CN112760399B - Major QTL site for controlling grain length of wheat grains, KASP primer closely linked with major QTL site and application of KASP primer - Google Patents
Major QTL site for controlling grain length of wheat grains, KASP primer closely linked with major QTL site and application of KASP primer Download PDFInfo
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Abstract
The invention discloses a main effect QTL site for controlling grain length of wheat grains, a KASP primer tightly linked with the main effect QTL site and application of the KASP primer, wherein the genetic position of the main effect QTL site QKl. sau-6A on a 6A chromosome of wheat is 16.78-31.64cM, SNP molecular markers on two sides of the main effect QTL site QKl. sau-6A are respectively AX-110126107 and AX-109894590, and the physical positions on an IWGSC RefSeq v1.0 reference genome in Chinese spring are respectively 13.35Mb and 617.82 Mb. The invention discloses a major QTL locus QKl. sau-6A for controlling grain length of grains for the first time, and the QTL can remarkably increase the grain length of the grains and improve the grain weight of wheat grains, and has higher value for high-yield breeding of the wheat.
Description
Technical Field
The invention relates to a major QTL site for controlling grain length of wheat grains, in particular to a major QTL site for controlling grain length of wheat grains, a KASP primer closely linked with the major QTL site and application of the KASP primer.
Background
Wheat is one of the three staple food crops in the world, providing approximately 20% of the protein and calories in human food. In recent years, with the reduction of the arable area, under the influence of a plurality of factors such as population growth and the like, the food yield in the future is difficult to meet the demand of people on food. It is predicted that by 2050, the grain yield per year will need at least 2.4% increase to meet the demand of people for grain. The wheat yield is mainly influenced by three factors of yield, namely the number of ears per unit area, thousand kernel weight and ear number. Thousand kernel weight, one of the three elements of yield, is mainly influenced by kernel traits, including grain length, grain width and grain thickness, which show extremely significant positive correlation with thousand kernel weight. Therefore, the identification and introduction of favorable alleles of the seed properties in the breeding engineering of wheat has important significance for improving the yield of wheat and ensuring national food safety.
Traditional breeding methods obtain many desirable traits by crossing different varieties or lines. This process is also a gradual integration of superior genes in different materials. However, the molecular mechanisms of inheritance for these traits are not known and it takes a lot of time and effort to identify the phenotype, which not only results in increased breeding costs but also in low breeding efficiency. With the continuous development of molecular sequencing technology, molecular marker-assisted breeding provides an effective solution for the bottleneck faced by the traditional breeding method, allows selection in early generations of target traits, shortens breeding process, and has accurate, stable and convenient detection result.
Single Nucleotide Polymorphism (SNP) molecular markers are one of the most abundant molecular markers in plants, and have the characteristics of large quantity, wide distribution, stable heredity, easiness in genotyping and the like. With the development of sequencing technology, more and more SNP molecular markers are discovered and used for genetic research of various agronomic traits of Wheat, including current commonly used SNP gene chips, such as 9K, 90K, 15K, 660K, 55K, 820K, 35K and 50KSNP gene chips (Sun C, Dong Z, ZHao L, et al.the Wheat 660K SNP array definitions for marker-assisted selection in polyploid Wheat [ J ]. Plant Biotechnology Journal,2020,18 (6)). The gene chips are integrated with thousands of SNP molecular markers, and the quality of a genetic map is improved.
Competitive Allele-Specific PCR (KASP) technology is based on the Specific matching of the base at the end of a primer to carry out accurate double-Allele judgment on SNP sites, and is one of the current SNP genotyping methods commonly used at home and abroad. The technology is based on two different fluorescent probes, and is combined with a plurality of SNP-PCR primers aiming at specific sites, so that the detection of the sites can be realized. And compared with a Taqman method, the method has the advantage that the cost is greatly reduced. Besides, the KASP technology has low detection error, extremely high stability and accuracy and obvious advantages in large-scale marker detection work. Therefore, the method has important significance for developing the KASP molecular marker linked with the major QTL or gene for controlling grain length of grains, and breeding wheat varieties with longer grains by using the molecular marker, laying theoretical foundation for wheat high-yield breeding and providing molecular marker assisted breeding means.
