CN113278724B - Molecular marker for identifying wheat leaf villus gene, primer set and application - Google Patents

Molecular marker for identifying wheat leaf villus gene, primer set and application Download PDF

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CN113278724B
CN113278724B CN202110607480.8A CN202110607480A CN113278724B CN 113278724 B CN113278724 B CN 113278724B CN 202110607480 A CN202110607480 A CN 202110607480A CN 113278724 B CN113278724 B CN 113278724B
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villus
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CN113278724A (en
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刘泽厚
王琴
万洪深
杨凡
李俊
杨武云
杨宁
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Crop Research Institute Of Sichuan Academy Of Agricultural Sciences
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Abstract

The invention provides a molecular marker for identifying wheat leaf villus genes, a primer group and application thereof. The gene is a new gene, is positioned on a 7BS chromosome of a wheat local variety Bai Maomai and is named QLP.saas-7BS; it was located between SNP chip probes AX-86175290 and AX-86172908, respectively, and KASP markers were developed according to the probe sequences, named KASP-AX-86175290 and KASP-AX-86172908, respectively. The invention can effectively detect and screen wheat villus character by using newly developed KASP molecular marker, and applies molecular marker technology in breeding wheat varieties with leaf villus phenotype character, thereby saving production cost, improving selection efficiency and shortening breeding period of wheat varieties; and the detection is convenient and quick, and is not influenced by the environment.

Description

Molecular marker for identifying wheat leaf villus gene, primer set and application
Technical Field
The invention relates to the field of molecular biology, in particular to a molecular marker for identifying new genes of wheat leaf villus, a primer group and application thereof.
Background
The main function of the wheat leaf is to carry out photosynthesis, produce organic matters and be an important organ for respiration and transpiration of wheat. There are 5 leaves of wheat, with scutellum (degenerated leaves), coleoptile (incomplete leaves), tillering sheath (incomplete leaves), husk (abnormal leaves) and green leaves (complete leaves). By leaf we mean a fully developed green leaf. The leaf consists of leaf body (leaf), leaf pillow, leaf sheath, leaf ear and leaf tongue. The joint of the leaf and the leaf sheath is called a leaf pillow. Leaf sheaths are grown on the nodes of the stems and surround all or part of the internodes, and the main function is to strengthen the strength of the stems and also to carry out photosynthesis. The blade tongue is positioned at the junction of the blade and the blade sheath, and has the main function of preventing rainwater, dust and pests from invading the blade sheath; the leaf ears are very small, and are planted on the left side and the right side of the basal part of the leaf blade, the stem is surrounded, and some varieties do not have leaf ears. The leaf ears have different colors of red, purple, green and the like, and can be used as marks for identifying varieties. The number of the wheat leaves varies to a certain extent according to the variety, environment and cultivation measures, but the number of the most suitable main stems and leaves is relatively stable in certain areas. Some varieties of wheat leaves are provided with villi, the villi of the wheat leaves is an important phenotype character related to stress resistance, and the wheat material with the villi of the wheat leaves has a plurality of stress resistances, such as drought resistance, insect resistance, ultraviolet injury resistance and the like. At present, leaves of cultivated wheat varieties have little or no fluff.
In the prior art, the research on the molecular genetic mechanism of the leaf villus gene is less, and particularly, the molecular basic research on the positioning of related genes and the like is not deep yet; molecular markers for identifying wheat villus genes have not been reported, and breeding selection of wheat villus by using molecular marker-assisted selection has not been reported.
