CN106755359B - Method for screening or auxiliary screening of wheat with different plant heights and special kit thereof - Google Patents
Method for screening or auxiliary screening of wheat with different plant heights and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for screening or assisting in screening wheat with different plant heights and a special kit thereof. The method for screening or assisting in screening wheat with different plant heights comprises the following steps: detecting whether the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapI, genotype HapII or genotype HapIII, wherein the plant height of the wheat with the genotype HapI is more than that of the wheat with the genotype HapIII is more than that of the wheat with the genotype HapII. Experiments prove that the method provided by the invention can screen the plant height character of the wheat by detecting the genotype of the wheat to be detected based on the TaSPL21-6A gene, and has important application value in wheat molecular breeding.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for screening or assisting in screening wheat with different plant heights and a special kit thereof.
Background
Wheat is one of the major food crops in the world, accounts for 17 percent of the planting area of the food crops in the world, lives 40 percent of the population in the world, provides 20 percent of food energy and protein for human beings, and is the main food source for 35 to 40 percent of the population in the world. Therefore, the wheat yield is directly related to the world's food safety. At present, under the condition of limited cultivated land area, breeding of new wheat varieties with outstanding yield per unit is the most economic and effective way for stabilizing and improving the wheat yield. Wheat yield is influenced by various factors (such as spike number, kernel number and kernel weight), and the coordination of all factors is key to high yield of wheat. Plant height is one of the important factors affecting wheat yield, and is expressed as follows: within a certain range, the yield of the wheat is correspondingly increased along with the increase of the plant height; however, beyond a certain range, the yield of wheat is reduced on the contrary along with the increase of plant height, mainly because the leaf area coefficient reaches the optimal value, the wheat is easy to fall down, the wheat is difficult to harvest and the like. Therefore, the mining and controlling of the excellent allelic variation of the wheat plant height related traits and the development of the functional markers of the corresponding allelic variation have important significance for the molecular marker-assisted breeding of high-yield wheat varieties.
Single Nucleotide Polymorphism (SNP) refers to a polymorphism of a DNA sequence caused by a Single nucleotide variation at the genome level. With the development of technology, the number of SNP detection methods is increased, and the detection cost is more and more economical, so that the SNP detection method becomes a new generation of molecular marker after SSR. At present, in the wheat genome research process, SNP markers are applied to the construction of wheat genetic maps, the positioning of important agronomic trait related genes of wheat, genome association analysis and the like.
The restriction amplified polymorphic sequence marker (CAPS) is mainly used for restriction analysis of the DNA fragment amplified by PCR. Designing specific primer according to gene sequence, combining specific PCR amplification and restriction enzyme cutting to detect nucleic acid sequence polymorphism. The basic principle is that a pair of specific PCR primers is designed by utilizing the DNA sequence of a known site, then a DNA fragment is specifically amplified by using the primers, then the obtained PCR amplification product is digested by using specific restriction enzyme, and finally the digestion product is separated and detected by gel electrophoresis.
Disclosure of Invention
The invention aims to solve the technical problem of how to screen or assist in screening wheat with different plant heights.
In order to solve the technical problems, the invention firstly provides a method for screening or assisting in screening wheat with different plant heights.
The method for screening or assisting in screening wheat with different plant heights, which is provided by the invention, can be specifically a method I, and can comprise the following steps: detecting whether the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapI, genotype HapII or genotype HapIII, wherein the plant height of the wheat with the genotype HapI is more than that of the wheat with the genotype HapIII;
the wheat with the genotype HapI is the wheat with the genotype based on the T125C SNP locus as TT homozygote, the genotype based on the T160C SNP locus as CC homozygote and the genotype based on the C169G SNP locus as CC homozygote; the wheat with the genotype HapII is the wheat with the genotype based on the T125C SNP locus being CC homozygous, the genotype based on the T160C SNP locus being TT homozygous and the genotype based on the C169G SNP locus being GG homozygous; the wheat with the genotype HapIII is the wheat with the genotype based on the T125C SNP locus being CC homozygous, the genotype based on the T160C SNP locus being TT homozygous and the genotype based on the C169G SNP locus being CC homozygous;
the T125C SNP site is the 125 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the T160C SNP site is the 160 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the C169G SNP site is 169 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome.
The method for screening or assisting in screening wheat with different plant heights, which is provided by the invention, can be specifically a method II, and can comprise the following steps: detecting whether the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapI, genotype HapII or genotype HapIII, wherein the plant height of the wheat with the genotype HapI is more than that of the wheat with the genotype HapIII;
the wheat with the genotype HapI is the wheat with the genotype based on the T125C SNP locus as TT homozygote, the genotype based on the T160C SNP locus as CC homozygote and the genotype based on the C169G SNP locus as CC homozygote; the wheat with the genotype HapII is the wheat with the genotype based on the T125C SNP locus being CC homozygous, the genotype based on the T160C SNP locus being TT homozygous and the genotype based on the C169G SNP locus being GG homozygous; the wheat with the genotype HapIII is the wheat with the genotype based on the T125C SNP locus being CC homozygous, the genotype based on the T160C SNP locus being TT homozygous and the genotype based on the C169G SNP locus being CC homozygous;
the T125C SNP site is the 125 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the T160C SNP site is the 160 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the C169G SNP site is 169 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome.
The method for screening or assisting in screening wheat with different plant heights, which is provided by the invention, can be specifically a method III, and sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) carrying out enzyme digestion on the PCR amplification product by using a restriction enzyme BglI to obtain an enzyme digestion product A; carrying out enzyme digestion on the PCR amplification product by using restriction enzyme BseDI or SecI to obtain an enzyme digestion product B;
(3) the following evaluations were carried out:
if the enzyme digestion product A is two DNA fragments and the enzyme digestion product B is one DNA fragment, the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapI; if the enzyme digestion product A is a DNA fragment and the enzyme digestion product B is two DNA fragments, the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapII; if the enzyme digestion product A is a DNA fragment and the enzyme digestion product B is a DNA fragment, the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapIII;
the plant height of the wheat with genotype HapI is greater than that of the wheat with genotype HapIII is greater than that of the wheat with genotype HapII;
the primer F is a1) or a 2):
a1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer R is a3) or a 4):
a3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5.
The method for screening or assisting in screening wheat with different plant heights, which is provided by the invention, can be specifically the fourth method, and sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) carrying out enzyme digestion on the PCR amplification product by using a restriction enzyme BglI to obtain an enzyme digestion product A; carrying out enzyme digestion on the PCR amplification product by using restriction enzyme BseDI or SecI to obtain an enzyme digestion product B;
(3) the following evaluations were carried out:
if the enzyme digestion product A has 176bp DNA fragments and 166bp DNA fragments and the enzyme digestion product B has 342bp DNA fragments, the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapI; if the enzyme digestion product A has a 342bp DNA fragment and the enzyme digestion product B has a 165bp DNA fragment and a 177bp DNA fragment, the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapII; if the enzyme digestion product A has a 342bp DNA fragment and the enzyme digestion product B has a 342bp DNA fragment, the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapIII;
the plant height of the wheat with genotype HapI is greater than that of the wheat with genotype HapIII is greater than that of the wheat with genotype HapII;
the primer F is a1) or a 2):
a1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer R is a3) or a 4):
a3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5.