Disclosure of Invention
The invention aims to provide a main effect QTL site for controlling grain length of wheat grains, a KASP primer closely linked with the main effect QTL site and application of the primer, wherein the main effect QTL site is QKl.
In order to achieve the purpose, the invention provides a main QTL locus QKl.sau-6A for controlling grain length of wheat grains, wherein the genetic position of the main QTL locus QKl.sau-6A on a 6A chromosome of wheat is 16.78-31.64cM, SNP molecular markers on two sides of the main QTL locus QKl.sau-6A are respectively AX-110126107 and AX-109894590, and the physical positions of the two molecular markers of AX-110126107 and AX-109894590 on an IWGSC RefSeq v1.0 of a Chinese spring reference genome are respectively 13.35Mb and 617.82 Mb.
Another objective of the invention is to provide KASP primers aiming at SNP molecular marker AX-109894590, wherein the nucleotide sequence of forward primer 1 of the KASP primers is shown as SEQ ID NO.2, the nucleotide sequence of forward primer 2 is shown as SEQ ID NO.3, and the nucleotide sequence of reverse primer is shown as SEQ ID NO. 4; wherein, the 5' ends of the forward primer 1 and the forward primer 2 are connected with different fluorescent label sequences.
Preferably, the 5' end of the forward primer 1 is connected with an F probe, and the nucleotide sequence of the F probe is shown as SEQ ID NO. 5; the 5' end of the forward primer 2 is connected with an H probe, and the nucleotide sequence of the H probe is shown in SEQ ID NO. 6.
The invention also aims to provide a kit for identifying the major QTL QKl. sau-6A of wheat grain length, wherein the primers in the kit are the KASP primers.
Another object of the present invention is to provide a method for identifying a major QTL qkl. sau-6A for wheat grain length, which comprises: the KASP primer is used for carrying out fluorescence quantitative PCR amplification, and the wheat material to be detected is genotyped according to the PCR amplification result, which comprises the following steps: if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer but does not contain the fluorescent signal of the fluorescent probe of the forward primer 1, the wheat material to be detected contains the major QTL site QKl. sau-6A for controlling the grain length of wheat grains; and if the wheat material to be detected has a fluorescence signal of the fluorescent probe of the forward primer 1 of the KASP primer but does not contain the fluorescence signal of the fluorescent probe of the forward primer 2, the wheat material to be detected does not contain the major QTL site QKl.
Preferably, the genotype of the wheat material to be tested containing the major QTL locus QKl. sau-6A for controlling the grain length of wheat grains is recorded as the genotype B, the genotype of the wheat material to be tested not containing the major QTL locus QKl. sau-6A for controlling the grain length of wheat grains is recorded as the genotype A, and the grain length of the wheat strain with the genotype B is significantly higher than that of the wheat strain with the genotype A.
Preferably, the reaction system for the fluorescent quantitative PCR amplification comprises: KASP Mastermix, the KASP primer, the genome DNA of the wheat material to be detected and RNase-free deionized water; wherein, in the KASP primer, the volume ratio of the forward primer 1 to the forward primer 2 to the reverse primer is 2:2: 5.
Preferably, the reaction procedure of the fluorescent quantitative PCR amplification comprises: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20 s; annealing and extending at 61 ℃ for 40s, circulating for 10 times, and reducing the annealing and extending temperature by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20 s; annealing and extending for 40s at 55 ℃; circulating for 30 times; keeping the temperature at 25 ℃ and collecting fluorescence signals.
Another object of the present invention is to provide KASP primers designed for SNP molecular marker AX-109894590, the use of which comprises any one of: (1) the application in detecting major QTLQKl. sau-6A of wheat grain length and gene positioning; (2) the application in breeding and creating wheat resources with different grain lengths; (3) the method is applied to popularization of the aggregate breeding between the grain length major QTL QKl. sau-6A and other excellent trait loci of wheat.