Disclosure of Invention
The invention aims to provide a molecular marker for identifying new genes of wheat leaf villus, a primer group and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the molecular marker for identifying wheat leaf villus genes provided by the invention is positioned on a 7BS chromosome of a local variety Bai Maomai, is named QLP.saas-7BS, and is a new gene locus related to wheat leaf villus; the QLP.saas-7BS localization interval is within the 0.48Mb interval between AX-86175290 (physical position 61106266-61106336 b) and AX-86172908 (physical position 60629908-60629979 b) according to the China spring reference genome (IWSSC RefSeq v 2.1) alignment analysis; two pairs of KASP specific linkage markers were developed using QLP.saas-7BS site flanking probes AX-86175290 and AX-86172908, the linkage markers being the KASP marker of KASP-AX-86175290 and the KASP marker of KASP-AX-86172908; the nucleotide sequences of the markers KASP-AX-86175290 and KASP-AX-86172908 are respectively,
KASP-AX-86172908：
FAM：GAAGGTGACCAAGTTCATGCTTGCACAACCAGTCAGCAGAAA
HEX：GAAGGTCGGAGTCAACGGATTTGCACAACCAGTCAGCAGAAG
COM：TCGGGATATTGAATTCTTGAGCTAC；
KASP-AX-86175290：
FAM: GAAGGTGACCAAGTTCATGCT TGCAGTGAAGTTATTAACACC，
HEX：GAAGGTCGGAGTCAACGGATT TGCAGTGAAGTTATTAACACT，
COM：ATATGCAGTACAGATCTTTCACAG。
further, the SNP probes of the two wings of the QLP.saas-7BS are AX-86175290 and AX-86172908, respectively, the KASP-AX-86172908 and KASP-AX-86175290 are designed according to the probe sequences of AX-86175290 and AX-86172908, respectively, the probe sequences of AX-86175290 and AX-86172908 are respectively,
AX-86172908: GGCTAGTGCTGTAAATGCACAACCAGTCAGCAGAA[A/G]CGTAAATGGTGTAGCTCAAGAATTCAATATCCCGA；
AX-86175290:
TCTAAATGGGAGACCTGCAGTGAAGTTATTAACAC[C/T]AACTGGAAGGACTGTGAAAGATCTGTACTGCATAT。
the invention provides a preparation method of a molecular marker for identifying wheat leaf villus genes, which comprises the following steps:
(1) Construction of a genetic population of high-generation recombinant inbred lines
Construction of a high-generation recombinant inbred genetic population F by using a commonly cultivated non-velvet Mao Xiaomai Chuan wheat 104 and a wheat local wheat variety Bai Maomai with high-density leaf fluff 8 Substitution;
(2) Construction of genetic linkage map
Carrying out whole genome scanning on the population by utilizing a 50K high-density SNP chip, constructing a genetic linkage map, and combining leaf villus phenotype characters of each strain of the population to locate a gene QLP.saas-7BS closely linked with the leaf villus characters;
(3) Genetic localization
The Bai Maomai leaf villus gene is genetically mapped on the 7BS chromosome of Bai Maomai, named QLP.saas-7BS, and analyzed according to the China spring reference genome (IWSSC RefSeq v 2.1) alignment, the probe interval is AX-86175290 (physical position is 61106266-61106336 b) and AX-86172908 (physical position is 60629908-60629979 b), the locus is located on the 7BS chromosome, and the physical distance of the locus is 0.48Mb;
(4) Obtaining molecular markers for identifying villus genes of wheat leaves
Two pairs of KASP-specific linkage markers, KASP markers of KASP-AX-86175290 and KASP markers of KASP-AX-86172908, were developed using QLP.saas-7BS site flanking probes AX-86175290 and AX-86172908.
Further, in the step (3), the genetic mapping method for Bai Maomai leaf villus gene is QTL mapping method.
The invention provides a primer group for identifying the molecular marker, wherein two groups of KASP function marker primers for detecting a gene QLP.saas-7BS are respectively as follows:
the first set, KASP-AX-86172908 primer sequences were as follows:
FAM: GAAGGTGACCAAGTTCATGCTTGCACAACCAGTCAGCAGAAA，
HEX：GAAGGTCGGAGTCAACGGATTTGCACAACCAGTCAGCAGAAG，
COM：TCGGGATATTGAATTCTTGAGCTAC；
the second set, KASP-AX-86175290 primer sequences were as follows:
FAM: GAAGGTGACCAAGTTCATGCT TGCAGTGAAGTTATTAACACC，
HEX：GAAGGTCGGAGTCAACGGATT TGCAGTGAAGTTATTAACACT，
COM：ATATGCAGTACAGATCTTTCACAG。
furthermore, competitive FAM and HEX forward primers are designed at the 5' end of the SNP position of the QLP.saas-7BS double-wing probe, a universal primer is designed at the 3' end, different fluorescent probe marking groups are respectively added to the 5' end of the primer sequence, and the fluorescent probe sequence of the FAM is GAAGGTGACCAAGTTCATGCT, HEX and the fluorescent probe sequence of the FAM is GAAGGTCGGAGTCAACGGATT.
