (1) (see FIG. 2 for detection results). In FIG. 2, A-F are the fluorescence detection results of the 6 sets of primers in Table 1, respectively.
According to the typing result of FIG. 2, a set of KASP primers (shown as A in FIG. 2) which have good amplification efficiency and high resolution and are completely consistent with the aggregation of the corresponding genotype parents are finally screened out, the applicant designates the primer set as KASP_IAAV2880, the nucleotide sequence of the primer set is shown as SEQ ID NO.1-SEQ ID NO.3, the primer set can be applied to the breeding selection of wheat grain heavy sites Qgw-4B, the dominant allelic variation is derived from Yangmai 158, and the non-dominant allelic variation is derived from Ningmai No. 9.
Example 3KASP_IAAV2880 molecular marker application
(1) Application of KASP_IAAV2880 in high-generation backcross population taking Ningmai No. 9 and Yangmai 158 as parents
Using the KASP primer combination KASP_IAAV2880 obtained in example 2, 213 strains of the BC1F7 population of Ningmai No. 9 XYangmai 158 (i.e., BC001-BC499 in Table 2) were randomly selected, DNA was extracted along with the two parents and subjected to fluorescent quantitative PCR genotyping. DNA extraction, KASP reaction system, amplification procedure and genotyping method were all identical to those described in example 2 (FIG. 3).
The end result was a genotype A for the same fluorescent signal as Yangmai 158 and a genotype B for Ningmai 9 (Table 2).
Table 2 genotype and phenotype of 213 strains in the ning wheat No. 9 x Yangmai 158 high generation backcross population
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Statistical analysis used SPSS 19.0. Comparing the A, B genotype in table 2, and the grain weight phenotype data carrying the two genotype materials, respectively, it was found that the grain weight of wheat carrying the a genotype was higher than the grain weight of wheat carrying the B genotype (fig. 4).
Further comparing the average particle weights of the test populations, as shown in table 3, the results found: the coincidence ratio of the high grain weight genotype (a genotype) to the high grain weight phenotype (i.e., grain weight higher than population average) was 64.47%, and the coincidence ratio of the low grain weight genotype (B genotype) to the low grain weight phenotype (i.e., grain weight lower than population average) was 73.77%.
TABLE 3 comparative analysis of test strain genotypes and phenotypes (grain weights) in high generation backcross populations
Statistical analysis (t-test) further showed that the phenotypic differences of the high grain weight genotype (a genotype) and the low grain weight genotype (B genotype) reached very significant levels (table 4).
TABLE 4 statistical analysis of genotype-phenotype (grain weight) of test lines in high-generation backcross populations
Note that: * Representing the difference in significance level 0.05, representing the difference in significance level 0.01
Therefore, the wheat grain weight can be obviously increased by carrying the genotype (A genotype) type of the provided QTL locus Qgw-4B, the KASP marking type related to the wheat grain weight is high in precision, and the high-yield molecular breeding method has high application value in conclusion.
(2) Application of KASP_IAAV2880 molecular marker in wheat high-generation strain material
DNA was extracted and fluorescent quantitative PCR genotyping was performed on 156 parts of wheat high-generation line material using the KASP primer combination KASP_IAAV2880 obtained in example 2. DNA extraction, KASP reaction system, amplification procedure and genotyping method were all identical to those described in example 2 (FIG. 3).
The end result was a genotype A for the same fluorescent signal as Yangmai 158 and a genotype B for Ningmai 9 (Table 5).
TABLE 5 genotypes and phenotypes of wheat high-generation strain materials
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Statistical analysis used SPSS 19.0. Comparing the A, B genotype in table 5, and the grain weight phenotype data carrying the two genotype materials, respectively, it was found that the grain weight of wheat carrying the genotype a was higher than the grain weight of wheat carrying the genotype B (fig. 4).
Further comparing the average particle weights of the test populations, as shown in table 6, the results found: the coincidence ratio of the high grain weight genotype (a genotype) to the high grain weight phenotype (i.e., grain weight higher than population average) was 63.04%, and the coincidence ratio of the low grain weight genotype (B genotype) to the low grain weight phenotype (i.e., grain weight lower than population average) was 64.06%.
TABLE 6 comparative analysis of genotypes and phenotypes (grain weights) of wheat high-generation lines
Statistical analysis (t-test) further showed that the phenotypic differences of the high grain weight genotype (a genotype) and the low grain weight genotype (B genotype) reached very significant levels (table 7).
TABLE 7 statistical analysis of genotype-phenotype (grain weight) of wheat high-generation lines
Note that: * Representing the difference in significance level 0.05, representing the difference in significance level 0.01
Taken together, it can be seen that carrying the genotype (genotype a) type of QTL locus Qgw-4B provided can significantly increase wheat grain weight, and the KASP markers associated therewith can accomplish accurate typing of dominant allelic variation. The tested wheat in the above embodiment comprises colony materials derived from Yangtze river downstream backbone wheat variety Yangmai 158 and Ningmai 9 serving as parents and wheat high-generation (F7 and F8) breeding lines obtained by hybridization of Yangtze river downstream wheat varieties, so that the obtained QTL locus Qgw-4B and developed KASP molecular markers have high application value for wheat, especially high-yield molecular breeding of wheat in Yangtze river downstream wheat areas.

Claims (2)

1. The application of a set of KASP primer groups related to the grain weight of wheat in detecting the grain weight of wheat is characterized in that the application refers to the fluorescent quantitative PCR amplification by taking genomic DNA of wheat to be detected as a template and utilizing the KASP primer groups to obtain an amplification product; then, carrying out fluorescent signal scanning on the amplified product by using a fluorescence parting instrument, and if the fluorescence signal of the forward primer KASP_IAAA2880_F1 appears in the wheat to be detected and the fluorescence signal of the forward primer KASP_IAA2880_F2 does not appear, marking the wheat to be detected as genotype A; if the wheat to be detected has a fluorescence signal of the forward primer KASP_IAA2880_F2 and has no fluorescence signal of the forward primer KASP_IAA2880_F1, the wheat to be detected is marked as a B genotype; the grain weight of the wheat with the genotype A is higher than that of the wheat with the genotype B;
the KASP primer group consists of a forward primer KASP_IAAF2880_F1 with a nucleotide sequence shown as SEQ ID NO.1, a forward primer KASP_IAAF2880_F2 with a nucleotide sequence shown as SEQ ID NO.2 and a reverse universal primer KASP_IAAF2880_R with a nucleotide sequence shown as SEQ ID NO. 3.
2. The use according to claim 1, wherein the PCR amplification is:
PCR amplification system: template DNA at a concentration of 120ng/ul 2.5. Mu.L, 2 XSKASP Master Mix 5. Mu.L, KASP Assay Mix 0.14. Mu.L, ddH 2 O was filled to 10. Mu.L;
the preparation method of each 100 mu L of KASP Assay Mix comprises the following steps: 100. Mu.M of the forward primer KASP_IAA2880_F112. Mu.L, 100. Mu.M of the forward primer KASP_IAA2880_F212. Mu.L, 100. Mu.M of the reverse universal primer KASP_IAA2880_R30mu. L, ddH 2 O is added to 100 mu L;
PCR amplification procedure: thermally activating at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing and extension at 61-55 ℃ for 1min, 10 times of circulation, and 0.6 ℃ of temperature reduction of each circulation annealing and extension; denaturation at 94℃for 20s, annealing at 55℃for 1min,28 cycles; preserving the temperature at 30 ℃ and collecting fluorescent signals.
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