CN109628469B - Molecular marker for identifying barley half-dwarf multi-tillering gene Hvhtd based on intron 2 and application thereof - Google Patents
Molecular marker for identifying barley half-dwarf multi-tillering gene Hvhtd based on intron 2 and application thereof Download PDFInfo
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Abstract
The invention discloses a gene Hvhtd for controlling a semi-dwarf multi-tillering character, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:1, the preparation method is as follows. The invention also provides a protein coded by the semi-short multi-tillering gene Hvhtd, and the amino acid sequence of the protein is shown as SEQ ID NO: 3, the preparation method is as follows. The invention also provides a molecular marker SNP62 for identifying the barley half-dwarf multi-tillering gene Hvhtd based on the intron 2; the molecular marker SNP62 obtained by the invention can identify whether the barley contains Hvhtd semi-short stalk multi-tillering gene. The invention utilizes the marker to carry out auxiliary selection of Hvhtd gene semi-short stalk multi-tillering breeding, can ensure 100% accuracy and accelerate the breeding progress of barley semi-short stalk multi-tillering breeding.
Description
Technical Field
The invention relates to a gene marking method and a corresponding primer for identifying a barley half-short-stalk multi-tillering gene Hvhtd, belongs to the technical field of agricultural biology, and can be used for rapid screening of barley half-short-stalk multi-tillering germplasm and molecular marker-assisted breeding containing Hvhtd mutant genes.
Background
The global food shortage is caused by a plurality of factors such as population growth, climate change and natural environment damage, and how to improve the yield of crops becomes a general concern of breeders. In the last 60 th century, the application of the semi-dwarf gene remarkably improves the crop yield, and is called as the 'green revolution' (Peng et al, 1999; Monna et al, 2002), and the 'green revolution gene' relates to the gibberellin metabolic pathway. The semi-dwarf crop increases the lodging resistance of the crop, but also increases the input amount of fertilizer, thus causing the problems of increase of production cost, environmental pollution and the like. Therefore, there is an urgent need to find a new genetic variation to improve the yield of crops.
Tillering and plant height are two very important agronomic traits that affect crop yield and plant type (Sakamoto and Matsuoka, 2004; Wang and Li, 2008; Alqudah et al, 2016), which, although affected by environmental factors such as light, temperature, humidity, nutrition and planting pattern, are largely determined by genetic factors. Therefore, the research on the genes for controlling tillering and plant height has important significance for improving the crop yield.
In barley, 5 semi-dwarf multi-tillering genes are currently reported, namely int-c, mnd, gra-a, int-m and fol-a (Druka et al, 2011; Hussen et al, 2014). However, only int-c and mnd were cloned, int-c being a homologous gene to the rice and maize TB1 genes, mnd encoding a cytochrome P450 protease (Ramsay et al, 2011; Mascher et al, 2014).
Disclosure of Invention
The invention aims to solve the technical problems that a semi-dwarf multi-tillering gene Hvhtd in barley is cloned and a molecular marker associated with the semi-dwarf multi-tillering character is provided; the molecular marker SNP62 obtained by the invention can identify whether the barley contains Hvhtd semi-short stalk multi-tillering gene.
In order to solve the technical problems, the invention provides a gene Hvhtd for controlling the semi-dwarf and multi-tillering character: the nucleotide sequence of the gene is shown as SEQ ID NO:1, the preparation method is as follows.
The invention also provides a protein coded by the semi-short multi-tillering gene Hvhtd, and the amino acid sequence of the protein is shown as SEQ ID NO: 3, the preparation method is as follows.
The invention also provides a molecular marker SNP62 for identifying the barley half-dwarf multi-tillering gene Hvhht d based on the intron 2, the barley is taken as a species, the molecular marker primer is selected from the following primer pair, the nucleotide sequence is 5 '→ 3',
the forward primer is:
htd allele-1 GAAGGTCGGAGTCAACGGATTGAATATATTGGTTCAGGCTCTCAGA
Hua30allele-2 GAAGGTGACCAAGTTCATGCTGAATATATTGGTTCAGGCTCTCAGG
reverse primer (common reverse primer) GGCAGCATTCACTGCTTCAA.
Note: underlined are adapter primers.
The invention also provides a method for identifying the barley half-dwarf multi-tillering gene Hvhtd by using the molecular marker SNP62, which comprises the following steps:
(1) extracting the genomic DNA of the barley variety to be detected (by adopting a CTAB method (Stein et al, 2001);
(2) performing PCR amplification on barley genome DNA by using a molecular marker SNP 62;
(3) and (3) analyzing by using QuantStaudio Real-Time PCR software according to the difference of the fluorescence signals of the PCR products, and further identifying the genotype of each barley to be detected (namely identifying the barley to be detected to belong to a homozygous wild type, a homozygous mutant type and a heterozygous type, and which type in the 3 types of genotypes).
As an improvement of the method for identifying the barley half-dwarf multi-tillering gene Hvhht, the KASPar PCR reaction system in the step 2) is as follows:
mu.L of genomic DNA (concentration 100 ng/. mu.L), 0.1. mu.L of primers, 1.4. mu.L of sterile water and 2.5. mu.L of KASP Buffer, the total amount of reaction being 5. mu.L; the PCR reaction is carried out on an ABI ViiA7 fluorescent quantitative PCR instrument, and the reaction program is as follows: 15min at 95 ℃; denaturation at 94 ℃ for 20s, annealing at 61 ℃ (-0.6 ℃/cycle) for 60s, 10 cycles; then denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 26 cycles;
of the 0.1. mu.L primers, 2 forward primers were each present at a concentration of 10pmol/L, and the reverse primer (common reverse primer) was present at a concentration of 30 pmol/L.
The invention uses EMS mutagenesis to obtain a semi-short stalk multi-tillering mutant material htd, and clones a gene Hvhtd for controlling the semi-short stalk multi-tillering character, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:1, the preparation method is as follows. The protein coded by the semi-dwarf multi-tillering gene Hvhtd has the amino acid sequence shown in SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof.