The method for screening or assisting in screening wheat with different plant heights, which is provided by the invention, can be specifically a method five, and sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) sequencing the PCR amplification product;
(3) the following evaluations were carried out:
if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of the sequence 1 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapI; if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of a sequence 2 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapII; if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of the sequence 3 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapIII;
the plant height of the wheat with genotype HapI is greater than that of the wheat with genotype HapIII is greater than that of the wheat with genotype HapII;
the primer F is a1) or a 2):
a1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer R is a3) or a 4):
a3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5.
In any of the above methods, the > may specifically be statistical >.
In order to solve the technical problems, the invention also provides a kit for identifying or assisting in identifying the plant height of wheat.
The kit for identifying or assisting in identifying the wheat plant height comprises a substance for detecting whether the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapI, genotype HapII or genotype HapIII;
the genotype HapI is TaSPL21-6A gene which is TT homozygous based on the genotype of the T125C SNP locus, CC homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus; the genotype HapII is TaSPL21-6A gene which is CC homozygous based on the genotype of T125C SNP locus, TT homozygous based on the genotype of T160C SNP locus and GG homozygous based on the genotype of C169G SNP locus; the genotype HapIII is TaSPL21-6A gene which is CC homozygous based on the genotype of the T125C SNP locus, TT homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus;
the T125C SNP site is the 125 th nucleotide from the 5' end of a sequence 9 in a sequence table or a sequence 10 in the sequence table in a wheat genome; the T160C SNP site is the 160 th nucleotide from the 5' end of a sequence 9 in a sequence table or a sequence 10 in the sequence table in a wheat genome; the C169G SNP site is 169 th nucleotide from 5' end of a sequence 9 in a sequence table or a sequence 10 in the sequence table in a wheat genome.
In the kit, the substance for detecting whether the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapI, the genotype HapII or the genotype HapIII can be a primer pair consisting of a primer F and a primer R;
the primer F is a1) or a 2):
a1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer R is a3) or a 4):
a3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5.
The kit can also comprise restriction enzyme BglI and/or restriction enzyme BseDI and/or restriction enzyme SecI.
In order to solve the technical problems, the invention also provides a molecular marker A and a molecular marker B.
The molecular marker A provided by the invention can be shown as a sequence 10 in a sequence table.
The molecular marker B provided by the invention can be shown as a sequence 9 in a sequence table.
(z1) or (z2) or (z3) or (z4) or (z5) also belong to the scope of protection of the present invention;
(z1) the kit according to any one of the above, or the molecular marker A, or the molecular marker B is applied to screening or auxiliary screening of wheat with different plant heights;
(z2) the application of the kit or the molecular marker A or the molecular marker B in the preparation of products for screening or auxiliary screening of wheat with different plant heights;
(z3) the use of the kit according to any one of the above, or the molecular marker A, or the molecular marker B, for genotyping the wheat TaSPL21-6A gene;
(z4) the use of any one of the kit or the molecular marker A or the molecular marker B in the preparation of a product for identifying the genotype of the wheat TaSPL21-6A gene;
(z5) use of the kit according to any one of the above, or the molecular marker A, or the molecular marker B in wheat breeding.
In the application, when the genotype of the T125C SNP site of the molecular marker A is TT homozygote, the genotype of the T160C SNP site is CC homozygote and the genotype of the C169G SNP site is CC homozygote, the wheat with the genotype HapI is judged; when the genotype of the T125C SNP site of the molecular marker A is CC homozygous, the genotype of the T160C SNP site is TT homozygous and the genotype of the C169G SNP site is GG homozygous, the wheat with the genotype HapI I is judged; when the genotype of the T125C SNP site of the molecular marker A is CC homozygous, the genotype of the T160C SNP site is TT homozygous and the genotype of the C169G SNP site is CC homozygous, the wheat with the genotype HapIII is judged; the T125C SNP site is the 125 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the T160C SNP site is the 160 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the C169G SNP site is 169 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome;
the plant height of wheat with genotype HapI is higher than that of wheat with genotype HapIII is higher than that of wheat with genotype HapII.
In the application, when the genotype of the T125C SNP site of the molecular marker B is TT homozygote, the genotype of the T160C SNP site is CC homozygote and the genotype of the C169G SNP site is CC homozygote, the wheat with the genotype HapI is judged; when the genotype of the T125C SNP site of the molecular marker B is CC homozygous, the genotype of the T160C SNP site is TT homozygous and the genotype of the C169G SNP site is GG homozygous, the wheat with the genotype HapII is judged; when the genotype of the T125C SNP site of the molecular marker B is CC homozygous, the genotype of the T160C SNP site is TT homozygous and the genotype of the C169G SNP site is CC homozygous, the wheat with the genotype HapIII is judged; the T125C SNP site is the 125 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the T160C SNP site is the 160 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the C169G SNP site is 169 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome;
the plant height of wheat with genotype HapI is higher than that of wheat with genotype HapIII is higher than that of wheat with genotype HapII.
In the above application, the > may specifically be statistical >.
Experiments prove that the method provided by the invention can screen or assist in screening the plant height character of the wheat by detecting the genotype of the wheat to be detected based on the TaSPL21-6A gene, and has important application value in wheat molecular breeding.
Drawings
FIG. 1 is the nucleotides of the three genotypes at the T125C SNP, the T160C SNP and the C169G SNP.
FIG. 2 shows the result of electrophoresis in step two, step 3) of example 2.
FIG. 3 shows the result of electrophoresis in step two, step 4) of example 2.
FIG. 4 is the correlation analysis of wheat of three genotypes under different planting conditions and plant height. E1 is 2009 beijing chang dry land, E2 is 2009 beijing chang flat paddy field, E3 is 2009 beijing cis-meaning dry land, E4 is 2009 beijing cis-meaning water paddy field, E5 is 2009 beijing cis-meaning dry land and heat stress is applied, E6 is 2009 beijing cis-meaning water paddy field and heat stress is applied, E7 is 2010 beijing jingzhang flat dry land, E8 is 2010 beijing chang flat paddy field, E9 is 2010 beijing cis-meaning dry land, E10 is 2010 beijing cis-meaning water paddy field, E11 is 2010 beijing cis-meaning dry land and heat stress is applied, E12 is 2010 beijing cis-meaning water paddy field and heat stress is applied, E13 is 2011 dry land, E14 is 2010 beijing cis-meaning water paddy field, E15 is Beijing cis-meaning water paddy field and heat stress is applied to Beijing 2011 water paddy field and 2012 heat stress is E19, E638 is Beijing cis-meaning water paddy field and heat stress is E2011 Beijing water paddy field; p < 0.5, P < 0.01, P < 0.001.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The wheat material used in the examples described below was obtained from the national germplasm resources pool (web site: http:// www.cgris.net /), publicly available from the national germplasm resources pool.
The 5 XPCR buffer and TransStart Fast Pfu DNA Polymerase are both products of Beijing Quanjin Biotechnology Limited, and the catalog number is AP 221. The dNTPs are products of Roche, and the product number is #316K 5S.
Example 1 obtaining of polymorphic sites and obtaining of haplotypes of the wheat TaSPL21-6A Gene
Firstly, obtaining the polymorphic site of the TaSPL21-6A gene of wheat
Genomic DNAs of 37 triticale species shown in Table 1 were each extracted and subjected to PCR amplification using the extracted genomic DNAs as templates and artificially synthesized primers 5'-GGATAGTGCAGTGAAGCGGT-3' and 5'-CCGTGTAAGAGGGTAACACTGA-3', respectively, to obtain PCR amplification products. Sequencing each PCR amplification product, and then aligning.
