CN117660691A - Molecular marker for identifying wheat spike length gene, primer group and application - Google Patents
Molecular marker for identifying wheat spike length gene, primer group and application Download PDFInfo
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- CN117660691A CN117660691A CN202410007535.5A CN202410007535A CN117660691A CN 117660691 A CN117660691 A CN 117660691A CN 202410007535 A CN202410007535 A CN 202410007535A CN 117660691 A CN117660691 A CN 117660691A
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- 241000209140 Triticum Species 0.000 title claims abstract description 64
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 64
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 25
- 239000003147 molecular marker Substances 0.000 title claims abstract description 18
- 230000002068 genetic effect Effects 0.000 claims abstract description 7
- 210000000349 chromosome Anatomy 0.000 claims abstract description 5
- 239000003550 marker Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 8
- 238000009395 breeding Methods 0.000 claims description 8
- 230000001488 breeding effect Effects 0.000 claims description 8
- 238000004925 denaturation Methods 0.000 claims description 8
- 230000036425 denaturation Effects 0.000 claims description 8
- 238000004153 renaturation Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 101150026303 HEX1 gene Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 210000001519 tissue Anatomy 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 230000000877 morphologic effect Effects 0.000 claims description 2
- 108700026220 vif Genes Proteins 0.000 abstract description 3
- 239000013077 target material Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 8
- 235000013339 cereals Nutrition 0.000 description 5
- 230000000694 effects Effects 0.000 description 3
- 230000009418 agronomic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001013934 Erigeron breviscapus Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a molecular marker for identifying wheat spike length genes, a primer group and application, wherein a 50K high-density SNP chip is utilized to carry out whole genome scanning on genetic groups and parents, a genetic linkage map is constructed, and field spike length phenotype data is combined, wherein the molecular marker is a Chuanmai 104 spike length character related QSL.saas-5DS interval marker which is positioned on a short arm of a Chuanmai 1045D chromosome, and a physical interval is 27261456-47532045 Mb. The KASP molecular marker primer group can be used for rapidly and accurately identifying whether the target material contains the gene locus or not, and is used for molecular marker assisted selection of wheat spike length genes.
Description
Technical Field
The invention relates to the technical field of molecular marker identification of wheat, in particular to a molecular marker for identifying wheat spike length genes, a primer group and application.
Background
Wheat yield has been the focus of attention by breeders. At present, due to frequent extreme weather and reduced arable area, the demand for wheat is also increasing with the increase of population number. Increasing wheat yield is an important measure for protecting grain safety. Besides being influenced by three factors of yield, the wheat yield is used as an important agronomic property of wheat, the wheat spike-producing space and spike density are influenced, the grain size and the grain number of wheat are indirectly influenced, and the grain size and the grain number are important factors influencing the wheat yield and are also important reference indexes of breeders in the process of variety breeding. Sichuan wheat 104 is a breakthrough wheat variety cultivated by using Sichuan wheat 42 and Sichuan agriculture 16 as parents in the crop institute of Sichuan agricultural sciences, is a wheat variety with the largest popularization area in Sichuan, and is one of important backbone parents in Sichuan wheat areas. Under the ecological environment condition of Sichuan, sichuan wheat 104 is one of the most representative wheat varieties with high and stable yield in Sichuan wheat regions and ideal synergistic expression of the high yield traits and the comprehensive agronomic traits. The development of the Sichuan wheat 104 ear length gene locus has important significance for molecular breeding of wheat.
