CN108866222B - Method for identifying corn kernel characters and special kit thereof - Google Patents
Method for identifying corn kernel characters and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying corn kernel traits and a special kit thereof. The kit comprises a substance for detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV; the kernel trait is kernel length and/or kernel width and/or kernel length/kernel width. Four haplotypes were typed based on the genotypes of SNPs S-1830 and Indel S-1558. SNP S-1830 is 189 th nucleotide from 5' end of target sequence of primer pair composed of primer F and primer R in maize genome; the nucleotide sequences of the primer F and the primer R are shown as a sequence 5 and a sequence 6 in sequence. Indel S-1558 is a deletion type I of a maize genome with 9 tandem repeats of a 2bp sequence, 9 tandem repeats of a 2bp sequence or a deletion type II of a 9 tandem repeats of a 2bp sequence. The kit has important application value in corn molecular breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for identifying corn kernel traits and a special kit thereof.
Background
Corn (Zea mays L.) is an important crop of food, feed and industrial materials in the world. With the increase of the world population, the demand of corn and other grains is increased day by day, and under the condition of continuously reducing the arable land area, the cultivation of a new corn variety with outstanding single yield is the most economic and effective way for stabilizing and improving the total yield of corn. The corn yield is influenced by three factors of the effective number of ears in unit area, the number of grains in ears and the grain weight, and the coordination of the three factors is the key for obtaining the high yield of the corn. Since the grain weight, which is one of yield-constituting factors, is relatively most stable and relatively less influenced by environmental conditions, has been considered by breeders, increasing the grain weight of maize is one of the important approaches for high-yield breeding of maize. The corn kernel properties (such as grain length and grain width) are important factors for determining grain weight. Therefore, the mining and control of the excellent allelic variation of the corn kernel 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 corn 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 the technology, the SNP detection method is economic and rapid, becomes a new generation of molecular marker following SSR, and is applied to the aspects of construction of corn genetic maps, positioning of corn important agronomic trait related genes, genome association analysis and the like.
Indel markers refer to the differences of parents across the entire genome, with respect to one material, where a certain number of nucleotides are inserted or deleted in the genome of another material, thereby forming a variation.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify the corn kernel traits.
In order to solve the technical problems, the invention firstly provides a method for identifying the corn kernel characters.
The method provided by the invention can be A1) or A2) or A3):
A1) a method for identifying corn kernel grain length, comprising the steps of: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain length of the haplotype HapII corn is more than that of the haplotype HapIV corn, and the grain length of the haplotype HapII corn is more than that of the haplotype HapI corn;
A2) a method for identifying the grain width of corn grains can comprise the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain width of the haplotype HapI corn is more than that of the haplotype HapII corn, and the grain width of the haplotype HapI corn is more than that of the haplotype HapIV corn;
A3) a method of identifying corn kernel shape, comprising the steps of: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain shape of the haplotype HapIII corn is more than that of the haplotype HapII corn; the shape of the kernel is kernel length/kernel width.
In the above method, the haplotype HapI maize may be AA homozygous based on the genotype of SNP S-1830 and has "deletion type II of 2bp sequence of 9 tandem repeats". The haplotype HapII maize can be a maize that is homozygous for GG based on the genotype of SNP S-1830 and has a "2 bp sequence of 9 tandem repeats". The haplotype Hap III maize can be GG homozygous based on SNP S-1830 genotype and has deletion type I of 2bp sequence with 9 tandem repeats. The haplotype Hap IV corn can be GG homozygous based on SNP S-1830 genotype and has 'deletion type II of 2bp sequence with 9 tandem repeats'.
In the above method, the SNP S-1830 may be the 189 nd nucleotide from the 5' end of the target sequence of the primer pair consisting of the primer F and the primer R in the maize genome. The primer F can be a1) or a2) as follows: a1) a single-stranded DNA molecule shown in sequence 5 of the sequence table; a2) 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 primer R can be a3) or a4) as follows: a3) a single-stranded DNA molecule shown in sequence 6 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 6 and has the same function as the sequence 6.
In the method, the '2 bp sequences with 9 tandem repeats' can be shown as a sequence 7 in a sequence table. The deletion type I of the 2bp sequence of the 9 tandem repeats can delete the 1 st and the 2 nd nucleotides of the 2bp sequence of the 9 tandem repeats. The deletion type II of the 2bp sequence of the 9 tandem repeats can be that the 1 st to 4 th nucleotides of the 2bp sequence of the 9 tandem repeats are deleted.