TABLE 1
| Numbering | Wheat variety | Numbering | Wheat variety |
| 1 | TAM-110 | 20 | Yumai 48 |
| 2 | Ann 85 middle 124-1 | 21 | Huai Mai 18 |
| 3 | Single R8108 | 22 | Double harvest |
| 4 | Yuandong 847 | 23 | Rougha 14 |
| 5 | Beijing No. 10 | 24 | Shannong Youmai No. 2 |
| 6 | Jing Ke 8922 | 25 | Long 6359 |
| 7 | Zhongyou 9507 | 26 | Jin wheat 16 |
| 8 | Cangmai 6005 | 27 | Jin wheat 25 |
| 9 | Heng Mai No. 2 | 28 | Jin wheat 39 |
| 10 | Handan 6050 | 29 | Dry election No. 10 |
| 11 | Balance 7228 | 30 | Jin wheat 51 |
| 12 | Weighing apparatus 35 | 31 | Linfeng 615 |
| 13 | Stone 4185 | 32 | Taiyuan 633 |
| 14 | Shimai No. 12 | 33 | Yuanhong 20410 |
| 15 | Bainong 3217 | 34 | PANDAS |
| 16 | Thinopyrum I | 35 | Opata 85 |
| 17 | Elytrigia 4110 | 36 | W7984 |
| 18 | Yumai No. 29 | 37 | Hometown 188 |
| 19 | Yumai 38 |
The alignment result shows that 3 SNP sites are found in the TaSPL21-6A gene of wheat and are respectively named as T125CSNP, T160CSNP and C169G SNP. T125CSNP is located at 125 th position from 5' tail end of sequence 1 in the sequence table, T160CSNP is located at 160 th position from 5' tail end of sequence 1 in the sequence table, and T169CSNP is located at 169 th position from 5' tail end of sequence 1 in the sequence table. The basic information of the 3 SNP sites is shown in Table 2 (since the genomic DNA is a double-stranded DNA molecule consisting of two single-stranded DNA molecules complementary to each other in the reverse direction, a DNA molecule encoding a protein, that is, a DNA molecule having a start codon to a stop codon, is generally designated as a sense DNA molecule; a DNA molecule complementary to the reverse direction of the sense DNA molecule is designated as an antisense DNA molecule; it is noted that the genotypes of the alleles at the T125C SNP, the T160C SNP and the C169G SNP are all the genotypes of the sense DNA).
TABLE 2
| SNP site name | Genotype of allele |
| T125C SNP | TT homozygote type and CC homozygote type |
| T160C SNP | TT homozygote type and CC homozygote type |
| C169G SNP | CC homozygote type and GG homozygote type |
II, obtaining haplotypes of the wheat TaSPL21-6A gene
According to the comparison result of the step one, only three haplotypes exist in the T125C SNP, the T160C SNP and the C169G SNP in the wheat natural variation population, so that the wheat is divided into three genotypes according to the wheat TaSPL21-6A gene: TaSPL21-6A-HapI (hereinafter referred to as genotype HapI), TaSPL21-6A-HapII (hereinafter referred to as genotype HapII) and TaSPL21-6A-HapIII (hereinafter referred to as genotype HapII). The nucleotides of the respective genotypes at the T125C SNP, the T160C SNP and the C169G SNP are shown in detail in FIG. 1. The genotypes of the alleles of the individual genotypes at the T125C SNP, T160C SNP and C169G SNP are shown in table 3. The nucleotide sequence of the TaSPL21-6A gene of wheat with genotype HapI is shown as a sequence 1 in a sequence table. The nucleotide sequence of the TaSPL21-6A gene of wheat with genotype HapII is shown as a sequence 2 in a sequence table. The nucleotide sequence of the TaSPL21-6A gene of the wheat with the genotype HapIII is shown as a sequence 3 in a sequence table.
TABLE 3
Example 2 establishment of TaSPL21-6A genotyping method based on the genotype of wheat TaSPL21-6A Gene
Preparation of specific primer pair
Specific primer pairs designed to amplify target sequences including the T125C SNP, the T160C SNP, and the C169G SNP were as follows:
and (3) primer F: 5'-GGATAGTGCAGTGAAGCGGT-3' (SEQ ID NO: 4 in the sequence Listing);
and (3) primer R: 5'-TGGCATCTCTTGGGCTCTT-3' (SEQ ID NO: 5 in the sequence Listing);
secondly, establishment of TaSPL21-6A gene typing method based on wheat TaSPL21-6A gene genotype
1. Method A
1) Extracting genome DNA of wheat to be detected;
2) and (2) performing PCR amplification by using the genome DNA obtained in the step 1) as a template and using a primer pair consisting of the primer F and the primer R obtained in the step one to obtain a PCR amplification product.
PCR amplification reaction (15. mu.L): ddH2O8.0. mu.L, 5 XPCR buffer 3.0. mu.L, each of 0.6. mu. L, dNTPs (2.5. mu. mol/L) 0.4. mu.L of primer F (5. mu. mol/L) and primer R (5. mu. mol/L), TransStart Fast Pfu DNApolymere0.3. mu.L (containing TransStart Fast Pfu DNA Polymerase 0.75U), and genomic DNA (20 ng/. mu.L) 2.1. mu.L.
The PCR amplification reaction condition is that the temperature is 95 ℃ for 5 min; 1min at 95 ℃, 45s at 59 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃; storing at 4 ℃.
3) Carrying out enzyme digestion on the PCR amplification product obtained in the step 2) by using a restriction enzyme BglI to obtain an enzyme digestion product A, and then carrying out agarose gel electrophoresis detection.
4) Digesting the PCR amplification product obtained in the step 2) by using restriction enzyme BseDI or SecI to obtain a digestion product B, and then carrying out agarose gel electrophoresis detection.
If the enzyme digestion product A shows a band type A1 (two bands are shown, 176bp and 166bp respectively) and the enzyme digestion product B shows a band type B2 (one band is shown, 342bp), the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapI; if the enzyme digestion product A shows a band type A2 (one band is shown and is 342bp) and the enzyme digestion product B shows a band type B1 (two bands are shown and are respectively 165bp and 177bp), the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapII; if the enzyme digestion product A shows a band type A2 (one band is shown and is 342bp) and the enzyme digestion product B shows a band type B2 (one band is shown and is 342bp), the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapIII.
2. Method B
1) Extracting genome DNA of wheat to be detected;
2) and (2) performing PCR amplification by using the genome DNA obtained in the step 1) as a template and using a primer pair consisting of the primer F and the primer R obtained in the step one to obtain a PCR amplification product.
PCR amplification reaction (15. mu.L): ddH2O8.0. mu.L, 5 XPCR buffer 3.0. mu.L, each of 0.6. mu. L, dNTPs (2.5. mu. mol/L) 0.4. mu.L of primer F (5. mu. mol/L) and primer R (5. mu. mol/L), TransStart Fast Pfu DNApolymere0.3. mu.L (containing TransStart Fast Pfu DNA Polymerase 0.75U), and genomic DNA (20 ng/. mu.L) 2.1. mu.L.
The PCR amplification reaction condition is that the temperature is 95 ℃ for 5 min; 1min at 95 ℃, 45s at 59 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃; storing at 4 ℃.
3) Sequencing the PCR amplification product obtained in the step 2).
If the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of the sequence 1 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapI; if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of a sequence 2 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is a genotype HapII; if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from the 5' end of the sequence 3 in the sequence table, the genotype of the wheat to be detected based on the TaSPL21-6A gene is the genotype HapIII.
Example 3 correlation analysis of wheat TaSPL21-6A Gene genotype and wheat plant height
The wheat to be tested is 262 wheat varieties (all are hexaploid wheat), and the specific name of the wheat varieties is shown in the 2 nd column in the table 4.