Correspondingly, the nucleotide sequence of the wild barley HORVU2Hr1G098820 gene is shown as SEQ ID NO:2, and the protein coded by the protein has the sequence shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.
The invention uses a chemical mutagen EMS to process barley variety flowers 30, obtains a semi-short stalk multi-tillering mutant htd with stable and hereditary characters through multi-generation screening and identification, clones a semi-short stalk multi-tillering gene Hvhhtd through BSR-Seq and a gene positioning method, researches find that the Hvhtd gene is that the encoded protein is changed to generate a semi-short stalk multi-tillering phenotype because the first base of intron 2 in a wild barley gene HORVU2Hr1G098820 is changed from G to A, so that the shearing mode of the gene is changed, and the gene encodes trypsin which is different from the barley semi-short stalk multi-tillering gene reported before. The invention designs a KASPar marker by utilizing the mutation site, and can quickly and accurately detect whether the barley material contains Hvhtd mutant genes.
Namely, the sequence analysis of the invention discovers that a single base mutation exists between the wild barley HORVU2Hr1G098820 gene and the Hvhtd gene of the semi-short stalk multi-tillering mutant htd on the nucleotide sequence, the first base of the 2 nd intron of the HORVU2Hr1G098820 gene is changed from G to A, and the mutation causes the gene shearing mode to be changed, thereby causing the encoded protein to be terminated early and generating the semi-short stalk multi-tillering phenotype.
The phenotype of the semi-dwarf multi-tillering mutant htd is as follows: the leaves are thin and narrow, and the color of the leaves is dark green; the tillering number is 23.3-27.8, which is 1.54-1.97 times of that of the corresponding wild type; the plant height is about 75cm, and is 5.6-11.1cm lower than that of the corresponding wild type. FIG. 5 shows mutant Htd at seedling stage on the left and wild type flower 30 at seedling stage on the right; in FIG. 6, mutant Htd is shown on the left side of heading date and wild type flower 30 is shown on the right side of heading date.
The invention clones a barley half-short-stalk multi-tillering gene Hvhtd by utilizing a biotechnology, which is different from the previous reported barley half-short-stalk multi-tillering gene, and designs a KASPar marker SNP62 by utilizing a single base mutation site, wherein the marker has the following advantages:
1) the gene marker obtained by the invention is designed according to the internal base mutation of the gene, so that genetic exchange does not exist, and further verification of phenotype is not required.
2) The KASPar marker designed by the invention directly utilizes the collected fluorescent signals to perform genotyping without electrophoresis, glue dyeing, band reading and other processes, and can simply, rapidly and high-flux identify whether the barley germplasm resources contain Hvhtd genes.
3) The method is utilized to carry out molecular marker-assisted selective breeding, and the selection efficiency of barley varieties is obviously improved.
4) The auxiliary selection of Hvhtd gene semi-short stalk multi-tillering breeding by using the marker can ensure 100% accuracy and accelerate the barley semi-short stalk multi-tillering breeding progress.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the Hvhtd gene structure and its site of differentiation from wild type;
FIG. 2 is an amino acid sequence alignment of the gene Hvhtd in the mutant htd and its wild-type flower 30;
in particular to the amino acid sequence comparison of a mutant htd semi-dwarf multi-tillering gene Hvhhtd and a corresponding gene HORVU2Hr1G098820 in a wild flower 30 thereof.
FIG. 3 shows the identification of Hvhtd genotypes of mutant htd and its wild-type flower 30 using the KASPar marker SNP62 designed in the present invention.
Upper left corner
Wild type flower 30 (shown as blue in the colour chart), lower right hand corner
Mutant htd (shown in red in the color chart), ■ as a negative control, were 2 replicates each.
FIG. 4 is a diagram showing the identification of barley germplasm resources by using the KASPar marker SNP62 designed by the present invention;
upper left corner
Is wild type (in color)Blue as shown in the color chart), ● in the lower right corner is the mutant htd (red as shown in the color chart), and in the middle
Heterozygous (green in color), ■ is a negative control.
Description of the drawings: in the automatically generated image of the software, red circles represent homozygous genotypes ALLEL1/ALLEL1, blue circles represent homozygous genotypes ALLEL2/ALLEL2, green circles represent heterozygous ALLEL1/ALLEL2, and Xs represent indeterminate genotypes.
FIG. 5 is a comparison graph of the semi-short multi-tillering mutant htd and the wild type flower at 30 seedling stages;
FIG. 6 is a comparison graph of the half-dwarf multi-tillering mutant htd and the wild type flower at the heading stage of 30.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 design of molecular marker SNP62
1) And test materials: mutant htd and its wild type flower 30 (i.e., flower 30, Hua 30);
the mutant htd is obtained by adopting EMS mutagenesis mode: a0.03 mol/L EMS treatment of the malting barley variety flower 30 was performed from 1710 individuals M2In the generations, a semi-short-stalk multi-tillering mutant is obtained according to phenotype observation, and through multi-generation observation, the semi-short-stalk multi-tillering mutant is found to be stably inherited, and is named as htd. Its appearance compared to its wild type flower 30 is: the leaf blade becomes thin and narrow, the leaf color becomes dark, the plant height becomes short, and the tillering is increased.
The mutant htd is cloned to a gene Hvhtd for controlling the semi-dwarf multi-tillering character, and the nucleotide sequence of the gene is shown as SEQ ID NO:1, the preparation method is as follows. The protein coded by the semi-dwarf multi-tillering gene Hvhtd has the amino acid sequence shown in SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof.
The nucleotide sequence of the wild type flower 30HORVU2Hr1G098820 gene is shown as SEQ ID NO:2, and the protein coded by the protein has the sequence shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.
2) And obtaining a molecular marker of the semi-short multi-tillering gene Hvhtd: according to the sequence alignment result, the gene Hvhtd is found to have a base mutation between the mutant htd and the wild flower 30 thereof, and a KASPar marker is designed by combining the single base mutation site by using Premier5 software and is named as SNP 62.