The invention provides a method for identifying wheat leaf villus genes, which comprises the following steps:
(1) Extracting the wheat genome DNA to be identified;
(2) PCR amplification of the DNA extracted in step (1) using the above designed KASP markers KASP-AX-86175290 and KASP-AX-86172908 primers;
further, in the step (2), the method of PCR amplification is as follows: preparing KASP genotyping mixed solution, adding the prepared KASP genotyping mixed solution to the PCR reaction plate which is packaged with DNA in the step (1), sealing the PCR reaction plate, and centrifuging; finally, carrying out PCR (polymerase chain reaction) circulation reaction, wherein the used instrument is a fluorescent quantitative PCR instrument;
PCR reaction system: 2. Mu.l of DNA; KASP Master mix 5 μl; KASP primer group 3 μl, and 3 primer concentrations of primer group are mixed according to the volume ratio FAM: HEX: COM=1:1:2; total reaction volume 10 μl;
PCR reaction procedure: pre-denaturation at 94℃for 15 min; denaturation at 94℃for 20sec, gradient renaturation/extension at 61-55℃for 60sec, 0.6℃decrease per cycle for 10 cycles; denaturation at 94℃for 20sec, renaturation/extension at 55℃for 60sec for 26 cycles; fluorescence data reading for KASP typing was performed below 40 ℃.
The molecular marker for identifying the wheat leaf villus gene is applied to breeding wheat varieties with leaf villus phenotype.
The primer group provided by the invention is applied to breeding wheat varieties with leaf villus phenotype.
The primer group provided by the invention is applied to breeding wheat varieties with leaf villus phenotype shapes.
Based on the technical scheme, the embodiment of the invention at least has the following technical effects:
(1) The molecular marker, the primer group and the application for identifying the wheat leaf villus gene provided by the invention are used for developing the molecular marker which can be used for assisting in the molecular assisted selection of the specific property of the wheat, so that the utilization efficiency of the important resource is greatly improved, the novel gene QLP.saas-7BS of the leaf villus in the wheat variety is positioned by using the KASP molecular marker for the first time, the molecular marker technology is applied to the breeding of the wheat variety with the leaf villus phenotype, the genotype of the wheat leaf villus phenotype can be directly and rapidly selected by means of the molecular marker, and the influence of the environment and external factors is eliminated;
(2) The molecular marker, the primer group and the application for identifying the wheat leaf villus gene provided by the invention have the advantages that the gene locus positioned by the molecular marker is clear in position, and the identification is convenient. The molecular marker linked with the gene locus can be detected to predict whether wheat has leaf villus phenotype, so that stress resistance, such as drought resistance, insect resistance, ultraviolet injury and the like, of wheat plants can be further predicted, the molecular marker is used for genotype detection of wheat varieties or strains to judge whether the varieties or strains have leaf villus and whether the varieties or strains contain leaf villus new genes QLP.saas-7BS, and by detecting the gene locus of the leaf villus of the wheat, single plants with leaf villus phenotype can be identified in a seedling stage, other plants are eliminated, and the varieties or strains with leaf villus phenotype are further rapidly screened for breeding utilization of the wheat villus phenotype, so that the wheat varieties containing the leaf villus genes are obtained, the production cost is saved, the selection efficiency of the wheat materials with the leaf villus phenotype is greatly improved, and the breeding period of the wheat varieties is greatly shortened; and the detection is convenient and quick, and is not influenced by the environment.