Genotyping of wheat to be tested
And (3) performing genotyping on each wheat to be detected by adopting the method A in the step two of the embodiment 2.
The results of part of the experiments with the restriction enzyme BglI are shown in FIG. 2(M is DNA Marker, C is band A1, and T is band A2).
The results of part of the experiments with restriction enzymes BseDI or SecI are shown in FIG. 3(M is DNA Marker, C is band B2, G is band B1)
The genotyping results for each wheat tested based on the genotype of wheat TaSPL21-6A gene are shown in table 4, column 3.
The results show that the genotype of the wheat to be detected based on the TaSPL21-6A gene is genotype HapI, genotype HapII or genotype HapIII based on T125C SNP, T160C SNP and C169G SNP. Since wheat varieties are cultivars and are therefore generally defaulted to highly homozygous plant material, the genotypes of the T125C SNP, T160C SNP, and C169G SNP are all homozygous.
Second, plant height character detection
Planting each wheat variety to be tested (all hexaploid wheat) in 2009 Beijing Changping dry land (hereinafter referred to as E1), 2009 Beijing Changping horizontal paddy land (hereinafter referred to as E2), 2009 Beijing cis-oriented dry land (hereinafter referred to as E3), 2009 Beijing cis-oriented paddy land (hereinafter referred to as E4), 2009 Beijing cis-oriented dry land and thermal stress (hereinafter referred to as E5), 2009 Beijing cis-oriented paddy land and thermal stress (hereinafter referred to as E6), 2010 Beijing Changping dry land (hereinafter referred to as E7), 2010 Beijing horizontal paddy land (hereinafter referred to as E8), 2010 Beijing cis-oriented dry land (hereinafter referred to as E9), 2010 Beijing cis-oriented dry land (hereinafter referred to as E6324), 2010 cis-oriented dry land (hereinafter referred to as E7), 2010 cis-oriented wet land and thermal stress (hereinafter referred to as E2011), 2010E 685-oriented dry land (hereinafter referred to as E685) and thermal stress, 2011 Beijing cisoid dry land is subjected to heat stress (hereinafter referred to as E15), 2011 Beijing cisoid paddy land is subjected to heat stress (hereinafter referred to as E16), 2012 Beijing Chang horizontal dry land (hereinafter referred to as E17), 2012 Beijing Chang horizontal paddy land (hereinafter referred to as E18), 2012 Beijing cisoid dry land (hereinafter referred to as E19) or 2012 Beijing cisoid paddy land (hereinafter referred to as E20), after maturation, the plant height of wheat is counted, and partial statistical results are shown in Table 4Columns 4 to 8. It should be noted that the dry land, the horizontal water land, the cis-trans land and the cis-trans land of Beijing are all experimental bases of the institute of crop science of Chinese academy of agricultural sciences. The dry land means that the whole growing period of the wheat to be tested is only irrigated by natural rainwater, and no water is artificially irrigated. The water field refers to that the whole growing period of the wheat to be tested is not only naturally irrigated by rainwater, but also artificially irrigated by water (the specific operation of artificial irrigation is that the wheat to be tested is irrigated respectively before the wintering period, the heading period, the flowering period and the filling period, and the water quantity of each irrigation is 750m3Hectare of water). And the heat stress is that when the wheat variety to be detected is in the flowering phase, the plastic greenhouse is covered until the wheat variety to be detected is mature. The heat stress is carried out in dry land, the average temperature outside the plastic greenhouse is 33 ℃, and the temperature inside the plastic greenhouse is 43 ℃. The plastic greenhouse is subjected to heat stress in the water, the average temperature outside the plastic greenhouse is 33 ℃, and the temperature inside the plastic greenhouse is 41 ℃.
Third, the correlation analysis of the genotype of the wheat TaSPL21-6A gene and the wheat plant height
The average plant height of wheat of three genotypes under different planting conditions is respectively counted, the genotype and the plant height are subjected to correlation analysis, and the results are shown in table 5 and fig. 4.
TABLE 4
Note: "-" indicates that no data was obtained.
TABLE 5
Note: p value is the significance level of the association analysis; PVE is the interpretation rate of phenotypic variation.
The result shows that the detection of the genotype of the wheat to be detected based on the TaSPL21-6A gene, the plant height of the 'genotype HapI wheat', the plant height of the 'genotype HapIII wheat', the plant height of the 'genotype HapII wheat'; the ">" is statistically >.
Fourthly, sequencing
The method B in the step II of the embodiment 2 is adopted to respectively sequence the wheat with the genotype HapI, the wheat with the genotype HapII and the wheat with the genotype HapII which are detected in the step I. Sequencing results show that the nucleotide sequences of the PCR amplification products of the wheat with genotype HapI are all shown as 1 st to 342 th sites from the 5' tail end of a sequence 1 in a sequence table, the nucleotide sequences of the PCR amplification products of the wheat with genotype HapII are all shown as 1 st to 342 th sites from the 5' tail end of a sequence 2 in the sequence table, and the nucleotide sequences of the PCR amplification products of the wheat with genotype HapIII are all shown as 1 st to 342 th sites from the 5' tail end of a sequence 3 in the sequence table. As can be seen, the detection results of the method A and the method B are completely consistent.
The results show that the plant height trait of the wheat to be detected can be screened or screened in an auxiliary manner by detecting the genotype of the wheat based on the TaSPL21-6A gene, and the method has important application value in wheat molecular breeding.