The forward primer is:
htd allele-1 GAAGGTCGGAGTCAACGGATTGAATATATTGGTTCAGGCTCTCAGA(5’-3’)
Hua30allele-2 GAAGGTGACCAAGTTCATGCTGAATATATTGGTTCAGGCTCTCAGG (5’-3’)
reverse primer (common reverse primer) GGCAGCATTCACTGCTTCAA (5 '-3')
Note: underlined are adapter primers.
Example 2 identification of sequence differences between mutant htd and its wild-type flower 30 using molecular markers:
1) and DNA extraction: taking 100mg of barley leaves, extracting DNA by using a CTAB method, and specifically comprising the following steps:
firstly, cutting leaves, grinding the leaves in liquid nitrogen, putting the leaves into a 1.5ml centrifuge tube, adding 600 mu L of CTAB into each tube, and carrying out warm water bath at 65 ℃ for 50-60min (taking out the leaves every 10min and shaking the leaves);
adding 600 microliters of chloroform/isoamylol (24: 1), fully mixing for 15min, slowly moving, weighing two by two, centrifuging for 9600 r after balancing, 10min, and centrifuging at 4 ℃;
③ sucking the supernatant to another 1.5ml new centrifuge tube, adding 2 times of anhydrous alcohol, and slightly mixing (placing in a refrigerator at-20 ℃ for 30-60min for precipitation);
thirdly, picking out the precipitate by using a gun head, placing the precipitate into a 1.5ml centrifugal tube, washing the precipitate with 70% ethanol for 2 times, and air-drying the precipitate;
adding 100 mu L of ultrapure water, dissolving (mother liquor) mother liquor in a refrigerator at 4 ℃: the working solution is 1: 20.
2) KASPar PCR reaction and analysis of results:
the primers were as described in example 1.
The KASPar PCR reaction system is as follows: mu.L of genomic DNA (concentration 100 ng/. mu.L), 0.1. mu.L of primers (2 forward primers each at 10pmol/L and reverse primer (common primer) at 30pmol/L), 1.4. mu.L of sterile water and 2.5. mu.L of KASP Buffer, the total reaction volume being 5. mu.L. The PCR reaction is carried out on an ABI ViiA7 fluorescent quantitative PCR instrument, and the reaction program is as follows: 15min at 95 ℃; denaturation at 94 ℃ for 20s, annealing at 61 ℃ (-0.6 ℃/cycle) for 60s, 10 cycles; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 26 cycles.
Note: KASP Buffer is a KASP Master mix kit from LGC company; -0.6 ℃/cycle represents 0.6 ℃ reduction per cycle.
The PCR reaction is directly carried out on an ABI ViiA7 fluorescent quantitative PCR instrument which is connected with Quantstrudio Real-Time PCR software, so as to directly obtain an analysis result, and the obtained result is shown in figure 3, namely, the software automatically divides a detection sample into a homozygous wild type, a homozygous mutant type and a heterozygous type according to different genotypes.
Analyzing by QuantStaudio Real-Time PCR software, when a dot with the same color as that of the mutant thd appears, indicating that the genotype of a material corresponding to the dot is the same as that of the htd, and the material is a homozygous mutant; when the dots with the same color as the wild type flower 30 appear, the genotype of the same flower 30 of the material corresponding to the dots is shown as the homozygous wild type; when a green dot appears, it indicates that the genotype of the material corresponding to the dot is heterozygous.
Remarks explanation: the method comprises the steps of respectively arranging respective fluorescent joints (with different colors) in front of 2 forward primers, wherein the fluorescent joints respectively correspond to ALLELE1 and ALLELE2, if a detected material is a homozygous genotype, only one corresponding primer is selected to amplify during amplification (for example, an htd mutant can only react with htd allole 1), distinguishing whether the detected material is allol 1/allol 1 or allol 2/allole 2 according to the difference of fluorescence, and if the detected material is a heterozygous type, using 2 primers during amplification, wherein the generated fluorescence is different from the material of the homozygous genotype, so that the heterozygous genotype is distinguished.
In the automatically generated image of the software, red circles represent homozygous genotypes ALLEL1/ALLEL1, blue circles represent homozygous genotypes ALLEL2/ALLEL2, green circles represent heterozygous ALLEL1/ALLEL2, and Xs represent indeterminate genotypes.
Example 3 detection of 32 barley germplasm resources (including mutant htd and flower) using molecular marker SNP62 according to the method described in example 230, 2 repeats) in the barley germplasm resource, wherein the barley germplasm resource comprises wild barley, local varieties, barley bred varieties from all over the world and F obtained by htd hybridization with Zhejiang peel No. 81(see table 1);
the results are shown in FIG. 4.
The detection result shows that the Htd/Zhejiang skin No. 8F in the materials1The hybrid is a heterozygote type, and the mutant htd is a mutant genotype; other materials are wild genotypes, and the phenotypes of the materials are different from the half-dwarf multi-tillering phenotype of the mutant htd by observation, which shows that the materials do not contain Hvthd mutant genes, and also proves that the molecular marker can be used for detecting barley germplasm resources.
TABLE 1 information of 32 barley germplasm resources detected using molecular marker SNP62
And (3) verification experiment: the barley described in table 1 was subjected to DNA detection, and the results were: the gene sequence shown in SEQ ID NO. 1, the Htd/Zhepi No. 8F are detected only in the mutant htd1The hybrid species detect the gene sequences as SEQ ID NO 1 and SEQ ID NO 2, while other materials only detect the gene sequence as SEQ ID NO 2; that is, the results obtained were completely the same as those obtained in example 3 above.
Example 4, the barley described in table 2 is tested according to the method described in example 3, and the test result shows that the materials (except htd) are all wild genotypes, and the phenotypes of the materials are observed to be different from the half-dwarf multi-tillering phenotype of mutant htd, which indicates that the materials do not contain Hvthd mutant genes, and also proves that the molecular marker can be used for testing barley germplasm resources.