The method provided by the invention can be B1) or B2) or B3):
B1) a method for identifying the grain length of corn kernels comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain length of the haplotype HapII corn is more than that of the haplotype HapIV corn, and the grain length of the haplotype HapII corn is more than that of the haplotype HapI corn;
B2) a method for identifying the grain width of corn grains comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain width of the haplotype HapI corn is more than that of the haplotype HapII corn, and the grain width of the haplotype HapI corn is more than that of the haplotype HapIV corn;
B3) a method for identifying the grain shape of corn kernels comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain shape of the haplotype HapIII corn is more than that of the haplotype HapII corn; the shape of the kernel is kernel length/kernel width.
In the above method, the method for detecting whether the haplotype of the corn to be detected is haplotype hapI, haplotype hapII, haplotype hapIII or haplotype Hap IV may comprise the following steps: detecting whether the genome DNA of the corn to be detected contains DNA fragments shown by a sequence 1 in a sequence table, a sequence 2 in the sequence table, a sequence 3 in the sequence table or a sequence 4 in the sequence table, and then judging as follows:
if the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 1 in the sequence table and does not contain the DNA segments shown in the sequence 2 in the sequence table, the sequence 3 in the sequence table and the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotype HapI;
if the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 2 in the sequence table and does not contain the DNA segments shown in the sequence 1 in the sequence table, the sequence 3 in the sequence table and the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotype HapII;
if the genome DNA of the corn to be detected contains a DNA segment shown in a sequence 3 in the sequence table and does not contain DNA fragments shown in a sequence 1 in the sequence table, a sequence 2 in the sequence table and a sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotype Hap III;
if the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 4 in the sequence table and does not contain the DNA segments shown in the sequence 1 in the sequence table, the sequence 2 in the sequence table and the sequence 3 in the sequence table, the haplotype of the corn to be detected is haplotype Hap IV.
The method provided by the invention can be C1) or C2) or C3):
C1) a method for identifying the grain length of corn kernels comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain length of the haplotype HapII corn is more than that of the haplotype HapIV corn, and the grain length of the haplotype HapII corn is more than that of the haplotype HapI corn;
C2) a method for identifying the grain width of corn grains comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain width of the haplotype HapI corn is more than that of the haplotype HapII corn, and the grain width of the haplotype HapI corn is more than that of the haplotype HapIV corn;
C3) a method for identifying the grain shape of corn kernels comprises the following steps: detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV, wherein the grain shape of the haplotype HapIII corn is more than that of the haplotype HapII corn; the shape of the kernel is kernel length/kernel width.
In the method, the method for detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV sequentially comprises the following steps:
(1) taking the genome DNA of the corn to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of the primer F and the primer R to obtain a PCR amplification product;
(2) sequencing the PCR amplification product, and then judging as follows:
if the nucleotide sequence of the PCR amplification product is shown as the sequence 1 in the sequence table, the haplotype of the corn to be detected is haplotype HapI; if the nucleotide sequence of the PCR amplification product is shown as the sequence 2 in the sequence table, the haplotype of the corn to be detected is haplotype HapII; if the nucleotide sequence of the PCR amplification product is shown as the sequence 3 in the sequence table, the haplotype of the corn to be detected is haplotype Hap III; and if the nucleotide sequence of the PCR amplification product is shown as the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotype Hap IV.
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 the corn kernel traits.
The kit for identifying the corn kernel traits provided by the invention can comprise a substance for detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV; the grain trait is f1) and/or f2) and/or f 3): f1) the grains are long; f2) the grain width is wide; f3) kernel length/kernel width.
In the kit, the haplotype HapI corn can be AA homozygous based on the genotype of the SNP S-1830 and has a deletion type II of 2bp sequences with 9 tandem repeats. The haplotype HapII maize can be a maize that is homozygous for GG based on the genotype of SNP S-1830 and has a "2 bp sequence of 9 tandem repeats". The haplotype Hap III maize can be GG homozygous based on SNP S-1830 genotype and has deletion type I of 2bp sequence with 9 tandem repeats. The haplotype Hap IV corn can be GG homozygous based on SNP S-1830 genotype and has 'deletion type II of 2bp sequence with 9 tandem repeats'.