<110> institute of crop science of Chinese academy of agricultural sciences
<120> method for screening or auxiliary screening of wheat with different plant heights and special kit thereof
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 3463
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 1
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatctgcccc agatttgggc tagatataag agatgtcggc cagcctggcg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc catcattgcc tctctccttc 360
tccggcctcc acgacctttt tagcctgaag gcacgcacaa ccgttcctgc tgcacagaca 420
catactccgg cggtccggcc tctttctttc ttacccatca gtcaggccgc gcgtcaccac 480
ctttctcatc cgcgctcgga atagccggcc atgatgagca gcaggctaag cggcggcacg 540
atggcaccgg ttagcgacat ggccgacttc ggttgcgctc ccatgcagtc ctaccccaac 600
tttgagccgg ccggcatggt catgcccggg gaccggcagc cgcccttcca gcaccaccac 660
ctctacgaca gcctcgactt caacgccgct gcattctcgt tccaagaccc ggtcgcgctc 720
ttctccagcg gctcggcgtt cagtaaccag ctccagcagc cgttcctcca gacgcaggtc 780
accacgccga cgatggcgtc gtcgtcgctg ctgcaggcgc cgatgatgac ccttccgggt 840
atgctgacgt cttcctcggc gtcgccggtg gacgcatgca ccttcggtgg cggcggcagt 900
gctgggttcc tgaagcagga ggagggcggc cccttctcag acgtcggcgg tggggggagg 960
atcgggctga acctcggccg caggacatac ttttccccgg cggacgtgct ggccgtggac 1020
cgcctgctga tgcgctcccg cttcggcgga gccggcgcaa tgggcatgct ggggctgggg 1080
ctcggcgccg ccgcccacca ccaccagacc ccgcggtgcc aggctgaggg ctgcaaggcc 1140
gacctctccg ccgccaagca ctaccaccgc cgccacaagg tctgcgagta ccacgccaag 1200
gccaccacag tcgccgcctc tggcaagcag cagcgcttct gccaacaatg cagccggtat 1260
gtacgtttat cctctgccat gggtggatcg ttgatgcatg cgaagctttg ctagcagacg 1320
aaaaaatcag cagtagaaag gctgcgagct ttcgatctag tggcattgac gctagcagct 1380
atatgcactg atcatatcat tcatgtcaac atgtcatata tcagaaatgg cgcacatcag 1440
atcagatctt gttcctctga gcgtatcggc atctgcatcc ctttgtccaa tgtactacta 1500
gcacgcccat catggcattg cagtggagtt cgatcagata cctaaaccta gtctgtgtag 1560
atacatcata catggcgtgt attacttaca acggtagaca tgtatatacc caagatccgg 1620
ttgtgtgaat ataactggtt cttgtttggg tacgtttgtt gggctaggtt tcatgtgctc 1680
gctgagtttg acgaggccaa gaggagctgc cggaagcggc tcacggagca caaccggcgc 1740
cgccgaaagc ctgccggcgc gcagggcaag gattcgccgc cgccttccaa gaaggcagac 1800
gctagcatca ccagctcata caccggcgat cacaagagta agaccactcc tctgatacat 1860
gtatagactt aaacactgga tatatgctta ttattggctt ttttaaagct tgatcacttt 1920
aagaagttgt gttttgattt tcggccgcaa aatgtggatt tcggccgaat ttcggtcatc 1980
tcggcttgag gcgaaaagta tatgtaggcc gaaattactc aaatttggta aattttggtc 2040
aaatttaagt caaattgcag tcaaaatttg atcaaatttc agccgaaaat tttgaaaatg 2100
gccgaaattc ggccatctcg gcctagggcg aaaaaaagct caaaccgaaa atcaaaacac 2160
agccagtaag ctagctagct acctagaaac tcctgaaact ttgttagagc ttagtgggta 2220
atttgaacta aaagcatgac acttcttttg caacaaggga acatttcaat aacgtatacc 2280
aagtaaagaa cacaacaaaa acagtttgcc taattcttaa ttgtccgaaa atttagtaaa 2340
tctattttgt cttgaaatgg cacatcactg ggctaaccta ataatgtact ccgtagcatc 2400
ttaagtgtgt aactgaagta agatataact gtagttttga cggtgcactt cagcacttgg 2460
ctgcagtaca caccggtaat tctatgtgcc agcaagcacg ttacaacgtt cggttggata 2520
gaaactagta gtacacgaca tgacatgcct agatgctact tctacatttt ggtcggtcga 2580
tctgacgtac gtacctacta ggagtactac tcagctcaat taacatgatg ttcatacgat 2640
ggcgccaaaa catcttatac gtacatacgt aggtgtacca tcgtactata cactagacgg 2700
taggtgcaac tcctgctaca tacatgtggc agtcacatct tgccgtcttg tctgacgccg 2760
cgacgtgtat atgatcagcc aacaagtcga cgacgggggc ggccttctcg ccgagcgccg 2820
gtggcttcag ctgccttcag cagcagcagc agcagcatga gatcgacaac ggcggccagt 2880
cgagcaacgc cacgccgact aacctgtccc tggcggcgcc accgccaccg ccgccgcctc 2940
aagacgacgc cggcttcggc gccggccttg acaccatgct gctgattcag cagcaagggc 3000
ccgatgaaca ggaggaggag gaggagcagc atttcatgat gacctctctc gtgcagtcgc 3060
atcgccagca gcagcagcac ggcgacagcg gcaacatctt atcgtgctcg acgacgtcgc 3120
cgtcggatca gcgtcgccat cagaacgacg gcgacagctg ctgcaacggc aacagcatgc 3180
agcatttctt tgaggtggag ttcatgtagc acgatgcatg catgcactgc atgaatgaac 3240
ctggcctagc ctagcctaga ggtggtttga ctgcattatc gtgaggtgtg tgtgcatgag 3300
gtgaaagaga aaacaagctg gttgcatgca ctaggctgtg catggagtat agctactagg 3360
gtacgtaggc gtgcaggctt tttctttcca gatttaattt cctgaccttt tctgcatact 3420
gtgtttttac ttgtactttt ctcagtgtta ccctcttaca cgg 3463
<210> 2
<211> 3463
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 2
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatccgcccc agatttgggc tagatataag agatgtcggt cagcctgggg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc catcattgcc tctctccttc 360
tccggcctcc acgacctttt tagcctgaag gcacgcacaa ccgttcctgc tgcacagaca 420
catactccgg cggtccggcc tctttctttc ttacccatca gtcaggccgc gcgtcaccac 480
ctttctcatc cgcgctcgga atagccggcc atgatgagca gcaggctaag cggcggcacg 540
atggcaccgg ttagcgacat ggccgacttc ggttgcgctc ccatgcagtc ctaccccaac 600
tttgagccgg ccggcatggt catgcccggg gaccggcagc cgcccttcca gcaccaccac 660
ctctacgaca gcctcgactt caacgccgct gcattctcgt tccaagaccc ggtcgcgctc 720
ttctccagcg gctcggcgtt cagtaaccag ctccagcagc cgttcctcca gacgcaggtc 780
accacgccga cgatggcgtc gtcgtcgctg ctgcaggcgc cgatgatgac ccttccgggt 840
atgctgacgt cttcctcggc gtcgccggtg gacgcatgca ccttcggtgg cggcggcagt 900
gctgggttcc tgaagcagga ggagggcggc cccttctcag acgtcggcgg tggggggagg 960
atcgggctga acctcggccg caggacatac ttttccccgg cggacgtgct ggccgtggac 1020
cgcctgctga tgcgctcccg cttcggcgga gccggcgcaa tgggcatgct ggggctgggg 1080
ctcggcgccg ccgcccacca ccaccagacc ccgcggtgcc aggctgaggg ctgcaaggcc 1140
gacctctccg ccgccaagca ctaccaccgc cgccacaagg tctgcgagta ccacgccaag 1200
gccaccacag tcgccgcctc tggcaagcag cagcgcttct gccaacaatg cagccggtat 1260
gtacgtttat cctctgccat gggtggatcg ttgatgcatg cgaagctttg ctagcagacg 1320
aaaaaatcag cagtagaaag gctgcgagct ttcgatctag tggcattgac gctagcagct 1380
atatgcactg atcatatcat tcatgtcaac atgtcatata tcagaaatgg cgcacatcag 1440
atcagatctt gttcctctga gcgtatcggc atctgcatcc ctttgtccaa tgtactacta 1500
gcacgcccat catggcattg cagtggagtt cgatcagata cctaaaccta gtctgtgtag 1560
atacatcata catggcgtgt attacttaca acggtagaca tgtatatacc caagatccgg 1620
ttgtgtgaat ataactggtt cttgtttggg tacgtttgtt gggctaggtt tcatgtgctc 1680
gctgagtttg acgaggccaa gaggagctgc cggaagcggc tcacggagca caaccggcgc 1740
cgccgaaagc ctgccggcgc gcagggcaag gattcgccgc cgccttccaa gaaggcagac 1800
gctagcatca ccagctcata caccggcgat cacaagagta agaccactcc tctgatacat 1860
gtatagactt aaacactgga tatatgctta ttattggctt ttttaaagct tgatcacttt 1920