Disclosure of Invention
The invention aims to provide a molecular marker for identifying wheat spike length genes, a primer group and application thereof, which can rapidly and accurately detect new gene locus QSL.saas-5DS of Chuanmai 104 spike length and solve the problems of rapid improvement of wheat spike length characters and the like in the breeding process.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the molecular marker for identifying the wheat spike length gene is a Chuanmai 104 spike length property related QSL.saas-5DS interval marker, is positioned on a short arm of a Chuanmai 1045D chromosome, has a physical interval of 27261456-47532045 Mb, corresponds to the 27261456 th and 47532045 th bases of a 5D chromosome of a China spring reference genome, is G or A, is AX-109321838 and AX-108932356, and has the following sequence:
GCTCAAGTTCATCTGTGTAAAGCAGCTGTCTCAGC [ A/G ] CATAGGTGTGAGCTCTGTGGCGTCCACCCTTGCTC; the AX-108932356 sequence is as follows:
TGACGAGGTGCATTTCAGATCTATCATCGACGGTT[A/G]CCAGGAAGCCGAGTGAGGACCCAATCAAAAACCTA。
the invention also provides a primer group for identifying wheat spike length genes, which comprises FAM primers, HEX primers and COM primers, wherein the primer group is designed for QSL.saas-5DS interval marks AX-109321838 and AX-108932356 related to Chuanmai 104 spike length characters, and the sequence KASP-AX-109321838 of the primer group marked by AX-109321838 is as follows:
FAM-1：GAAGGTGACCAAGTTCATGCTGTAAAGCAGCTGTCTCAGCA
HEX-1：GAAGGTCGGAGTCAACGGATTGTAAAGCAGCTGTCTCAGCG
COM-1：GAGCAAGGGTGGACGCCAC；
the primer group sequence KASP-AX-108932356 marked by AX-108932356 is as follows:
FAM-2：GAAGGTGACCAAGTTCATGCTCAGATCTATCATCGACGGTTA
HEX-2：GAAGGTCGGAGTCAACGGATTCAGATCTATCATCGACGGTTG
COM-2：TAGGTTTTTGATTGGGTCCTC。
furthermore, the primer group for identifying the wheat spike length gene is applied to wheat gene identification or wheat molecular marker assisted breeding.
Furthermore, the application of the primer group for identifying the wheat spike length genes in the identification of the wheat genes comprises the following steps:
s1, extracting DNA of leaf tissue of a wheat sample to be detected as a template;
s2, performing PCR amplification on the DNA template by using KASP-AX-109321838 and KASP-AX-108932356;
s3, performing fluorescence scanning on the PCR amplification product, and performing genotype analysis:
AX-109321838: the plant genotype is AA, and the wheat contains a Sichuan wheat 104 ear length locus; the plant genotype is GG, and the wheat does not contain a Chuanmai 104 spike length site;
AX-108932356: the plant genotype is AA, and the wheat does not contain the scion length locus of Chuanmai 104; the plant genotype is GG, and the wheat contains a Sichuan wheat 104 ear length locus.
Furthermore, the application of the primer group for identifying wheat spike length genes in wheat gene identification is that S2
The PCR reaction system is as follows: in a total volume of 2. Mu.L, 1. Mu.L of 2 XMaster Mix, 0.004. Mu.L of upstream primer FAM, 0.004. Mu.L of upstream primer HEX, 0.012. Mu.L of universal primer COM, 1. Mu.L of DNA template, and ultrapure water was supplemented to 2. Mu.L;
the PCR reaction conditions were: firstly, pre-denaturing for 10 min at 95 ℃; followed by 10 cycles of drop PCR, denaturation at 95℃for 20 s, renaturation extension at 61-55℃for 45s (0.6℃decrease per cycle), followed by a conventional PCR process of 35 cycles, denaturation at 95℃for 20 s, renaturation extension at 55℃for 45s, and finally reading of fluorescent signal at 30℃for 30 s. If the sample clustering effect is not significant, the denaturation at 95℃for 20 s, renaturation at 55℃for 45s 5-10 cycles can be repeated, and the fluorescent signal 30 s can be read at 30 ℃.