In the kit, the SNP S-1830 may be 189 th nucleotide from the 5' end of a target sequence of a primer pair consisting of a primer F and a primer R in the maize genome. The primer F can be a1) or a2) as follows: a1) a single-stranded DNA molecule shown in sequence 5 of the sequence table; a2) 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 primer R can be a3) or a4) as follows: a3) a single-stranded DNA molecule shown in sequence 6 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 6 and has the same function as the sequence 6.
In the kit, the "2 bp sequences with 9 tandem repeats" can be shown as a sequence 7 in a sequence table. The deletion type I of the 2bp sequence of the 9 tandem repeats can delete the 1 st and the 2 nd nucleotides of the 2bp sequence of the 9 tandem repeats. The deletion type II of the 2bp sequence of the 9 tandem repeats can be that the 1 st to 4 th nucleotides of the 2bp sequence of the 9 tandem repeats are deleted.
In the kit, the substance for detecting whether the haplotype of the corn to be detected is haplotype HapI, haplotype HapII, haplotype HapIII or haplotype HapIV can be a primer pair consisting of the primer F and the primer R.
A primer pair consisting of the primer F and the primer R also belongs to the protection scope of the invention.
The invention also protects a molecular marker A shown as a sequence 1 in the sequence table, a molecular marker B shown as a sequence 2 in the sequence table, a molecular marker C shown as a sequence 3 in the sequence table or a molecular marker D shown as a sequence 4 in the sequence table.
The application of any one of the kit, the primer pair, the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D in the identification of the corn kernel traits also belongs to the protection scope of the invention; the grain trait may be f1) and/or f2) and/or f 3): f1) the grains are long; f2) the grain width is wide; f3) kernel length/kernel width.
The application of any one of the kit, the primer pair, the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D in the preparation of a product for identifying the corn kernel trait also belongs to the protection scope of the invention; the grain trait may be f1) and/or f2) and/or f 3): f1) the grains are long; f2) the grain width is wide; f3) kernel length/kernel width.
The application of any one of the kit, the primer pair, the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D in corn breeding also belongs to the protection scope of the invention.
Any of the above > may specifically be statistical >.
Any one of the grain lengths can be specifically 10 grain lengths. Any one of the grain widths can be specifically 10 grain widths. Any one of the grain length and the grain width is the ratio of the grain length to the grain width. Any one of the grain length/grain width may specifically be a ratio of the grain length (10 grain length) of 10 grains to the grain width (10 grain width) of 10 grains.
In actual plant breeding, the grain shape of the corn with any one of the haplotypes Hap III can be expressed as long grains. The grain shape of any haplotype HapII corn can be represented as partial long grain. The grain shape of any one haplotype Hap IV corn can be represented as a partial circular grain. The grain shape of any one haplotype HapI corn can be represented as a round grain.
Experiments prove that the kit provided by the invention can identify the corn kernel traits by detecting the haplotype of the corn to be detected based on the SNP S-1830 and the Indel S-1558, and has important application value in corn molecular breeding.
Drawings
FIG. 1 shows the correlation analysis results of grain length and haplotype.
FIG. 2 shows the correlation analysis of grain width and haplotype.
FIG. 3 shows the results of association analysis of the particle shape and the haplotype.
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 206 maize varieties in the following examples were all derived from the national germplasm resources pool (web site: http:// www.cgris.net /) and were publicly available from the institute for crop science, the national academy of agricultural sciences (i.e., at the applicant) to repeat the experiment.
Both 5 XPCR buffer and TransStart Fast Pfu DNA Polymerase are products of Beijing Quanjin Biotechnology, Inc.
Example 1 discovery of SNP site and Indel marker and haplotype acquisition
Discovery of SNP sites and Indel markers
Through a large number of experiments, the inventors of the present invention analyzed the genomic DNA of 206 maize varieties shown in column 2 of Table 3, and found 1 SNP site (this SNP site was named SNP S-1830) and 1 Indel marker (this Indel marker was named Indel S-1558).
SNP S-1830 is located at position 189 from the 5 'end of sequence 1 in the sequence table, at position 189 from the 5' end of sequence 2 in the sequence table, at position 189 from the 5 'end of sequence 3 in the sequence table, or at position 189 from the 5' end of sequence 4 in the sequence table.
Indel S-1558 has three genotypes, namely a "2 bp sequence with 9 tandem repeats", "deletion type I of 2bp sequence with 9 tandem repeats", and "deletion type II of 2bp sequence with 9 tandem repeats".
The '2 bp sequences with 9 tandem repeats' are shown as a sequence 7 in a sequence table.