aagaagttgt gttttgattt tcggccgcaa aatgtggatt tcggccgaat ttcggtcatc 1980
tcggcttgag gcgaaaagta tatgtaggcc gaaattactc aaatttggta aattttggtc 2040
aaatttaagt caaattgcag tcaaaatttg atcaaatttc agccgaaaat tttgaaaatg 2100
gccgaaattc ggccatctcg gcctagggcg aaaaaaagct caaaccgaaa atcaaaacac 2160
agccagtaag ctagctagct acctagaaac tcctgaaact ttgttagagc ttagtgggta 2220
atttgaacta aaagcatgac acttcttttg caacaaggga acatttcaat aacgtatacc 2280
aagtaaagaa cacaacaaaa acagtttgcc taattcttaa ttgtccgaaa atttagtaaa 2340
tctattttgt cttgaaatgg cacatcactg ggctaaccta ataatgtact ccgtagcatc 2400
ttaagtgtgt aactgaagta agatataact gtagttttga cggtgcactt cagcacttgg 2460
ctgcagtaca caccggtaat tctatgtgcc agcaagcacg ttacaacgtt cggttggata 2520
gaaactagta gtacacgaca tgacatgcct agatgctact tctacatttt ggtcggtcga 2580
tctgacgtac gtacctacta ggagtactac tcagctcaat taacatgatg ttcatacgat 2640
ggcgccaaaa catcttatac gtacatacgt aggtgtacca tcgtactata cactagacgg 2700
taggtgcaac tcctgctaca tacatgtggc agtcacatct tgccgtcttg tctgacgccg 2760
cgacgtgtat atgatcagcc aacaagtcga cgacgggggc ggccttctcg ccgagcgccg 2820
gtggcttcag ctgccttcag cagcagcagc agcagcatga gatcgacaac ggcggccagt 2880
cgagcaacgc cacgccgact aacctgtccc tggcggcgcc accgccaccg ccgccgcctc 2940
aagacgacgc cggcttcggc gccggccttg acaccatgct gctgattcag cagcaagggc 3000
ccgatgaaca ggaggaggag gaggagcagc atttcatgat gacctctctc gtgcagtcgc 3060
atcgccagca gcagcagcac ggcgacagcg gcaacatctt atcgtgctcg acgacgtcgc 3120
cgtcggatca gcgtcgccat cagaacgacg gcgacagctg ctgcaacggc aacagcatgc 3180
agcatttctt tgaggtggag ttcatgtagc acgatgcatg catgcactgc atgaatgaac 3240
ctggcctagc ctagcctaga ggtggtttga ctgcattatc gtgaggtgtg tgtgcatgag 3300
gtgaaagaga aaacaagctg gttgcatgca ctaggctgtg catggagtat agctactagg 3360
gtacgtaggc gtgcaggctt tttctttcca gatttaattt cctgaccttt tctgcatact 3420
gtgtttttac ttgtactttt ctcagtgtta ccctcttaca cgg 3463
<210> 3
<211> 3463
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 3
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatccgcccc agatttgggc tagatataag agatgtcggt cagcctggcg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc catcattgcc tctctccttc 360
tccggcctcc acgacctttt tagcctgaag gcacgcacaa ccgttcctgc tgcacagaca 420
catactccgg cggtccggcc tctttctttc ttacccatca gtcaggccgc gcgtcaccac 480
ctttctcatc cgcgctcgga atagccggcc atgatgagca gcaggctaag cggcggcacg 540
atggcaccgg ttagcgacat ggccgacttc ggttgcgctc ccatgcagtc ctaccccaac 600
tttgagccgg ccggcatggt catgcccggg gaccggcagc cgcccttcca gcaccaccac 660
ctctacgaca gcctcgactt caacgccgct gcattctcgt tccaagaccc ggtcgcgctc 720
ttctccagcg gctcggcgtt cagtaaccag ctccagcagc cgttcctcca gacgcaggtc 780
accacgccga cgatggcgtc gtcgtcgctg ctgcaggcgc cgatgatgac ccttccgggt 840
atgctgacgt cttcctcggc gtcgccggtg gacgcatgca ccttcggtgg cggcggcagt 900
gctgggttcc tgaagcagga ggagggcggc cccttctcag acgtcggcgg tggggggagg 960
atcgggctga acctcggccg caggacatac ttttccccgg cggacgtgct ggccgtggac 1020
cgcctgctga tgcgctcccg cttcggcgga gccggcgcaa tgggcatgct ggggctgggg 1080
ctcggcgccg ccgcccacca ccaccagacc ccgcggtgcc aggctgaggg ctgcaaggcc 1140
gacctctccg ccgccaagca ctaccaccgc cgccacaagg tctgcgagta ccacgccaag 1200
gccaccacag tcgccgcctc tggcaagcag cagcgcttct gccaacaatg cagccggtat 1260
gtacgtttat cctctgccat gggtggatcg ttgatgcatg cgaagctttg ctagcagacg 1320
aaaaaatcag cagtagaaag gctgcgagct ttcgatctag tggcattgac gctagcagct 1380
atatgcactg atcatatcat tcatgtcaac atgtcatata tcagaaatgg cgcacatcag 1440
atcagatctt gttcctctga gcgtatcggc atctgcatcc ctttgtccaa tgtactacta 1500
gcacgcccat catggcattg cagtggagtt cgatcagata cctaaaccta gtctgtgtag 1560
atacatcata catggcgtgt attacttaca acggtagaca tgtatatacc caagatccgg 1620
ttgtgtgaat ataactggtt cttgtttggg tacgtttgtt gggctaggtt tcatgtgctc 1680
gctgagtttg acgaggccaa gaggagctgc cggaagcggc tcacggagca caaccggcgc 1740
cgccgaaagc ctgccggcgc gcagggcaag gattcgccgc cgccttccaa gaaggcagac 1800
gctagcatca ccagctcata caccggcgat cacaagagta agaccactcc tctgatacat 1860
gtatagactt aaacactgga tatatgctta ttattggctt ttttaaagct tgatcacttt 1920
aagaagttgt gttttgattt tcggccgcaa aatgtggatt tcggccgaat ttcggtcatc 1980
tcggcttgag gcgaaaagta tatgtaggcc gaaattactc aaatttggta aattttggtc 2040
aaatttaagt caaattgcag tcaaaatttg atcaaatttc agccgaaaat tttgaaaatg 2100
gccgaaattc ggccatctcg gcctagggcg aaaaaaagct caaaccgaaa atcaaaacac 2160
agccagtaag ctagctagct acctagaaac tcctgaaact ttgttagagc ttagtgggta 2220
atttgaacta aaagcatgac acttcttttg caacaaggga acatttcaat aacgtatacc 2280
aagtaaagaa cacaacaaaa acagtttgcc taattcttaa ttgtccgaaa atttagtaaa 2340
tctattttgt cttgaaatgg cacatcactg ggctaaccta ataatgtact ccgtagcatc 2400
ttaagtgtgt aactgaagta agatataact gtagttttga cggtgcactt cagcacttgg 2460
ctgcagtaca caccggtaat tctatgtgcc agcaagcacg ttacaacgtt cggttggata 2520
gaaactagta gtacacgaca tgacatgcct agatgctact tctacatttt ggtcggtcga 2580
tctgacgtac gtacctacta ggagtactac tcagctcaat taacatgatg ttcatacgat 2640
ggcgccaaaa catcttatac gtacatacgt aggtgtacca tcgtactata cactagacgg 2700
taggtgcaac tcctgctaca tacatgtggc agtcacatct tgccgtcttg tctgacgccg 2760
cgacgtgtat atgatcagcc aacaagtcga cgacgggggc ggccttctcg ccgagcgccg 2820
gtggcttcag ctgccttcag cagcagcagc agcagcatga gatcgacaac ggcggccagt 2880
cgagcaacgc cacgccgact aacctgtccc tggcggcgcc accgccaccg ccgccgcctc 2940
aagacgacgc cggcttcggc gccggccttg acaccatgct gctgattcag cagcaagggc 3000
ccgatgaaca ggaggaggag gaggagcagc atttcatgat gacctctctc gtgcagtcgc 3060
atcgccagca gcagcagcac ggcgacagcg gcaacatctt atcgtgctcg acgacgtcgc 3120
cgtcggatca gcgtcgccat cagaacgacg gcgacagctg ctgcaacggc aacagcatgc 3180
agcatttctt tgaggtggag ttcatgtagc acgatgcatg catgcactgc atgaatgaac 3240
ctggcctagc ctagcctaga ggtggtttga ctgcattatc gtgaggtgtg tgtgcatgag 3300
gtgaaagaga aaacaagctg gttgcatgca ctaggctgtg catggagtat agctactagg 3360
gtacgtaggc gtgcaggctt tttctttcca gatttaattt cctgaccttt tctgcatact 3420
gtgtttttac ttgtactttt ctcagtgtta ccctcttaca cgg 3463
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 4
ggatagtgca gtgaagcggt 20
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 5
tggcatctct tgggctctt 19
<210> 6
<211> 342
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 6
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatctgcccc agatttgggc tagatataag agatgtcggc cagcctggcg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc ca 342
<210> 7
<211> 342
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 7