TABLE 2 information of 83 barley germplasm resources detected using molecular marker SNP62
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> intron 2-based molecular marker for identifying barley half-dwarf multi-tillering gene Hvhtd and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4945
<212> DNA
<213> barley (Hordeum vulgare L.)
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gccgggctgc tcgtcgtccc tctttggtgg ttgagttgcc aatctggtat gccaggttgt 60
tgctccctgc cagtgccatg ggagttccgt gatgcctgca cagtaacagc ttcaggcgtt 120
ctcttcttat ttccttccgc cttccttgtt ggcctcgctt ccggggccaa atgccacgga 180
aagcttagca gctcggcctc caagaatctt cgccacggga gcagggagct ccattgccgc 240
tctgcaaccg gcatcaatcc aagctcgcgt gccttcagct gcaggctctc ttctgcggcc 300
agataaaagt tgcattgctc cagcaaaacc gatttcaggc ctgctattgg atgtaacaca 360
aggggagagt gcttgccgtt tgttcccccc gatttcttgt cggcgggttg tttcttcttt 420
tgattatgcc ttttctcttt cagcaaagtg cacgatgcct ttgccaaact tcacccgcgg 480
tgccgcctgc ttaatttgcg cgtaggccct acccttttcg cgtcgatatt tttgttatca 540
tcacaagtac tccgtgtggt agttgtcatg tggatttatg ccaattccct gtgtttaaag 600
gcagaatttg tgccatgcct ttcgccgaac aaacggatgg agacttcctc caccctctgg 660
cttttgcaga ttccactctc ctgtgatatt ctctgcttgg ttctacggcg gatgcgcaac 720
aagaatatac atatacaatg ggtcatattc gtcgtacaga ggattcctct tatgatatta 780
tctgcttttg tttgcgtggt tgcaatcctg caagaaccta ttccccgaga atatcaggct 840
actccaccag cacttgttta tttattttct tgtgtataat agcttggatg ctagctagcc 900
tcacactcat acattgctta atttcaccat acatttccca gtctgatgta ggtctattag 960
ggtgcaaaca catttggtag attgctgagt gctaactttc cagtgtctcc tctcctgaca 1020
tttttgcctt ggggcaagat tatgttctca gacttgtttg ctctaatttt tagggccata 1080
gcttctcctg tccactacgc ctgtcaactg ttcggcacga tgaagccttc agatgacagg 1140
atgcagctct caggtttgac gcggtcggaa gagtcgtctc ttgatgtgga ggggcattgc 1200
tctcaccatg aggcatttcc ttgttctccg tcgatgcaac cggttgcttc tgggtgcgtg 1260
cacacagaaa acagcgcggc atacttctta tggccgactt caaacctgca gcattgtgcg 1320
gcggagggac gggcaaacta ctttgggaac ctccagaaag gattactgcc ggtactccct 1380
ggaaagctgc ccaagggtca gcaagcaaat agcttgcttg atttgatgac cataagagct 1440
ttccacagca agatattgcg gcgtttcagc cttgggacgg cagtgggctt ccgcataaca 1500
aaaggggttc tcacagaaac ccctgccatt cttgtcttcg ttgctcgaaa ggttcacaag 1560
aaatggctta atccgaacca atgccttcct gcaattcttg cggtatgaga acctcatttt 1620
atctcttgtc ttatattatt ggacaggctt gttttcagac ttggagtcgt ttttgtcttt 1680
gactaatatc tttcacttgg ctggatgttt acagggtcca ggaggtgttt ggtgtgatgt 1740
tgatgtcgta gaattttcat actatggtgc gccggctcaa acacctaaag aacagacgtt 1800
tagtgagctt gttaataagc tgtgtggcag tgacgaatat attggttcag gctctcagat 1860
ttatacatct atgctcttat ctgtattggt ttgcattacc tagatatcat cttcttgaag 1920
cagtgaatgc tgccatgtgt acaaattgga ttgggcacaa acatgcacta ctagttattt 1980
tctctgtctt gccagtgggc tgttcaaatt ttgtgaagat tctttggaca aagtgaataa 2040
aattgcagtg cccataaggt tatgacaatg gctgataggt actatgtcag ttcacactgt 2100
tatgcataac aatgagctac tagaagtcct gccagtgcta actttgttct tgtttagttt 2160
aaataaatct ttgttcgttt ttcttgcggt cgtctctaga gtgccaactg gatgtaactt 2220
aagttaatcc cattgtgaac tgatcagcaa tcagaagtaa ctttcttaac ggcagtctct 2280
agagtgtcat gtcatttaat gactaagtac ttgaatggtc taagttatca tatgcagctg 2340
aacaattggg tccctgcaca aatctagaca ccacttatct ttaaaactca tgtaccattt 2400
tgaatgacag tttttttttc tttccaaaac agaactaaat tatatactct agaagttaga 2460
gcagcatata gtgcattgaa tgattcaaga gggcatgctc tcatttcctg cttgagagtt 2520
ctctatacct gtttcactgt ctcaggttgc aagccaggat acatttggaa ctttgggtgc 2580
aattgtgaaa cgacgcacca acaacaagca agttggtttc ctcacgaacc gacatgttgc 2640
agttgatttg gactatccta accagaagat gtttcacccg ttgccgccga atcttgggcc 2700
tggtgtttat cttggagctg tcgagagggc aacatctttc atcacagatg atgtttggta 2760
tggaatttat gctggaacaa acccaggtag agcagctaca aattatgcgg ttgatatagg 2820
ggattcatgt attattcgca gaattgaact ttaaagtata tgttatagag gaaggcctag 2880
ttaatgtgtc gtctatttaa acacaatgtc ttaaatgcat aaaatgaata aattgatgca 2940
ttccacaaac acaaacataa gtttgtgatg caggacttgt ctttctgatt gtaccagttt 3000
ttgtcagtca ggtcaacaac aacttgtgct ttagactaca taggaacaaa acaatatctg 3060
tttatggaac aaaaaactta gtttatgttt tatatatgta aatttataat ttatgtttta 3120