The main effect QTL locus for controlling the grain length of wheat grains, the KASP primer closely linked with the main effect QTL locus and the application of the KASP primer have the following advantages:
the invention discloses a major QTL locus QKl. sau-6A for controlling grain length for the first time, which is a new major QTL locus located at 16.78-31.64cM on the 6A chromosome of wheat, and the QTL can significantly increase the grain length of grains and improve the grain weight of wheat grains, and has higher value for high-yield breeding of wheat.
In addition, the invention also provides a KASP molecular marker KASP-AX-109894590 for accurately detecting the QTL QKl.sau-6A based on a fluorescent quantitative PCR platform, wherein the molecular marker is closely linked with the QKl.sau-6A, presents the characteristic of coseparation marker, has the characteristics of stable amplification and convenient and quick detection, and can be used for molecular marker assisted breeding. Moreover, the molecular marker can greatly reduce phenotype identification work and can be utilized in the seedling stage of wheat, so that non-target plants are eliminated, the breeding cost is saved, and the breeding efficiency is improved.
Drawings
Fig. 1 is the location of major QTL qkl. sau-6A on the chromosome that controls grain length.
FIG. 2 shows the result of genotyping the parents Chuannon 18 and T1208 by fluorescence quantitative PCR using KASP molecular marker KASP-AX-109894590 linked to grain length major QTL in Experimental example 2 of the present invention.
FIG. 3 shows the result of genotyping of 18 XT 1208 high generation inbred progeny of Chuannon by fluorescence quantitative PCR using KASP molecular marker KASP-AX-109894590 in Experimental example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Experimental example 1 acquisition of major QTL QKl. sau-6A and KASP-AX-109894590 molecular markers for controlling grain length
(1) Hybridizing by using a variety Chuannong 18 with shorter grain length as a female parent and using a wheat material T1208 with longer grain length as a male parent to obtain F1Generation, F1Selfing to obtain F2Generation, obtaining a high-generation self-bred genetic population containing 371 lines by a single-seed transmission method;
(2) 371 strains (F) constructed by Chuannong 18 XT 1208 using 55K SNP gene chip11-F13) The genome of the recombinant inbred population is scanned, and a linkage genetic map is constructed, wherein the genetic linkage map covers 21 chromosomes of wheat and has the total length of 4192.62 cM;
(3) the qigong and qigong wheat breeding base is planted in qigong and qigong wheat breeding base of Sichuan agriculture university (qigong and qigong wheat breeding base in Sichuan province, 30 degrees 25 'in northern latitude, 103 degrees 28' in east longitude and 493.3m in altitude) in 2016, 2018, 2019 and 2020. Adopting a random block design, wherein the row length of a cell is 2m, 10 plants are planted in each row, the row spacing is 0.25m, 4 rows are planted in each plant, the three times of the design are repeated, the cultivation management is the same as a field standard management method, and a herbicide and an insecticide are used to ensure that no insect pest and no disease exist in the field in the growth process;
(4) threshing after wheat is mature and harvested, naturally drying to 12% of water content, measuring grain length by using a ten thousand deep SC-G automatic seed test analysis and thousand kernel weight analyzer (produced by Hangzhou ten thousand deep detection science and technology Co., Ltd.), carrying out QTL positioning on phenotype data of grain length of three environments by using software IcMapping 4.1, finally positioning a stable main effect QTL QKl.sau-6A which is derived from a parent T1208 and is detected under three environments on a wheat 6A chromosome, wherein the average LOD value is 32.10, and the average interpreted phenotype variation rate is 23.88%. The molecular markers AX-110126107 and AX-109894590 flanking the QTL are located on the 6A chromosome at 16.78-31.64 cM;
(5) by searching SNP molecular markers positioned in the QTL molecular marker interval, 4 SNP molecular markers are searched, namely AX-109894590(SEQ ID NO.1, the 36 th base has C or T polymorphism and is shown by n in the sequence table), AX-110139825(SEQ ID NO.16, the 36 th base has A or G polymorphism and is shown by n in the sequence table), AX-110714735(SEQ ID NO.17, the 36 th base has C or T polymorphism and is shown by n in the sequence table) and AX-110126107(SEQ ID NO.18, the 36 th base has C or T polymorphism and is shown by n in the sequence table), then the sequence marked by SNP molecules is designed into KASP primers on a PolyMarker website, the sequences of the KASP primers are converted into the KASP primers aiming at 4 SNP marks in total, the related primer sequences are shown in the following table 1, and each pair of primers consists of 3 sequences, namely: forward primer 1: f probe + amplification primer sequence; forward primer 2: h probe + amplification primer sequence; reverse primer: the nucleotide sequences of the amplification primer sequence, the F probe and the H probe are as follows:
f probe (SEQ ID NO. 5): 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H probe (SEQ ID NO. 6): 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached).