Based on the technical scheme, the embodiment of the invention at least has the following technical effects:
the molecular marker provided by the invention can be used for carrying out molecular marker auxiliary selection on the important characteristics of the spike length in the wheat breeding process, is used for carrying out morphological genetic improvement on the spike length of the wheat, can improve the detection efficiency, shortens the breeding process, and lays a molecular foundation for breeding high-yield stable-yield wheat varieties with excellent spike length genes.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings that are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained from the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the molecular detection results of KASP-AX-109321838 on the ear length of a group material according to the embodiment of the present invention;
FIG. 2 is a graph showing the molecular detection results of KASP-AX-108932356 on the ear length of a group material according to the embodiment of the present invention;
in the figure, 1, chuanmai 104; 2. sichuan wheat 104 spike length gene QSL.saas-5DS derivative offspring material; 3. bai Maomai; 4. bai Maomai derivative offspring populations; 5. negative control.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The genetic population and parents are subjected to genome-wide scanning by using a 50K high-density SNP chip to construct a genetic linkage map, the genetic linkage map is combined with field spike length phenotype data, a new spike length gene locus QSL.saas-5DS from Chuanmai 104 is positioned on a short arm of a 5D chromosome of Chuanmai 104, a physical interval is 27261456-47532045 Mb, and a molecular marker primer set is designed by analyzing the SNP locus.
AX-109321838：
GCTCAAGTTCATCTGTGTAAAGCAGCTGTCTCAGC[A/G]CATAGGTGTGAGCTCTGTGGCGTCCACCCTTGCTC
AX-108932356：
TGACGAGGTGCATTTCAGATCTATCATCGACGGTT[A/G]CCAGGAAGCCGAGTGAGGACCCAATCAAAAACCTA
The sequences of the primer labeled with KASP are:
a first pair of KASP tagged primers: KASP-AX-109321838
FAM-1：GAAGGTGACCAAGTTCATGCTGTAAAGCAGCTGTCTCAGCA
HEX-1：GAAGGTCGGAGTCAACGGATTGTAAAGCAGCTGTCTCAGCG
COM-1：GAGCAAGGGTGGACGCCAC
A second pair of KASP tagged primers: KASP-AX-108932356
FAM-2：GAAGGTGACCAAGTTCATGCTCAGATCTATCATCGACGGTTA
HEX-2：GAAGGTCGGAGTCAACGGATTCAGATCTATCATCGACGGTTG
COM-2：TAGGTTTTTGATTGGGTCCTC
And (2) performing KASP molecular marker detection on the parent Chuanmai 104 and Bai Maomai, 32 parts of Chuanmai 104 derived offspring material and 26 parts of white hair wheat derived offspring material by using a KASP-AX-109321838 primer group and a KASP-AX-108932356 primer group, wherein as shown in figures 1 and 2, whether the test material contains a Chuanmai 104 spike length gene locus QSL.saas-5DS can be efficiently and rapidly detected by the two primer groups.
In fig. 1 and 2: 1,2 represents a detection result consistent with the genotype of Chuanmai 104 and has a phenotype of spike length, wherein 1 is parent Chuanmai 104,2 which represents 32 parts of Chuanmai 104 derivative offspring material; 3,4 represents a detection result consistent with Bai Maomai genotype, has a phenotype of spike length, does not carry Chuanmai 104 spike length genotype, wherein 3 represents erigeron breviscapus, and 4 represents 26 Bai Maomai derivative offspring which do not carry Chuanmai 104 spike length locus; 5 is a negative control, and water is used as a detection sample.
The detection method comprises the following steps:
s1, extracting Chuanmai 104, bai Maomai, DNA of leaf tissue of a derivative progeny material of Chuanmai 104 and Bai Maomai as a template;
s2, performing PCR amplification on the DNA template by using KASP-AX-109321838 and KASP-AX-108932356;
the PCR reaction system is as follows: in a total volume of 2. Mu.L, 1. Mu.L of 2 XMaster Mix, 0.004. Mu.L of upstream primer FAM, 0.004. Mu.L of upstream primer HEX, 0.012. Mu.L of universal primer COM, 1. Mu.L of DNA template, and ultrapure water was supplemented to 2. Mu.L;
the PCR reaction conditions were: firstly, pre-denaturing for 10 min at 95 ℃; followed by 10 cycles of drop PCR, denaturation at 95℃for 20 s, renaturation extension at 61-55℃for 45s (0.6℃decrease per cycle), followed by a conventional PCR process of 35 cycles, denaturation at 95℃for 20 s, renaturation extension at 55℃for 45s, and finally reading of fluorescent signal at 30℃for 30 s. If the sample clustering effect is not significant, the denaturation at 95℃for 20 s, renaturation at 55℃for 45s 5-10 cycles can be repeated, and the fluorescent signal 30 s can be read at 30 ℃.