Drawings
FIG. 1 is a technical roadmap of an embodiment of the invention;
FIG. 2 is a photograph of the side of the leaf nap of Chuanmai 104 and Bai Maomai, as examined by LEICA EZ 4HD stereoscopic microscope, in example 1 of the present invention, chuanmai 104 leaves without nap, bai Maomai leaves with long and dense nap;
FIG. 3 is a photograph of front view of leaf villi of Chuanmai 104 and Bai Maomai leaves detected by LEICA EZ 4HD stereoscopic microscope in example 1 of the invention, wherein leaf villi of Chuanmai 104 is free from villi and Bai Maomai leaves are long and dense;
FIG. 4 is a typing chart of detection of a specific molecular marker KASP-AX-86175290 by a fluorescent quantitative PCR apparatus in example 2 of the present invention;
FIG. 5A typing chart of detection of specific molecular marker KASP-AX-86172908 by a fluorescent quantitative PCR apparatus in example 2 of the present invention.
FIG. 6 wheat leaf villus novel gene QLP.saas-7BS.
Detailed Description
Example 1:
a molecular marker for identifying wheat leaf villus gene, which is located on the 7BS chromosome of Bai Maomai and is named qlp.saas-7BS (as shown in fig. 6); the QLP.saas-7BS comprises two linked molecular markers, a KASP marker of KASP-AX-86175290 and a KASP marker of AX-86172908, and the QLP.saas-7BS is located within a physical interval of 0.48Mb between AX-86175290 (physical location 61106266-61106336 b) and AX-86172908 (physical location 60629908-60629979 b).
Example 2:
the preparation method of the molecular marker in the embodiment 1 is shown in the technical route in fig. 1, and the preparation method comprises the following steps:
(1) Construction of a genetic population of high-generation recombinant inbred lines
Construction of a high-generation recombinant inbred genetic population F by using a commonly cultivated non-velvet Mao Xiaomai Chuanmai 104 and a wheat local variety Bai Maomai with high-density leaf villi 8 Substitution;
as shown in fig. 2, the side photographs of the villus of the leaves of Chuanmai 104 and Bai Maomai show that Chuanmai 104 has no villus and Bai Maomai long villus;
as shown in fig. 3, the front view of the nap of the Chuanmai 104 and Bai Maomai leaves, the Chuanmai 104 leaves have no nap, and the nap of the Bai Maomai leaves are long and dense;
(2) Construction of genetic linkage map
Carrying out whole genome scanning on the population by using a 50K high-density SNP chip, constructing a high-density genetic linkage map, and identifying that the leaf villus phenotype character of each strain of the population is genetically stable in multiple environments through multi-year and multi-point environment planting;
(3) Genetic localization
Genetic mapping is carried out on Bai Maomai leaf villus genes (a 50K high-density SNP chip is utilized to carry out gene mapping on leaf villus characters of wheat local varieties with high-density leaf villus characteristics) by adopting a QTL mapping method, the genes are mapped on a 7BS chromosome of Bai Maomai and named as QLP.saas-7BS, the loci are positioned on the 7BS chromosome according to the comparison analysis of China spring reference genome (IWSSC RefSeq v 2.1), the probe interval is AX-86175290 (physical position is 61106266-61106336 b) and AX-86172908 (physical position is 60629908-60629979 b), and the physical distance of the loci is 0.48Mb;
(4) Obtaining molecular markers for identifying villus genes of wheat leaves
Two pairs of KASP specific markers closely linked to the gene locus were developed using QLP.saas-7BS site flanking probes AX-86175290 and AX-86172908, respectively, the linked markers being KASP markers, KASP-AX-86175290 and KASP-AX-86172908, respectively.
The sequence information of the primer group of the specific molecular marker KASP-AX-86175290 is as follows:
the sequence of the primer set for the specific marker KASP-AX-86172908 is as follows:
FAM： GAAGGTGACCAAGTTCATGCTTGCACAACCAGTCAGCAGAAA
HEX：GAAGGTCGGAGTCAACGGATTTGCACAACCAGTCAGCAGAAG
COM：TCGGGATATTGAATTCTTGAGCTAC
the sequence of the primer set of the specifically labeled KASP-AX-86175290 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTTGCAGTGAAGTTATTAACACC
HEX：GAAGGTCGGAGTCAACGGATTTGCAGTGAAGTTATTAACACT
COM：ATATGCAGTACAGATCTTTCACAG
in the FAM and HEX primer sequences in the KASP-labeled primer group sequences, different fluorescent probe labeling groups are respectively contained at the 5' end, and the fluorescent probe labeling group sequence of the FAM primer is GAAGGTGACCAAGTTCATGCT, HEX primer and the fluorescent probe labeling group sequence of the FAM primer is GAAGGTCGGAGTCAACGGATT.