TABLE 1 four pairs of KASP primer sequences
Experimental example 2 screening of KASP molecular marker polymorphic primers between parents
(1) Extracting DNA of parent Chuannong 18 and T1208 trefoil stages by adopting a CTAB method;
(2) and performing fluorescent quantitative PCR amplification by using the synthesized primers, and performing polymorphism analysis on the parents according to the PCR amplification result. Selected reaction system for fluorescent quantitative PCR amplification: KASP Mastermix 4.5. mu.L, KASP Assay Mix 2. mu.L, 50ng template DNA, RNase-free deionized water to a total amount of 10. mu.L, wherein the KASP Assay Mix contains nucleotide sequences for each pair of primers as shown in Table 1, and the volume ratio of the three primers is 2:2: 5;
the fluorescent quantitative PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20 s; annealing and extending at 61 ℃ for 40s, circulating for 10 times, and reducing the annealing and extending temperature by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20 s; annealing and extending for 40s at 55 ℃; circulating for 30 times; preserving the temperature at 25 ℃, and collecting a fluorescence signal;
(3) screening of polymorphic primers between parents, finally screening a pair of primers which have polymorphism between the parents Chuannon 18 and T1208 and are named as KASP-AX-109894590, wherein the result of genotyping the parents is shown in figure 1, the genotype of the parent Chuannon 18 has a fluorescence type of FAM fluorophore, and the genotype of the parent T1208 has a fluorescence type of HEX fluorophore.
Experimental example 3 application of KASP-AX-109894590 molecular marker in Chuannong 18 XT 1208 high-generation inbred population
The same method as the above is adopted to randomly select 127 strains (including two parents) in the Chuan nong 18X T1208 high-generation selfing population to extract DNA for carrying out fluorescence quantitative PCR genotyping, wherein the genotyping result is the same as that of the Chuan nong 18 and is marked as an A genotype, the genotyping result is the same as that of the T1208 and is marked as a B genotype, finally, 33 strains with the A genotypes are seen, 94 strains with the B genotypes are seen, and the grain length of the strain with the B genotypes is obviously higher than that of the strain with the A genotypes, and the results are shown in tables 2 and 3.
Table 2 genotyping 127 lines in Chuannong 18 XT 1208 high generation inbred population
Note: a is the same as Chuannong 18; b is the same as T1208.
Table 3 shows the results of t test
Note: the numbers in brackets indicate the number of lines carrying the corresponding genotypic material.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
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Claims (7)
1. The KASP primer aiming at the SNP molecular marker AX-109894590 is characterized in that the nucleotide sequence of a forward primer 1 of the KASP primer is shown as SEQ ID NO.2, the nucleotide sequence of a forward primer 2 of the KASP primer is shown as SEQ ID NO.3, and the nucleotide sequence of a reverse primer of the KASP primer is shown as SEQ ID NO. 4; wherein, the 5' ends of the forward primer 1 and the forward primer 2 are connected with different fluorescent label sequences.
2. A kit for identifying major QTL qkl. sau-6A of wheat grain length, wherein the primers in the kit are KASP primers according to claim 1; the genetic position of the major QTL QKl. sau-6A on the 6A chromosome of wheat is 16.78-31.64 cM.