tatatgtaga tttataaact tataagtttc tccttctgtt tagtgttgtt atatatgatg 3180
catatgtgct gttctaatcc ccaaacatat atgctgcccc tctgtggacc tgcagagaca 3240
tttgtacgag ctgacggcgc attcatccca tttgctgatg actttgacat ttccacagtc 3300
acaactgtag ttagggaagt tggtgagatt ggggatgtta agattataga tctgcagtgt 3360
cctatcaaga gcctcatcgg gaggcaagtt tgcaaagtcg gcagaagttc cggtcacaca 3420
actgggactg tgatggcata tgcccttgag tacaatgatg agaaaggaat atgcttcttc 3480
actgacctcc tcgttgttgg tgagaatcgc caaacatttg atttggaggg tgacagcgga 3540
agccttatta tcctgaccag ccaggatggg gagaagccac gtcctattgg gataatatgg 3600
ggtggcacag caaaccgtgg gaggataaag ctcacaagtg gctatggtcc tgaaaactgg 3660
actacagggg ttgatcttgg ccgccttctt gatcgcctag aacttgatct tatcataaac 3720
gacgaatcgc tcaaaggtga gcactgcaac agtttccttc atattccagt gttgtcctgg 3780
aaatacattt gccatgcaaa cttttattat gttattggca tttttcatat ctcattgttg 3840
aatcccgttt gctccaatgt tacaaggctg acgtaattca tctataataa agatgctgtg 3900
caggagcaaa ggaatgcttt tgtggctgca attaactctg ctattgggga gtcctctgcg 3960
gtgactgtta ctgccccaga agccacccca gcagagaagg ttgaagagat ctttgagcct 4020
cttgggatcc aaattcagca gctgcctcgt cacgacccga caagcgccgc agacgaaggg 4080
gagggcgcag ccaacacacc gtccgacatg gaagagcgtc agttcatctc gaacttcgtt 4140
ggcatgtctc cggtgcgccg tgaccatgat gctcggagga ccattgccaa cctgaacaac 4200
ccgtcagagg aagagcttgc catgtcgctg cacctaggcg accgggagcc caagcggcta 4260
cgtttggacc cggaatcaaa cctagacctc gagaagcagc ctcacccgga ccaggaaccg 4320
agcctagatc tggagaagca gcctcgttcg gacccggaaa tgatgagcct agacctggag 4380
aagcagcctc gctcggaccc ggaaccgggc ctggacctgg agaagcgcct tccagctgac 4440
ccggagccga gcatagactt ggagaagtga actcgctgat tcgaatgacg tctttggaat 4500
gagcttgcaa acgataaaat cgagtagatg gaagaacccc tgtggttcag caaactggac 4560
ctgcgatgtt agcttaagtt aactactact gctggccttg tggggaagca tatcatttca 4620
tatcatgtac tagtatggca cttgattcag ctccttgtag gatattgttg ggtggtatag 4680
ccctgcagtt ttgctgtgct cataagattt gatatacaga atgctgaatc tgaatctctt 4740
ctcttggaga acaatatgcc ggcactgatg gatgagactc tgggatgatt gatgtctctt 4800
tccaaatcac tgatggagta attgtttgat cttacgatgt atttcttcca ttccataatt 4860
tttgtcgtgt ttttagttca aggggagtac ttctcaaatc tagcccttgt tcaggatctt 4920
tttttaaaac ccaaattcag aatct 4945
<210> 2
<211> 4945
<212> DNA
<213> barley (Hordeum vulgare L.)
<400> 2
gccgggctgc tcgtcgtccc tctttggtgg ttgagttgcc aatctggtat gccaggttgt 60
tgctccctgc cagtgccatg ggagttccgt gatgcctgca cagtaacagc ttcaggcgtt 120
ctcttcttat ttccttccgc cttccttgtt ggcctcgctt ccggggccaa atgccacgga 180
aagcttagca gctcggcctc caagaatctt cgccacggga gcagggagct ccattgccgc 240
tctgcaaccg gcatcaatcc aagctcgcgt gccttcagct gcaggctctc ttctgcggcc 300
agataaaagt tgcattgctc cagcaaaacc gatttcaggc ctgctattgg atgtaacaca 360
aggggagagt gcttgccgtt tgttcccccc gatttcttgt cggcgggttg tttcttcttt 420
tgattatgcc ttttctcttt cagcaaagtg cacgatgcct ttgccaaact tcacccgcgg 480
tgccgcctgc ttaatttgcg cgtaggccct acccttttcg cgtcgatatt tttgttatca 540
tcacaagtac tccgtgtggt agttgtcatg tggatttatg ccaattccct gtgtttaaag 600
gcagaatttg tgccatgcct ttcgccgaac aaacggatgg agacttcctc caccctctgg 660
cttttgcaga ttccactctc ctgtgatatt ctctgcttgg ttctacggcg gatgcgcaac 720
aagaatatac atatacaatg ggtcatattc gtcgtacaga ggattcctct tatgatatta 780
tctgcttttg tttgcgtggt tgcaatcctg caagaaccta ttccccgaga atatcaggct 840
actccaccag cacttgttta tttattttct tgtgtataat agcttggatg ctagctagcc 900
tcacactcat acattgctta atttcaccat acatttccca gtctgatgta ggtctattag 960
ggtgcaaaca catttggtag attgctgagt gctaactttc cagtgtctcc tctcctgaca 1020
tttttgcctt ggggcaagat tatgttctca gacttgtttg ctctaatttt tagggccata 1080
gcttctcctg tccactacgc ctgtcaactg ttcggcacga tgaagccttc agatgacagg 1140
atgcagctct caggtttgac gcggtcggaa gagtcgtctc ttgatgtgga ggggcattgc 1200
tctcaccatg aggcatttcc ttgttctccg tcgatgcaac cggttgcttc tgggtgcgtg 1260
cacacagaaa acagcgcggc atacttctta tggccgactt caaacctgca gcattgtgcg 1320
gcggagggac gggcaaacta ctttgggaac ctccagaaag gattactgcc ggtactccct 1380
ggaaagctgc ccaagggtca gcaagcaaat agcttgcttg atttgatgac cataagagct 1440
ttccacagca agatattgcg gcgtttcagc cttgggacgg cagtgggctt ccgcataaca 1500
aaaggggttc tcacagaaac ccctgccatt cttgtcttcg ttgctcgaaa ggttcacaag 1560