"deletion type I of 2bp sequence of 9 tandem repeats" means that the 1 st and 2 nd nucleotides are deleted from the 2bp sequence of 9 tandem repeats.
"deletion type II of 2bp sequence of 9 tandem repeats" means that 1 st to 4 th nucleotides are deleted from "2 bp sequence of 9 tandem repeats".
The basic information of SNPs S-1830 and Indel S-1558 is shown in Table 1 (since genomic DNA is a double-stranded DNA molecule consisting of two single-stranded DNA molecules complementary to each other in the opposite directions, a DNA molecule encoding a protein, that is, a DNA molecule having an initiation codon to a termination codon, is generally designated as a sense DNA molecule, and a DNA molecule complementary to the opposite direction of the sense DNA molecule is designated as an antisense DNA molecule.
TABLE 1
Obtaining of haplotype
According to the experimental result of the first step, only 4 haplotypes exist in the natural variation population of the corn based on the SNPs S-1830 and Indel S-1558: haplotype Hap I, haplotype Hap II, haplotype Hap III and haplotype Hap IV. The genotypes of the four haplotypes at SNP S-1830 and Indel S-1558 are detailed in Table 2.
TABLE 2
Example 2 establishment of haplotype typing methods based on the genotypes of SNP S-1830 and Indel S-1558
Preparation of specific primer pair
The specific primer pairs designed to amplify the target sequence including SNPs S-1830 and Indel S-1558 were as follows:
and (3) primer F: 5'-CTCACCCCCACAAGTCAAGA-3' (SEQ ID NO: 5 in the sequence Listing);
and (3) primer R: 5'-CCGACCGAACCAAACAAACA-3' (SEQ ID NO: 6 in the sequence Listing).
Establishing haplotype typing method based on SNP S-1830 and Indel S-1558 genotypes
1) Extracting the genome DNA of the corn to be detected;
2) taking the genome DNA in the step 1) as a template, and carrying out PCR amplification by adopting a specific primer pair consisting of a primer F and a primer R to obtain a PCR amplification product.
PCR amplification reaction (15. mu.L): ddH2mu.L of O8.0. mu.L, 3.0. mu.L of 5 XPCR buffer, 0.6. mu. L, dNTPs (concentration of 2.5. mu. mol/L; i.e., 2.5. mu. mol/L for each of dATP, dTTP, dCTP and dGTP) for each of primer F (concentration of 5. mu. mol/L), 0.4. mu.L of TransStart Fast Pfu DNA Polymerase (containing 0.75U of TransStart Fast Pfu DNA Polymerase), and 2.1. mu.L of genomic DNA (20 ng/. mu.L).
The PCR amplification reaction conditions are as follows: 5min at 95 ℃; 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 sequence 1 in the sequence table, the haplotype of the corn to be detected is haplotype Hap I; if the nucleotide sequence of the PCR amplification product is shown as sequence 2 in the sequence table, the haplotype of the corn to be detected is haplotype Hap II; if the nucleotide sequence of the PCR amplification product is shown as sequence 3 in the sequence table, the haplotype of the corn to be detected is haplotype Hap III; if the nucleotide sequence of the PCR amplification product is shown as sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotype Hap IV.
Example 3 correlation analysis of four haplotypes and maize grain traits
The corn to be detected is 206 corn varieties, and the specific corn variety names are shown in the 2 nd column in the table 3 in detail.
Genotyping of maize to be tested
And (3) performing haplotype classification on each corn to be detected by adopting the method of the second step in the embodiment 2.
The haplotype results for each maize tested are shown in Table 3, column 3.
The results show that based on SNP S-1830 and Indel S-1558, the haplotype of the corn to be detected is haplotype Hap I, haplotype Hap II, haplotype Hap III or haplotype Hap IV.
Second, investigation of corn kernel traits
In 2015, each corn variety to be tested (diploid corn) is planted in the field (normal field management during planting), after the corn variety is mature, the 10 grain length (cm), the 10 grain width (cm) and the 10 grain length/10 grain width of the seeds are investigated, and the investigation results are shown in the 4 th to 6 th columns in table 3 in sequence.
TABLE 3
Third, correlation analysis
Respectively counting the average 10 grain length, the average 10 grain width and the average 10 grain length/10 grain width of the corn varieties of the four haplotypes, and then performing association analysis (by adopting SAS9.0 software), wherein the counting results are shown in fig. 1, fig. 2 and fig. 3 in sequence.