S3, performing fluorescence scanning on the PCR amplification product, and performing genotype analysis.
Finally, it should be noted that:
the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (5)
1. The molecular marker for identifying the wheat spike length gene is characterized in that the molecular marker is a Chuanmai 104 spike length character related QSL.saas-5DS interval marker which is positioned on a short arm of a Chuanmai 1045D chromosome, the physical interval is 27261456-47532045 Mb, the molecular markers are AX-109321838 and AX-108932356, and the sequence of AX-109321838 is as follows: GCTCAAGTTCATCTGTGTAAAGCAGCTGTCTCAGC [ A/G ] CATAGGTGTGAGCTCTGTGGCGTCCACCCTTGCTC; the AX-108932356 sequence is as follows:
TGACGAGGTGCATTTCAGATCTATCATCGACGGTT[A/G]CCAGGAAGCCGAGTGAGGACCCAATCAAAAACCTA。
2. a primer set for identifying wheat spike length genes, which is characterized by comprising FAM primers, HEX primers and COM primers, wherein the primer set is designed for Sichuan wheat 104 spike length property related QSL.saas-5DS interval markers AX-109321838 and AX-108932356, and the sequence KASP-AX-109321838 of the primer set marked by AX-109321838 is as follows:
FAM-1：GAAGGTGACCAAGTTCATGCTGTAAAGCAGCTGTCTCAGCA
HEX-1：GAAGGTCGGAGTCAACGGATTGTAAAGCAGCTGTCTCAGCG
COM-1：GAGCAAGGGTGGACGCCAC；
the primer group sequence KASP-AX-108932356 marked by AX-108932356 is as follows:
FAM-2：GAAGGTGACCAAGTTCATGCTCAGATCTATCATCGACGGTTA
HEX-2：GAAGGTCGGAGTCAACGGATTCAGATCTATCATCGACGGTTG
COM-2：TAGGTTTTTGATTGGGTCCTC。
3. the use of the primer set for identifying wheat spike length genes according to claim 2 in wheat gene identification, wheat spike length morphological genetic improvement or wheat molecular marker assisted breeding.
4. Use of a primer set for identifying wheat spike length genes according to claim 3 for wheat gene identification, comprising the steps of:
s1, extracting DNA of leaf tissue of a wheat sample to be detected as a template;
s2, performing PCR amplification on the DNA template by using the KASP-AX-109321838 and KASP-AX-108932356 as described in claim 2;
s3, performing fluorescence scanning on the PCR amplification product, and performing genotype analysis:
AX-109321838: the plant genotype is AA, and the wheat contains a Sichuan wheat 104 ear length locus; the plant genotype is GG, and the wheat does not contain a Chuanmai 104 spike length site;
AX-108932356: the plant genotype is AA, and the wheat does not contain the scion length locus of Chuanmai 104; the plant genotype is GG, and the wheat contains a Sichuan wheat 104 ear length locus.
5. The use of the primer set for identifying wheat spike length gene according to claim 4, wherein in S2
The PCR reaction system is as follows: in a total volume of 2. Mu.L, 1. Mu.L of 2 XMaster Mix, 0.004. Mu.L of upstream primer FAM, 0.004. Mu.L of upstream primer HEX, 0.012. Mu.L of universal primer COM, 1. Mu.L of DNA template, and ultrapure water was supplemented to 2. Mu.L;
the PCR reaction conditions were: firstly, pre-denaturing for 10 min at 95 ℃; followed by 10 cycles of drop PCR, denaturation at 95℃for 20 s, renaturation extension at 61-55℃for 45s, followed by a conventional PCR process of 35 cycles, denaturation at 95℃for 20 s, renaturation extension at 55℃for 45s, and finally reading of the fluorescent signal at 30℃for 30 s.
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