Example 3:
verifying the KASP tag of AX-86175290 and the KASP tag of AX-86172908 in example 1, the KASP tag primer detection reagent was a mix reagent of Ai Ji analytical technology (Shanghai) Co., ltd, and the quantitative PCR apparatus was a CFX384 Real-Time System of BIO-RAD Co.; phenotype of leaf villi was identified by "observations with stereo microscope LEICA E24 HD"; the detection of the specific mark is parting detection by a fluorescent quantitative PCR instrument; the method specifically comprises the following steps:
(1) Extracting the wheat genome DNA to be identified and sub-packaging the wheat genome DNA on a PCR reaction plate;
(2) PCR amplification of the DNA extracted in step (1) using a set of labeled primers KASP-AX-86175290 and KASP-AX-86172908;
the PCR amplification method comprises the following steps: preparing KASP genotyping mixed solution, adding the prepared KASP genotyping mixed solution to the PCR reaction plate which is packaged with DNA in the step (1), sealing the PCR reaction plate, and centrifuging; finally, carrying out PCR circulation reaction;
PCR reaction system: 2. Mu.l of DNA; KASP Master mix 5 μl; KASP primer set 3 μl (primer set 3 primers concentrations are 10 μm, mixed in a ratio of FAM: HEX: com=1:1:2 by volume); total reaction volume 10 μl;
PCR reaction procedure: pre-denaturation at 94℃for 15 min; denaturation at 94℃for 20sec, gradient renaturation/extension at 61-55℃for 60sec, 0.6℃decrease per cycle for 10 cycles; denaturation at 94℃for 20sec, renaturation/extension at 55℃for 60sec for 26 cycles; the detection results of the fluorescence data reading of KASP typing at 40℃or below are shown in FIGS. 4 and 5.
(3) The detection result is shown as figure 2 and figure 3, the characteristic of villus of wheat leaves is identified, and the detection result of KASP mark is stable and reliable according to the phenotypic analysis and the positioning result, and can be used for molecular detection of gene locus QLP.saas-7BS.
SEQUENCE LISTING
<110> institute of crop and agricultural sciences of Sichuan province
<120> a molecular marker for identifying wheat leaf villus gene, primer set and application
<130> AJ2133803
<160> 8
<170> PatentIn version 3.5
<210> 1
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<213> artificial sequence
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gaaggtgacc aagttcatgc ttgcacaacc agtcagcaga aa 42
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gaaggtcgga gtcaacggat ttgcacaacc agtcagcaga ag 42
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tcgggatatt gaattcttga gctac 25
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gaaggtgacc aagttcatgc ttgcagtgaa gttattaaca cc 42
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gaaggtcgga gtcaacggat ttgcagtgaa gttattaaca ct 42
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atatgcagta cagatctttc acag 24
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tctaaatggg agacctgcag tgaagttatt aacacctaac tggaaggact gtgaaagatc 60
tgtactgcat at 72

Claims (8)

1. A molecular marker for identifying wheat leaf villus genes, which is characterized in that: the gene is located on 7BS chromosome of local variety Bai Maomai and named QLP.saas-7BS; the QLP.saas-7BS location interval is within a 0.48Mb interval between AX-86175290 and AX-86172908, the physical position of AX-86175290 is 61106266-61106336b, and the physical position of AX-86172908 is 60629908-60629979b; two pairs of KASP specific linkage markers, namely a KASP marker of KASP-AX-86175290 and a KASP marker of KASP-AX-86172908, were developed by using the SNP probes AX-86175290 and AX-86172908 flanking the QLP.saas-7BS site; the nucleotide sequences of the primer sets of the markers KASP-AX-86175290 and KASP-AX-86172908 are respectively,