The result shows that the haplotype of the corn to be detected based on the SNP S-1830 and Indel S-1558, the 10 grain length of the 'haplotype HapII corn', the 10 grain length of the 'haplotype HapIV', the 10 grain length of the 'haplotype HapIII', and the 10 grain length of the 'haplotype HapI corn' are detected; 10-fold width of "haplotype HapI maize >" 10-fold width of haplotype HapII maize > "10-fold width of haplotype HapIV >" 10-fold width of haplotype HapIII "; "10 grains long/10 grains wide of" haplotype Hap III maize ">" 10 grains long/10 grains wide of "haplotype HapI maize" > "10 grains long/10 grains wide of" haplotype Hap IV maize ">" 10 grains long/10 grains wide of "haplotype HapI maize". The ">" is all statistically >.
The results show that the corn kernel trait can be identified by detecting the haplotype of the corn to be detected based on the SNP S-1830 and the Indel S-1558, and the method has important application value in corn molecular breeding.
<110> institute of crop science of Chinese academy of agricultural sciences
<120> method for identifying corn kernel traits and special kit thereof
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 2351
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 1
ctcaccccca caagtcaaga acaggtacca caggatgagg cgcatggagg atgctgcgat 60
gtgttcgtga gaggtctagg ccgtcgtctc ctagtcaact ttgggttgct ggatcgttgt 120
ctccttacca tgtaattatt tatttatttt gtacagaact cctattatat agtaaagtta 180
ttacattcat ttctgtacca tgatttatca tatgtgtgag acttggtccc agcacacctg 240
gtgattatgt tcgcgcctgg gtccctaaaa ctcgagtgtg acagagggcg tgcggagatg 300
gccggaaaac gcgtgaacgt ggacgcatcc acgatggggg cgtgggtggg aggttaggga 360
cgaggtctga cgggtgggtc cgcaggacag agagagagga tgagcgcgtg cgaggggatc 420
agcaccgaca ggccgacccc acagagcaga gagagagaga gaaggggtgc gtgggctggc 480
gccgataggc cgggtccgtc tgtccgactt gggctgaaat ggttttttct atttttcagg 540
gaatttctag tgtttttcta tttatgttct ctagggtttt gaattcaaat tcaaactaaa 600
tcaaacatgt gcaacaattt aaaaaatatt tggagctcag cacgatgcaa catttcatga 660
ctcatattat tttgacaaaa taaaataatc aacccctcac taattaagct aattctacta 720
aaaagaaaaa gagagagaaa ctagagagag aggagtaaca cctgaatttg gtaggtaatt 780
agaaagaaat tttatacccc caaatttagg gtgttacaac ctctgcggac aaggcaaaat 840
gcgcagttat ttgaaaggaa tattcgaacc aaggttcgat ctaccacggc cacggcccgc 900
cggcggcggc ggcacgcgcg tgtgtggtcg tctttcattc ttttcccaac ttcttaatag 960
atgcaccaat tggtgcacct atttaagttg attgattgat ctcttaaact tacaatatgg 1020
tactaaatta ttagtacacc atatcattaa agtggaccac tagcattgac tattattgaa 1080
tattaattgg gccaagccaa cattaatcca atataaacaa tgataattag gtaatatttt 1140
gaataatatg gatgacataa atcttgaaaa tataggatac atggagatta tgtattgaac 1200
ttgagaaatc tatagacaga gtttctgaat tgaactaggt aaatctgtaa acgggaatac 1260
tgaattatac tccctctctt tagaaaatta acaaattctt acaatacttg atgtatgtat 1320
tatatatatg tgtatagatt tattatcatt catttgaata tagacataaa accaagatct 1380
aaaacgaata ctattttaga cggagagagt atagatttgt aagaatctat ttagctgatg 1440
tatcctttca gttaggattc aatttttttt taataggagg attcaaattt tttgatagta 1500