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatccgcccc agatttgggc tagatataag agatgtcggt cagcctgggg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc ca 342
<210> 8
<211> 342
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 8
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatccgcccc agatttgggc tagatataag agatgtcggt cagcctggcg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc ca 342
<210> 9
<211> 3463
<212> DNA
<213> Artificial sequence
<220>
<221>
<222> (125)…(125) (160)…(160) (169)…(169)
<223> n is c or t r is c or g
<400> 9
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatcngcccc agatttgggc tagatataag agatgtcggn cagcctggrg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc catcattgcc tctctccttc 360
tccggcctcc acgacctttt tagcctgaag gcacgcacaa ccgttcctgc tgcacagaca 420
catactccgg cggtccggcc tctttctttc ttacccatca gtcaggccgc gcgtcaccac 480
ctttctcatc cgcgctcgga atagccggcc atgatgagca gcaggctaag cggcggcacg 540
atggcaccgg ttagcgacat ggccgacttc ggttgcgctc ccatgcagtc ctaccccaac 600
tttgagccgg ccggcatggt catgcccggg gaccggcagc cgcccttcca gcaccaccac 660
ctctacgaca gcctcgactt caacgccgct gcattctcgt tccaagaccc ggtcgcgctc 720
ttctccagcg gctcggcgtt cagtaaccag ctccagcagc cgttcctcca gacgcaggtc 780
accacgccga cgatggcgtc gtcgtcgctg ctgcaggcgc cgatgatgac ccttccgggt 840
atgctgacgt cttcctcggc gtcgccggtg gacgcatgca ccttcggtgg cggcggcagt 900
gctgggttcc tgaagcagga ggagggcggc cccttctcag acgtcggcgg tggggggagg 960
atcgggctga acctcggccg caggacatac ttttccccgg cggacgtgct ggccgtggac 1020
cgcctgctga tgcgctcccg cttcggcgga gccggcgcaa tgggcatgct ggggctgggg 1080
ctcggcgccg ccgcccacca ccaccagacc ccgcggtgcc aggctgaggg ctgcaaggcc 1140
gacctctccg ccgccaagca ctaccaccgc cgccacaagg tctgcgagta ccacgccaag 1200
gccaccacag tcgccgcctc tggcaagcag cagcgcttct gccaacaatg cagccggtat 1260
gtacgtttat cctctgccat gggtggatcg ttgatgcatg cgaagctttg ctagcagacg 1320
aaaaaatcag cagtagaaag gctgcgagct ttcgatctag tggcattgac gctagcagct 1380
atatgcactg atcatatcat tcatgtcaac atgtcatata tcagaaatgg cgcacatcag 1440
atcagatctt gttcctctga gcgtatcggc atctgcatcc ctttgtccaa tgtactacta 1500
gcacgcccat catggcattg cagtggagtt cgatcagata cctaaaccta gtctgtgtag 1560
atacatcata catggcgtgt attacttaca acggtagaca tgtatatacc caagatccgg 1620
ttgtgtgaat ataactggtt cttgtttggg tacgtttgtt gggctaggtt tcatgtgctc 1680
gctgagtttg acgaggccaa gaggagctgc cggaagcggc tcacggagca caaccggcgc 1740
cgccgaaagc ctgccggcgc gcagggcaag gattcgccgc cgccttccaa gaaggcagac 1800
gctagcatca ccagctcata caccggcgat cacaagagta agaccactcc tctgatacat 1860
gtatagactt aaacactgga tatatgctta ttattggctt ttttaaagct tgatcacttt 1920
aagaagttgt gttttgattt tcggccgcaa aatgtggatt tcggccgaat ttcggtcatc 1980
tcggcttgag gcgaaaagta tatgtaggcc gaaattactc aaatttggta aattttggtc 2040
aaatttaagt caaattgcag tcaaaatttg atcaaatttc agccgaaaat tttgaaaatg 2100
gccgaaattc ggccatctcg gcctagggcg aaaaaaagct caaaccgaaa atcaaaacac 2160
agccagtaag ctagctagct acctagaaac tcctgaaact ttgttagagc ttagtgggta 2220
atttgaacta aaagcatgac acttcttttg caacaaggga acatttcaat aacgtatacc 2280
aagtaaagaa cacaacaaaa acagtttgcc taattcttaa ttgtccgaaa atttagtaaa 2340
tctattttgt cttgaaatgg cacatcactg ggctaaccta ataatgtact ccgtagcatc 2400
ttaagtgtgt aactgaagta agatataact gtagttttga cggtgcactt cagcacttgg 2460
ctgcagtaca caccggtaat tctatgtgcc agcaagcacg ttacaacgtt cggttggata 2520
gaaactagta gtacacgaca tgacatgcct agatgctact tctacatttt ggtcggtcga 2580
tctgacgtac gtacctacta ggagtactac tcagctcaat taacatgatg ttcatacgat 2640
ggcgccaaaa catcttatac gtacatacgt aggtgtacca tcgtactata cactagacgg 2700
taggtgcaac tcctgctaca tacatgtggc agtcacatct tgccgtcttg tctgacgccg 2760
cgacgtgtat atgatcagcc aacaagtcga cgacgggggc ggccttctcg ccgagcgccg 2820
gtggcttcag ctgccttcag cagcagcagc agcagcatga gatcgacaac ggcggccagt 2880
cgagcaacgc cacgccgact aacctgtccc tggcggcgcc accgccaccg ccgccgcctc 2940
aagacgacgc cggcttcggc gccggccttg acaccatgct gctgattcag cagcaagggc 3000
ccgatgaaca ggaggaggag gaggagcagc atttcatgat gacctctctc gtgcagtcgc 3060
atcgccagca gcagcagcac ggcgacagcg gcaacatctt atcgtgctcg acgacgtcgc 3120
cgtcggatca gcgtcgccat cagaacgacg gcgacagctg ctgcaacggc aacagcatgc 3180
agcatttctt tgaggtggag ttcatgtagc acgatgcatg catgcactgc atgaatgaac 3240
ctggcctagc ctagcctaga ggtggtttga ctgcattatc gtgaggtgtg tgtgcatgag 3300
gtgaaagaga aaacaagctg gttgcatgca ctaggctgtg catggagtat agctactagg 3360
gtacgtaggc gtgcaggctt tttctttcca gatttaattt cctgaccttt tctgcatact 3420
gtgtttttac ttgtactttt ctcagtgtta ccctcttaca cgg 3463
<210> 10
<211> 342
<212> DNA
<213> Artificial sequence
<220>
<221>
<222> (125)…(125) (160)…(160) (169)…(169)
<223> n is c or t r is c or g
<400> 10
ggatagtgca gtgaagcggt tagggtttgg tccgacgaat ggatgaaaag gaatagacgt 60
ggggtcgggt gggctagcgt ggaccgggtc cgacgtggcg gacacgcccg agcgtcccct 120
tatcngcccc agatttgggc tagatataag agatgtcggn cagcctggrg tttgagatcc 180
gtttaagacg ttcgtgtggg ttgaattttt gtgaccaggt cgttcgtccg cacatttaga 240
acgggttcga ggcgtcccag cgtggatgct ggagttgaga tttaggtggg gtcgtttgcg 300
cagataaagg acgggagaag gccaagagcc caagagatgc ca 342
Claims (6)
1. A method for screening or assisting in screening wheat with different plant heights comprises the following steps: detecting the wheat to be detected based onTaSPL21-6AThe genotype of the gene is genotypeHapI. Genotype(s)HapII is also genotypeHapIII, genotypeHapPlant height of wheat of I>Genotype(s)HapPlant height of wheat of III>Genotype(s)HapII plant height of wheat;
the genotypeHapThe wheat of I is the wheat which is TT homozygous based on the genotype of the T125C SNP locus, CC homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus;
the genotypeHapThe wheat II is the wheat which is homozygous for CC based on the genotype of the T125C SNP locus, homozygous for TT based on the genotype of the T160C SNP locus and homozygous for GG based on the genotype of the C169G SNP locus;
the genotypeHapThe wheat of III is the wheat which is CC homozygous based on the genotype of the T125C SNP locus, TT homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus;
the ' T125C SNP site ' is 125 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the "T160C SNP site" is the 160 th nucleotide from the 5' end of a sequence 9 in a sequence table in a wheat genome; the "C169G SNP site" is the 169 th nucleotide from the 5' end of sequence 9 in the sequence table in the wheat genome.