the sequence of the KASP-AX-86172908 primer group is as follows:
FAM：GAAGGTGACCAAGTTCATGCTTGCACAACCAGTCAGCAGAAA，
HEX：GAAGGTCGGAGTCAACGGATTTGCACAACCAGTCAGCAGAAG，
COM：TCGGGATATTGAATTCTTGAGCTAC；
the sequence of the KASP-AX-86175290 primer group is as follows:
FAM：GAAGGTGACCAAGTTCATGCT TGCAGTGAAGTTATTAACACC，
HEX：GAAGGTCGGAGTCAACGGATT TGCAGTGAAGTTATTAACACT，
COM：ATATGCAGTACAGATCTTTCACAG。
2. the molecular marker for identifying wheat leaf villus gene according to claim 1, wherein: SNP probes of two wings of the QLP.saas-7BS are respectively AX-86175290 and AX-86172908, KASP-AX-86172908 and KASP-AX-86175290 are respectively designed according to the probe sequences of AX-86175290 and AX-86172908, the probe sequences of AX-86175290 and AX-86172908 are respectively,
AX-86172908: GGCTAGTGCTGTAAATGCACAACCAGTCAGCAGAA[A/G]CGTAAATGGTGTAGCTCAAGAATTCAATATCCCGA；
AX-86175290:
TCTAAATGGGAGACCTGCAGTGAAGTTATTAACAC[C/T]AACTGGAAGGACTGTGAAAGATCTGTACTGCATAT。
3. primer set for identifying a molecular marker according to claim 2, characterized in that there are two sets of KASP function-labeling primers for detecting the gene qlp.saas-7BS, respectively as follows:
the first set, KASP-AX-86172908 primer sequences were as follows:
FAM: GAAGGTGACCAAGTTCATGCTTGCACAACCAGTCAGCAGAAA，
HEX：GAAGGTCGGAGTCAACGGATTTGCACAACCAGTCAGCAGAAG，
COM：TCGGGATATTGAATTCTTGAGCTAC；
the second set, KASP-AX-86175290 primer sequences were as follows:
FAM: GAAGGTGACCAAGTTCATGCT TGCAGTGAAGTTATTAACACC，
HEX：GAAGGTCGGAGTCAACGGATT TGCAGTGAAGTTATTAACACT，
COM：ATATGCAGTACAGATCTTTCACAG。
4. the primer set according to claim 3, wherein competitive FAM and HEX forward primers are designed at the 5' end and universal primers are designed at the 3' end according to the SNP position of the QLP.saas-7BS double-wing probe, different fluorescent probe labeling groups are respectively added to the primer sequences at the 5' end, and the fluorescent probe sequence of FAM is GAAGGTGACCAAGTTCATGCT, HEX and the fluorescent probe sequence of FAM is GAAGGTCGGAGTCAACGGATT.
5. A method for identifying villus genes of wheat leaves, which is characterized in that: comprising the following steps:
(1) Extracting the wheat genome DNA to be identified;
(2) PCR amplification of the DNA extracted in step (1) was performed using the KASP markers KASP-AX-86175290 and KASP-AX-86172908 primers designed in claim 3.
6. The method for identifying wheat leaf villus gene according to claim 5, wherein: in the step (2), the PCR amplification method comprises the following steps: preparing KASP genotyping mixed solution, adding the prepared KASP genotyping mixed solution to the PCR reaction plate which is packaged with DNA in the step (1), sealing the PCR reaction plate, and centrifuging; finally, carrying out PCR (polymerase chain reaction) circulation reaction, wherein the used instrument is a fluorescent quantitative PCR instrument;
PCR reaction system: 2. Mu.l of DNA; KASP Master mix 5 μl; KASP primer group 3 μl, and 3 primer concentrations of primer group are mixed according to the volume ratio FAM: HEX: COM=1:1:2; total reaction volume 10 μl;
PCR reaction procedure: pre-denaturation at 94℃for 15 min; denaturation at 94℃for 20sec, gradient renaturation/extension at 61-55℃for 60sec, 0.6℃decrease per cycle for 10 cycles; denaturation at 94℃for 20sec, renaturation/extension at 55℃for 60sec for 26 cycles; fluorescence data reading for KASP typing was performed below 40 ℃.
7. The use of a molecular marker for identifying wheat leaf villus genes according to claim 1 or 2 for breeding wheat varieties with leaf villus phenotype.
8. The use of the primer set according to claim 3 or 4 for breeding wheat varieties with leaf villus phenotype.
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