catctagatt ctagaccgtc gatctttcaa acgttgaaaa gaggaagaag agcttgacgt 1560
tttgcttagc ttaacgctag cccaacgaac ttggcgaaac cgatggagcc tcaaacgggc 1620
cgtatccgtc gtgcacagca tgctaaagcc cagtgctgac gacgacgaac aacactcatc 1680
cggcccggtc atgagcccaa tgcggggcca cttcgtcgtt tttgacaagt agacccacag 1740
tggccccacg tgcttgcccc tccctttctc ttctcccctt gtgtcgcgct cgcgcatctg 1800
aagcaagggg gggaagggcc aagggggatc ggtcttgttc tcggagaggg ttgagctgct 1860
tgacggtttt gactcggatc atggtgtgca gctctatcag atagagagct gacgtgaggt 1920
gtgagacgcg gatcgtggag tactactcag taggagaggt ggggaggggg aagaaggcga 1980
tagccgggga ggatggtgct tgtgcttgcg ctgggggatc tgcacatccc gcaccgggcg 2040
cccgacctcc ccgccaaatt caagtccatg ctcgtgcccg gcaagatcca acacatcatc 2100
tgcactggca atctctgcat caaggtaacc ttaccttccg cgcctggccg gccggccggg 2160
taaccggcgt ccctcggcga tcgattttac aatcctttcc tctcgcctca tgtgtccaat 2220
ttttgcgctg gctaggtcgt actgccgcgg atagttgcat cacgtgagaa atcgttatgg 2280
ttcttaggtc gtgctgggat cagccgcatc gctgtattag tctactgcag ttgtttgttt 2340
ggttcggtcg g 2351
<210> 2
<211> 2355
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 2
ctcaccccca caagtcaaga acaggtacca caggatgagg cgcatggagg atgctgcgat 60
gtgttcgtga gaggtctagg ccgtcgtctc ctagtcaact ttgggttgct ggatcgttgt 120
ctccttacca tgtaattatt tatttatttt gtacagaact cctattatat agtaaagtta 180
ttacattcgt ttctgtacca tgatttatca tatgtgtgag acttggtccc agcacacctg 240
gtgattatgt tcgcgcctgg gtccctaaaa ctcgagtgtg acagagggcg tgcggagatg 300
gccggaaaac gcgtgaacgt ggacgcatcc acgatggggg cgtgggtggg aggttaggga 360
cgaggtctga cgggtgggtc cgcaggacag agagagagga tgagcgcgtg cgaggggatc 420
agcaccgaca ggccgacccc acagagcaga gagagagaga gagagaaggg gtgcgtgggc 480
tggcgccgat aggccgggtc cgtctgtccg acttgggctg aaatggtttt ttctattttt 540
cagggaattt ctaatgtttt tctatttatg ttctctaggg ttttgaattc aaattcaaac 600
taaatcaaac atgtgcaaca atttaaaaaa tatttggagc tcagcacgat gcaacatttc 660
atgactcata ttattttgac aaaataaaat aatcaacccc tcactaatta agctaattct 720
actaaaaaga aaaagagaga gaaactagag agagaggagt aacacctgaa tttggtaggt 780
aattagaaag aaattttata cccccaaatt tagggtgtta caacctctgc ggacaaggca 840
aaatgcgcag ttatttgaaa ggaatattcg aaccaaggtt cgatctacca cggccacggc 900
ccgccggcgg cggcggcacg cgcgtgtgtg gtcgtctttc attcttttcc caacttctta 960
atagatgcac caattggtgc acctatttaa gttgattgat tgatctctta aacttacaat 1020
atggtactaa attattagta caccatatca ttaaagtgga ccactagcat tgactattat 1080
tgaatattaa ttgggccaag ccaacattaa tccaatataa acaatgataa ttaggtaata 1140
ttttgaataa tatggatgac ataaatcttg aaaatatagg atacatggag attatgtatt 1200
gaacttgaga aatctataga cagagtttct gaattgaact aggtaaatct gtaaacggga 1260
atactgaatt atactccctc tctttagaaa attaacaaat tcttacaata cttgatgtat 1320
gtattatata tatgtgtata gatttattat cattcatttg aatatagaca taaaaccaag 1380
atctaaaacg aatactattt tagacggaga gagtatagat ttgtaagaat ctatttagct 1440
gatgtatcct ttcagttagg attcaatttt tttttaatag gaggattcaa attttttgat 1500
agtacatcta gattctagac cgtcgatctt tcaaacgttg aaaagaggaa gaagagcttg 1560