aaatggctta atccgaacca atgccttcct gcaattcttg cggtatgaga acctcatttt 1620
atctcttgtc ttatattatt ggacaggctt gttttcagac ttggagtcgt ttttgtcttt 1680
gactaatatc tttcacttgg ctggatgttt acagggtcca ggaggtgttt ggtgtgatgt 1740
tgatgtcgta gaattttcat actatggtgc gccggctcaa acacctaaag aacagacgtt 1800
tagtgagctt gttaataagc tgtgtggcag tgacgaatat attggttcag gctctcaggt 1860
ttatacatct atgctcttat ctgtattggt ttgcattacc tagatatcat cttcttgaag 1920
cagtgaatgc tgccatgtgt acaaattgga ttgggcacaa acatgcacta ctagttattt 1980
tctctgtctt gccagtgggc tgttcaaatt ttgtgaagat tctttggaca aagtgaataa 2040
aattgcagtg cccataaggt tatgacaatg gctgataggt actatgtcag ttcacactgt 2100
tatgcataac aatgagctac tagaagtcct gccagtgcta actttgttct tgtttagttt 2160
aaataaatct ttgttcgttt ttcttgcggt cgtctctaga gtgccaactg gatgtaactt 2220
aagttaatcc cattgtgaac tgatcagcaa tcagaagtaa ctttcttaac ggcagtctct 2280
agagtgtcat gtcatttaat gactaagtac ttgaatggtc taagttatca tatgcagctg 2340
aacaattggg tccctgcaca aatctagaca ccacttatct ttaaaactca tgtaccattt 2400
tgaatgacag tttttttttc tttccaaaac agaactaaat tatatactct agaagttaga 2460
gcagcatata gtgcattgaa tgattcaaga gggcatgctc tcatttcctg cttgagagtt 2520
ctctatacct gtttcactgt ctcaggttgc aagccaggat acatttggaa ctttgggtgc 2580
aattgtgaaa cgacgcacca acaacaagca agttggtttc ctcacgaacc gacatgttgc 2640
agttgatttg gactatccta accagaagat gtttcacccg ttgccgccga atcttgggcc 2700
tggtgtttat cttggagctg tcgagagggc aacatctttc atcacagatg atgtttggta 2760
tggaatttat gctggaacaa acccaggtag agcagctaca aattatgcgg ttgatatagg 2820
ggattcatgt attattcgca gaattgaact ttaaagtata tgttatagag gaaggcctag 2880
ttaatgtgtc gtctatttaa acacaatgtc ttaaatgcat aaaatgaata aattgatgca 2940
ttccacaaac acaaacataa gtttgtgatg caggacttgt ctttctgatt gtaccagttt 3000
ttgtcagtca ggtcaacaac aacttgtgct ttagactaca taggaacaaa acaatatctg 3060
tttatggaac aaaaaactta gtttatgttt tatatatgta aatttataat ttatgtttta 3120
tatatgtaga tttataaact tataagtttc tccttctgtt tagtgttgtt atatatgatg 3180
catatgtgct gttctaatcc ccaaacatat atgctgcccc tctgtggacc tgcagagaca 3240
tttgtacgag ctgacggcgc attcatccca tttgctgatg actttgacat ttccacagtc 3300
acaactgtag ttagggaagt tggtgagatt ggggatgtta agattataga tctgcagtgt 3360
cctatcaaga gcctcatcgg gaggcaagtt tgcaaagtcg gcagaagttc cggtcacaca 3420
actgggactg tgatggcata tgcccttgag tacaatgatg agaaaggaat atgcttcttc 3480
actgacctcc tcgttgttgg tgagaatcgc caaacatttg atttggaggg tgacagcgga 3540
agccttatta tcctgaccag ccaggatggg gagaagccac gtcctattgg gataatatgg 3600
ggtggcacag caaaccgtgg gaggataaag ctcacaagtg gctatggtcc tgaaaactgg 3660
actacagggg ttgatcttgg ccgccttctt gatcgcctag aacttgatct tatcataaac 3720
gacgaatcgc tcaaaggtga gcactgcaac agtttccttc atattccagt gttgtcctgg 3780
aaatacattt gccatgcaaa cttttattat gttattggca tttttcatat ctcattgttg 3840
aatcccgttt gctccaatgt tacaaggctg acgtaattca tctataataa agatgctgtg 3900
caggagcaaa ggaatgcttt tgtggctgca attaactctg ctattgggga gtcctctgcg 3960
gtgactgtta ctgccccaga agccacccca gcagagaagg ttgaagagat ctttgagcct 4020
cttgggatcc aaattcagca gctgcctcgt cacgacccga caagcgccgc agacgaaggg 4080
gagggcgcag ccaacacacc gtccgacatg gaagagcgtc agttcatctc gaacttcgtt 4140
ggcatgtctc cggtgcgccg tgaccatgat gctcggagga ccattgccaa cctgaacaac 4200
ccgtcagagg aagagcttgc catgtcgctg cacctaggcg accgggagcc caagcggcta 4260
cgtttggacc cggaatcaaa cctagacctc gagaagcagc ctcacccgga ccaggaaccg 4320
agcctagatc tggagaagca gcctcgttcg gacccggaaa tgatgagcct agacctggag 4380
aagcagcctc gctcggaccc ggaaccgggc ctggacctgg agaagcgcct tccagctgac 4440
ccggagccga gcatagactt ggagaagtga actcgctgat tcgaatgacg tctttggaat 4500
gagcttgcaa acgataaaat cgagtagatg gaagaacccc tgtggttcag caaactggac 4560
ctgcgatgtt agcttaagtt aactactact gctggccttg tggggaagca tatcatttca 4620
tatcatgtac tagtatggca cttgattcag ctccttgtag gatattgttg ggtggtatag 4680
ccctgcagtt ttgctgtgct cataagattt gatatacaga atgctgaatc tgaatctctt 4740
ctcttggaga acaatatgcc ggcactgatg gatgagactc tgggatgatt gatgtctctt 4800
tccaaatcac tgatggagta attgtttgat cttacgatgt atttcttcca ttccataatt 4860
tttgtcgtgt ttttagttca aggggagtac ttctcaaatc tagcccttgt tcaggatctt 4920
tttttaaaac ccaaattcag aatct 4945
<210> 3
<211> 249
<212> PRT
<213> barley (Hordeum vulgare L.)