2. A method for screening or assisting in screening wheat with different plant heights comprises the following steps: detection of wheat substrate to be detectedIn thatTaSPL21-6AThe genotype of the gene is genotypeHapI. Genotype(s)HapII is also genotypeHapIII, genotypeHapPlant height of wheat of I>Genotype(s)HapPlant height of wheat of III>Genotype(s)HapII plant height of wheat;
the genotypeHapThe wheat of I is the wheat which is TT homozygous based on the genotype of the T125C SNP locus, CC homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus;
the genotypeHapThe wheat II is the wheat which is homozygous for CC based on the genotype of the T125C SNP locus, homozygous for TT based on the genotype of the T160C SNP locus and homozygous for GG based on the genotype of the C169G SNP locus;
the genotypeHapThe wheat of III is the wheat which is CC homozygous based on the genotype of the T125C SNP locus, TT homozygous based on the genotype of the T160C SNP locus and CC homozygous based on the genotype of the C169G SNP locus;
the ' T125C SNP site ' is 125 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the "T160C SNP site" is the 160 th nucleotide from the 5' end of a sequence 10 in a sequence table in a wheat genome; the "C169G SNP site" is the 169 th nucleotide from the 5' end of sequence 10 in the sequence table in the wheat genome.
3. A method for screening or assisting in screening wheat with different plant heights sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) using restriction enzyme to amplify the PCR productBglIPerforming enzyme digestion to obtain an enzyme digestion product A; using restriction enzyme to amplify the PCR productBseDI orSecI, carrying out enzyme digestion to obtain an enzyme digestion product B;
(3) the following evaluations were carried out:
if the enzyme digestion product A is two DNA fragments and the enzyme digestion product B is one DNAFragment, the wheat to be tested is based onTaSPL21-6AThe genotype of the gene is the genotype HapI;
if the enzyme digestion product A is one DNA fragment and the enzyme digestion product B is two DNA fragments, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is the genotype HapII;
if the enzyme digestion product A is a DNA fragment and the enzyme digestion product B is a DNA fragment, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is the genotype HapIII;
genotype(s)HapPlant height of wheat of I>Genotype(s)HapPlant height of wheat of III>Genotype(s)HapII plant height of wheat;
the primer F is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 5 of a sequence table.
4. A method for screening or assisting in screening wheat with different plant heights sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) using restriction enzyme to amplify the PCR productBglIPerforming enzyme digestion to obtain an enzyme digestion product A; using restriction enzyme to amplify the PCR productBseDI orSecI, carrying out enzyme digestion to obtain an enzyme digestion product B;
(3) the following evaluations were carried out:
if the enzyme digestion product A has 176bp DNA fragments and 166bp DNA fragments and the enzyme digestion product B has 342bp DNA fragments, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is the genotype HapI;
if the enzyme digestion product A has 342bp DNA fragments and the enzyme digestion product B has 165bp DNA fragments and 177bp DNA fragments, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is the genotype HapII;
if the enzyme digestion product A has a DNA fragment of 342bp and the enzyme digestion product B has a DNA fragment of 342bp, waiting for the enzyme digestion product A to have the DNA fragment of 342bpWheat is measured based onTaSPL21-6AThe genotype of the gene is the genotype HapIII;
genotype(s)HapPlant height of wheat of I>Genotype(s)HapPlant height of wheat of III>Genotype(s)HapII plant height of wheat;
the primer F is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 5 of a sequence table.
5. A method for screening or assisting in screening wheat with different plant heights sequentially comprises the following steps:
(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a PCR amplification product;
(2) sequencing the PCR amplification product;
(3) the following evaluations were carried out:
if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from 5' end of sequence 1 in the sequence table, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is the genotype HapI;
if the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from 5' end of sequence 2 in the sequence table, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is genotypeHapII；
If the nucleotide sequence of the PCR amplification product is shown as 1 st to 342 th bits from 5' end of a sequence 3 in the sequence table, the wheat to be detected is based onTaSPL21-6AThe genotype of the gene is genotypeHapIII；
Genotype(s)HapPlant height of wheat of I>Genotype(s)HapPlant height of wheat of III>Genotype(s)HapII plant height of wheat;
the primer F is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 5 of a sequence table.
(z1) or (z 2):
(z1) application of the molecular marker A or the molecular marker B in screening or auxiliary screening of wheat with different plant heights;
(z2) application of the molecular marker A or the molecular marker B in preparation of products for screening or auxiliary screening of wheat with different plant heights;
the molecular marker A is shown as a sequence 10 in a sequence table;
the molecular marker B is shown as a sequence 9 in a sequence table.
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| CN107858445B (en) * | 2017-11-20 | 2021-06-29 | 东北农业大学 | Method for screening soybean varieties with different growth periods and plant heights and special kit thereof |
| CN108546776B (en) * | 2018-07-16 | 2020-03-31 | 四川农业大学 | InDel marker of poland dwarf wheat, identification method and application |
| CN109439788B (en) * | 2018-11-20 | 2022-05-13 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | KASP molecular marker closely linked with major gene locus of wheat plant height and application thereof |
| CN115992290A (en) * | 2022-12-15 | 2023-04-21 | 河北师范大学 | Wheat plant height related SNP locus and application thereof in auxiliary screening of wheat with different plant heights |
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