acgttttgct tagcttaacg ctagcccaac gaacttggcg aaaccgatgg agcctcaaac 1620
gggccgtatc cgtcgtgcac agcatgctaa agcccagtgc tgacgacgac gaacaacact 1680
catccggccc ggtcatgagc ccaatgcggg gccacttcgt cgtttttgac aagtagaccc 1740
acagtggccc cacgtgcttg cccctccctt tctcttctcc ccttgtgtcg cgctcgcgca 1800
tctgaagcaa gggggggaag ggccaagggg gatcggtctt gttctcggag agggttgagc 1860
tgcttgacgg ttttgactcg gatcatggtg tgcagctcta tcagatagag agctgacgtg 1920
aggtgtgaga cgcggatcgt ggagtactac tcagtaggag aggtggggag ggggaagaag 1980
gcgatagccg gggaggatgg tgcttgtgct tgcgctgggg gatctgcaca tcccgcaccg 2040
ggcgcccgac ctccccgcca aattcaagtc catgctcgtg cccggcaaga tccaacacat 2100
catctgcact ggcaatctct gcatcaaggt aaccttacct tccgcgcctg gccggccggc 2160
cgggtaaccg gcgtccctcg gcgatcgatt ttacaatcct ttcctctcgc ctcatgtgtc 2220
caatttttgc gctggctagg tcgtactgcc gcggatagtt gcatcacgtg agaaatcgtt 2280
atggttctta ggtcgtgctg ggatcagccg catcgctgta ttagtctact gcagttgttt 2340
gtttggttcg gtcgg 2355
<210> 3
<211> 2367
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 3
ctcaccccca caagtcaaga acaggtacca caggatgagg cgcatggagg atgctgcgat 60
gtgttcgtga gaggtctagg ccgtcgtctc ctagtcaact ttgggttgat ggatcgttgt 120
ctccttacca tgtatttatt tatttatttt gtacagaact cctattatat agtaaagtta 180
ttacattcgt ttctgtacca tgatttatca tatgtgtgag acttggtccc agcgcacctg 240
gtgattatgt tcgcgcccgg gtccctaaaa cccgagtgtg acagagggcg tgcggagatg 300
gccggaaaac gcgtgaacgt ggacgcatcc acgatggggg cgtgggtggg aggttaggga 360
cgaggtctga cgggtgggtc cgcagggcag agagagagga tgagcgcgtg cgaggggatc 420
agcaccgaca ggccgacccc acagagcaga gagagagaga gagaaggggt gcgtgggctg 480
gcgccgatag gccgggtccg tctgtccgac ttgggctgaa atggtttttt ctatttttca 540
gggaatttct aatgtttttc tatttatgtt ctctagggtt ttgaattcaa attcaaacta 600
aatcaaacat gtgcaacaat ttaaaaaata tttggagctc accacgatgc aacatttcat 660
gactcatatt gttttgacaa aataaaataa tcaacccctc actaattaag ctaattctac 720
taaaaagaaa aagagagaga aactagagag agaggagtaa cacctgaatt tggtaggtaa 780
ttagaaagaa attttatacc cccaaattta gggtgttaca acctctgcgg acaaggcaaa 840
atgcgcagtt atttgaaagg aatattcgaa ccaaggttcg atctaccacg gccacggccc 900
gccggcggcg gcggcacgcg cgcgtgtggt cgtttttcat tcttttccca acttcttaat 960
agatgcacca attggtgcac ctatttaagt tgattgattg atctcttgaa cttacaatat 1020
gatactaaat tattagtata ccatatcatt aaagtggacc actagcattg actattattg 1080
aatattaatt gggccaagcc aacattaatc caatataaac aatgataatt aggtaatatt 1140
ttgaataata tggatggcat aaatcttgaa aatataggat acatggagat tatgtattga 1200
acttgagaaa tctatagaca gagtttctga attgaactag gtaaatctgt aaacgggaat 1260
actgaattat actccctctc tttagaaaat taacaaattc ttacaatact tgatgtatgt 1320
attatatata tgtgtataga tttattatca ttcatttgaa tatagacata aaaaccaaga 1380
tctaaaacga atactatttt agacggagag agtatagatt tgtaagaatc tatttagctg 1440
atgtatcctt tcagttagga ttcaattttt ttttaatagg aggattcaaa ttttttgata 1500
gtacatctag attctagacc gtcgatcttt caaacgttga aaagaggaag aagagcttga 1560
cgttttgctt agcttaacgc tagcccaacg aacttggcga aaccgatgga gcctcaaacg 1620
ggccgtatcc gtcgtgcaca gcatgctaaa gcccagtgct gacgacgacg aacaacactc 1680