<400> 3
Met Phe Ser Asp Leu Phe Ala Leu Ile Phe Arg Ala Ile Ala Ser Pro
1 5 10 15
Val His Tyr Ala Cys Gln Leu Phe Gly Thr Met Lys Pro Ser Asp Asp
20 25 30
Arg Met Gln Leu Ser Gly Leu Thr Arg Ser Glu Glu Ser Ser Leu Asp
35 40 45
Val Glu Gly His Cys Ser His His Glu Ala Phe Pro Cys Ser Pro Ser
50 55 60
Met Gln Pro Val Ala Ser Gly Cys Val His Thr Glu Asn Ser Ala Ala
65 70 75 80
Tyr Phe Leu Trp Pro Thr Ser Asn Leu Gln His Cys Ala Ala Glu Gly
85 90 95
Arg Ala Asn Tyr Phe Gly Asn Leu Gln Lys Gly Leu Leu Pro Val Leu
100 105 110
Pro Gly Lys Leu Pro Lys Gly Gln Gln Ala Asn Ser Leu Leu Asp Leu
115 120 125
Met Thr Ile Arg Ala Phe His Ser Lys Ile Leu Arg Arg Phe Ser Leu
130 135 140
Gly Thr Ala Val Gly Phe Arg Ile Thr Lys Gly Val Leu Thr Glu Thr
145 150 155 160
Pro Ala Ile Leu Val Phe Val Ala Arg Lys Val His Lys Lys Trp Leu
165 170 175
Asn Pro Asn Gln Cys Leu Pro Ala Ile Leu Ala Gly Pro Gly Gly Val
180 185 190
Trp Cys Asp Val Asp Val Val Glu Phe Ser Tyr Tyr Gly Ala Pro Ala
195 200 205
Gln Thr Pro Lys Glu Gln Thr Phe Ser Glu Leu Val Asn Lys Leu Cys
210 215 220
Gly Ser Asp Glu Tyr Ile Gly Ser Gly Ser Gln Ile Tyr Thr Ser Met
225 230 235 240
Leu Leu Ser Val Leu Val Cys Ile Thr
245
<210> 4
<211> 674
<212> PRT
<213> barley (Hordeum vulgare L.)
<400> 4
Met Phe Ser Asp Leu Phe Ala Leu Ile Phe Arg Ala Ile Ala Ser Pro
1 5 10 15
Val His Tyr Ala Cys Gln Leu Phe Gly Thr Met Lys Pro Ser Asp Asp
20 25 30
Arg Met Gln Leu Ser Gly Leu Thr Arg Ser Glu Glu Ser Ser Leu Asp
35 40 45
Val Glu Gly His Cys Ser His His Glu Ala Phe Pro Cys Ser Pro Ser
50 55 60
Met Gln Pro Val Ala Ser Gly Cys Val His Thr Glu Asn Ser Ala Ala
65 70 75 80
Tyr Phe Leu Trp Pro Thr Ser Asn Leu Gln His Cys Ala Ala Glu Gly
85 90 95
Arg Ala Asn Tyr Phe Gly Asn Leu Gln Lys Gly Leu Leu Pro Val Leu
100 105 110
Pro Gly Lys Leu Pro Lys Gly Gln Gln Ala Asn Ser Leu Leu Asp Leu
115 120 125
Met Thr Ile Arg Ala Phe His Ser Lys Ile Leu Arg Arg Phe Ser Leu
130 135 140
Gly Thr Ala Val Gly Phe Arg Ile Thr Lys Gly Val Leu Thr Glu Thr
145 150 155 160
Pro Ala Ile Leu Val Phe Val Ala Arg Lys Val His Lys Lys Trp Leu
165 170 175
Asn Pro Asn Gln Cys Leu Pro Ala Ile Leu Ala Gly Pro Gly Gly Val
180 185 190
Trp Cys Asp Val Asp Val Val Glu Phe Ser Tyr Tyr Gly Ala Pro Ala
195 200 205
Gln Thr Pro Lys Glu Gln Thr Phe Ser Glu Leu Val Asn Lys Leu Cys
210 215 220
Gly Ser Asp Glu Tyr Ile Gly Ser Gly Ser Gln Val Ala Ser Gln Asp
225 230 235 240
Thr Phe Gly Thr Leu Gly Ala Ile Val Lys Arg Arg Thr Asn Asn Lys
245 250 255
Gln Val Gly Phe Leu Thr Asn Arg His Val Ala Val Asp Leu Asp Tyr
260 265 270
Pro Asn Gln Lys Met Phe His Pro Leu Pro Pro Asn Leu Gly Pro Gly
275 280 285
Val Tyr Leu Gly Ala Val Glu Arg Ala Thr Ser Phe Ile Thr Asp Asp
290 295 300
Val Trp Tyr Gly Ile Tyr Ala Gly Thr Asn Pro Glu Thr Phe Val Arg
305 310 315 320
Ala Asp Gly Ala Phe Ile Pro Phe Ala Asp Asp Phe Asp Ile Ser Thr
325 330 335
Val Thr Thr Val Val Arg Glu Val Gly Glu Ile Gly Asp Val Lys Ile
340 345 350
Ile Asp Leu Gln Cys Pro Ile Lys Ser Leu Ile Gly Arg Gln Val Cys
355 360 365
Lys Val Gly Arg Ser Ser Gly His Thr Thr Gly Thr Val Met Ala Tyr
370 375 380
Ala Leu Glu Tyr Asn Asp Glu Lys Gly Ile Cys Phe Phe Thr Asp Leu
385 390 395 400
Leu Val Val Gly Glu Asn Arg Gln Thr Phe Asp Leu Glu Gly Asp Ser
405 410 415
Gly Ser Leu Ile Ile Leu Thr Ser Gln Asp Gly Glu Lys Pro Arg Pro
420 425 430
Ile Gly Ile Ile Trp Gly Gly Thr Ala Asn Arg Gly Arg Ile Lys Leu
435 440 445
Thr Ser Gly Tyr Gly Pro Glu Asn Trp Thr Thr Gly Val Asp Leu Gly
450 455 460
Arg Leu Leu Asp Arg Leu Glu Leu Asp Leu Ile Ile Asn Asp Glu Ser
465 470 475 480
Leu Lys Asp Ala Val Gln Glu Gln Arg Asn Ala Phe Val Ala Ala Ile
485 490 495
Asn Ser Ala Ile Gly Glu Ser Ser Ala Val Thr Val Thr Ala Pro Glu
500 505 510
Ala Thr Pro Ala Glu Lys Val Glu Glu Ile Phe Glu Pro Leu Gly Ile
515 520 525
Gln Ile Gln Gln Leu Pro Arg His Asp Pro Thr Ser Ala Ala Asp Glu
530 535 540
Gly Glu Gly Ala Ala Asn Thr Pro Ser Asp Met Glu Glu Arg Gln Phe