3. A method for identifying a major QTL qkl. sau-6A for wheat grain length, the method comprising:
the KASP primer of claim 1, used for fluorescent quantitative PCR amplification, and genotyping the wheat material to be detected according to the PCR amplification result, wherein the method comprises the following steps:
if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer but does not contain the fluorescent signal of the fluorescent probe of the forward primer 1, the wheat material to be detected contains the wheat grain length major QTL QKl.
If the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer but does not contain the fluorescent signal of the fluorescent probe of the forward primer 2, the wheat material to be detected does not contain the wheat grain length major QTL QKl.sau-6A;
wherein the genetic position of the major QTL QKl. sau-6A on the 6A chromosome of wheat is 16.78-31.64 cM;
the variety of the wheat is T1208 or Chuannong 18.
4. The method according to claim 3, wherein the genotype of the wheat material to be tested containing the wheat grain length major QTL QKl.sau-6A is recorded as genotype B, the genotype of the wheat material to be tested not containing the wheat grain length major QTL QKl.sau-6A is recorded as genotype A, and the grain length of the wheat strain with genotype B is significantly higher than that of the wheat strain with genotype A.
5. The method of claim 3, wherein the reaction system for the fluorescent quantitative PCR amplification comprises: KASP Mastermix, KASP primer as claimed in claim 1, genomic DNA of wheat material to be tested, RNase-free deionized water; wherein, in the KASP primer, the volume ratio of the forward primer 1 to the forward primer 2 to the reverse primer is 2:2: 5.
6. The method of claim 3, wherein the reaction sequence of the fluorescent quantitative PCR amplification comprises: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing and extension at 61 ℃ for 40s, and circulation for 10 times, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20s, annealing and extension at 55 ℃ for 40s, and circulating for 30 times; keeping the temperature at 25 ℃ and collecting fluorescence signals.
7. Use of the method for identifying wheat grain length major QTL qkl. sau-6A according to claim 3, wherein the use comprises any of:
(1) the application in breeding and creating wheat resources with different grain lengths;
(2) the method is applied to popularization of the aggregate breeding between the grain length major QTL QKl. sau-6A and other excellent trait loci of wheat.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106434953A (en) * | 2016-10-27 | 2017-02-22 | 宁波大学 | Detection and application of novel molecular marker hsa-circ-0074362 for gastric cancer |
| CN108998562A (en) * | 2018-09-10 | 2018-12-14 | 中国农业科学院作物科学研究所 | Based on grain length genetic marker and application under 895 genetic background of wheat in wheat breed |
| CN111471790A (en) * | 2020-04-26 | 2020-07-31 | 四川农业大学 | Molecular marker closely linked with wheat grain filling rate QT L QGfr. sicau-7D.1 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017053247A1 (en) * | 2015-09-21 | 2017-03-30 | Dow Agrosciences Llc | Wheat with elevated fructan, arabinoxylan |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434953A (en) * | 2016-10-27 | 2017-02-22 | 宁波大学 | Detection and application of novel molecular marker hsa-circ-0074362 for gastric cancer |
| CN108998562A (en) * | 2018-09-10 | 2018-12-14 | 中国农业科学院作物科学研究所 | Based on grain length genetic marker and application under 895 genetic background of wheat in wheat breed |
| CN111471790A (en) * | 2020-04-26 | 2020-07-31 | 四川农业大学 | Molecular marker closely linked with wheat grain filling rate QT L QGfr. sicau-7D.1 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| "QTL Mapping and Validation for Kernel Area and Circumference in Common Wheat via High-Density SNP-Based Genotyping";Tianheng Ren等;《Front. Plant Sci》;20210817;第12卷;第1-13页 * |
| "利用SNP基因芯片技术进行小麦遗传图谱构建及重要农艺性状QTL分析";高尚等;《应用与环境生物学报》;20160225;第22卷(第1期);第85-94页 * |
| "小麦单位面积穗数和粒长主效QTL紧密连锁KASP标记的开发及其效应评价";范涛等;《中国农业科学》;20210716;第54卷(第14期);第2941-2951页 * |
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