545 550 555 560
Ile Ser Asn Phe Val Gly Met Ser Pro Val Arg Arg Asp His Asp Ala
565 570 575
Arg Arg Thr Ile Ala Asn Leu Asn Asn Pro Ser Glu Glu Glu Leu Ala
580 585 590
Met Ser Leu His Leu Gly Asp Arg Glu Pro Lys Arg Leu Arg Leu Asp
595 600 605
Pro Glu Ser Asn Leu Asp Leu Glu Lys Gln Pro His Pro Asp Gln Glu
610 615 620
Pro Ser Leu Asp Leu Glu Lys Gln Pro Arg Ser Asp Pro Glu Met Met
625 630 635 640
Ser Leu Asp Leu Glu Lys Gln Pro Arg Ser Asp Pro Glu Pro Gly Leu
645 650 655
Asp Leu Glu Lys Arg Leu Pro Ala Asp Pro Glu Pro Ser Ile Asp Leu
660 665 670
Glu Lys
Claims (5)
1. The gene Hvhtd for controlling the multi-tillering character of the barley semi-dwarf is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO:1, the preparation method is as follows.
2. The protein encoded by the gene Hvhtd for controlling the barley half-dwarf multi-tillering trait according to claim 1, is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO: 3, the preparation method is as follows.
3. A molecular marker SNP62 for identifying a barley half-dwarf multi-tillering gene Hvhht based on an intron 2 takes barley as a species, and is characterized in that: the molecular marker primer is selected from the following primer pairs, wherein the nucleotide sequence is 5 '→ 3',
the forward primer is:
htd allele-1 GAAGGTCGGAGTCAACGGATTGAATATATTGGTTCAGGCTCTCAGA
Hua30 allele-2 GAAGGTGACCAAGTTCATGCTGAATATATTGGTTCAGGCTCTCAGG
the reverse primer GGCAGCATTCACTGCTTCAA.
4. The method for identifying the barley half-dwarf multi-tillering gene Hvhtd by using the primer corresponding to the molecular marker SNP62 as claimed in claim 3 is characterized by comprising the following steps:
(1) extracting the genomic DNA of the barley variety to be detected;
(2) performing PCR amplification on barley genome DNA by using a primer corresponding to the molecular marker SNP 62;
(3) and (3) analyzing by using QuantStaudio Real-Time PCR software according to the difference of the fluorescence signals of the PCR products, and further identifying the genotype of each barley to be detected.
5. The method for identifying the barley half-dwarf multi-tillering gene Hvhtd as claimed in claim 4, wherein the KASPar PCR reaction system used for the PCR amplification in the step 2) is as follows:
1 u L genome DNA, 0.1 u L primer, 1.4 u L sterilized water and 2.5 u L KASP Buffer, the total reaction volume is 5 u L; the PCR reaction was performed on an ABIViA 7 fluorescent quantitative PCR instrument and the reaction procedure was: 15min at 95 ℃; denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s with-0.6 ℃/cycle, 10 cycles; then denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 26 cycles;
of the 0.1. mu.L primer, 2 forward primers were each present at a concentration of 10pmol/L and the reverse primer at a concentration of 30 pmol/L.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008097197A1 (en) * | 2007-02-05 | 2008-08-14 | National University Of Singapore | Putative cytokinin receptor and methods for use thereof |
| CN107164347A (en) * | 2017-06-16 | 2017-09-15 | 中国科学院遗传与发育生物学研究所 | Control Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and the ideotype gene NPT1 of yield and its application |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008097197A1 (en) * | 2007-02-05 | 2008-08-14 | National University Of Singapore | Putative cytokinin receptor and methods for use thereof |
| CN107164347A (en) * | 2017-06-16 | 2017-09-15 | 中国科学院遗传与发育生物学研究所 | Control Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and the ideotype gene NPT1 of yield and its application |
Non-Patent Citations (1)
| Title |
|---|
| Expression level of a gibberellin 20-oxidase gene is associated with multiple agronomic and quality traits in barley;Qiaojun Jia等;《Theoretical and Applied Genetics》;20110212;第122卷(第8期);第1451–1460页 * |
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