atccggcccg gtcatgagcc caatgcgggg ccacttcgtc gtttttgaca agtagaccca 1740
cagtggcccc acgtgcttgc ccctcccttt ctcttctccc cttgtgtcgc gctcgcgcat 1800
ctgaagcaag ggggggaagg gccaaggggg atcggtcttg ttctcggaga gggttgagct 1860
gcttgacggt tttgactcgg atcatggtgt gcagctctat cagatagaga gctgacgtga 1920
ggtgtgagac gcggatcgtg gagtactact cagtaggaga tcaagaggaa ggaggtgggg 1980
agggggaaga aggcgatagc cggggaggat ggtgcttgtg cttgcgctgg gggatctgca 2040
catcccgcac cgggcgcccg acctccccgc caaattcaag tccatgctcg tgcccggcaa 2100
gatccaacac atcatctgca ctggcaatct ctgcatcaag gtaaccttac cttccgcgcc 2160
tggccggccg gccgggtaac cggcgtccct cggcgatcga ttttacaatc ctttcctctc 2220
gcctcatgtg tccaattttt gcgctggcta ggtcgtactg ccgcggatag ttgcatcacg 2280
tgagaaatcg ttatggttct taggtcgtgc tgggatcagc cgcatcgctg tattagtcta 2340
ctgcagttgt ttgtttggtt cggtcgg 2367
<210> 4
<211> 2351
<212> DNA
<213> Artificial sequence
<220>
<221>
<400> 4
ctcaccccca caagtcaaga acaggtacca caggatgagg cgcatggagg atgctgcgat 60
gtgttcgtga gaggtctagg ccgtcgtctc ctagtcaact ttgggttgct ggatcgttgt 120
ctccttacca tgtaattatt tatttatttt gtacagaact cctattatat agtaaagtta 180
ttacattcgt ttctgtacca tgatttatca tatgtgtgag acttggtccc agcacacctg 240
gtgattatgt tcgcgcccgg gtccctaaaa ctcgagtgtg acagagggcg tgcggagatg 300
gccggaaaac gcgtgaacgt ggacgcatcc acgatggggg cgtgggtggg aggttaggga 360
cgaggtctga cgggtgggtc cgcaggacag agagagagga tgagcgcgtg cgaggggatc 420
agcaccgaca ggccgacccc acagagcaga gagagagaga gaaggggtgc gtgggctggc 480
gccgataggc cgggtccgtc tgtccgactt gggctgaaat ggttttttct atttttcagg 540
gaatttctaa tgtttttcta tttatgttct ctagggtttt gaattcaaat tcaaactaaa 600
tcaaacatgt gcaacaattt aaaaaatatt tggagctcag cacgatgcaa catttcatga 660
ctcatattat tttgacaaaa taaaataatc aacccctcac taattaagct aattctacta 720
aaaagaaaaa gagagagaaa ctagagagag aggagtaaca cctgaatttg gtaggtaatt 780
agaaagaaat tttatacccc caaatttagg gtgttacaac ctctgcggac aaggcaaaat 840
gcgtagttat ttgaaaggaa tattcgaacc aaggttcgat ctaccacggc cacggcccgc 900
cggcggcggc ggcacgcgcg tgtgtggtcg tctttcattc ttttcccaac ttcttaatag 960
atgcaccaat tggtgcacct atttaagttg attgattgat ctcttaaact tacaatatgg 1020
tactaaatta ttagtacacc atatcattaa agtggaccac tagcattgac tattattgaa 1080
tattaattgg gccaagccaa cattaatcca atataaacaa tgataattag gtaatatttt 1140
gaataatatg gatgacataa atcttgaaaa tataggatac atggagatta tgtattgaac 1200
ttgagaaatc tatagacaga gtttctgaat tgaactaggt aaatctgtaa acgggaatac 1260
tgaattatac tccctctctt tagaaaatta acaaattctt acaatacttg atgtatgtat 1320
tatatatatg tgtatagatt tattatcatt catttgaata tagacataaa accaagatct 1380
aaaacgaata ctattttaga cggagagagt atagatttgt aagaatctat ttagctgatg 1440
tatcctttca gttaggattc aatttttttt taataggagg attcaaattt tttgatagta 1500
catctagatt ctagaccgtc gatctttcaa acgttgaaaa gaggaagaag agcttgacgt 1560
tttgcttagc ttaacgctag cccaacgaac ttggcgaaac cgatggagcc tcaaacgggc 1620
cgtatccgtc gtgcacagca tgctaaagcc cagtgctgac gacgacgaac aacactcatc 1680
cggcccggtc atgagcccaa tgcggggcca cttcgtcgtt tttgacaagt agacccacag 1740
tggccccacg tgcttgcccc tccctttctc ttctcccctt gtgtcgcgct cgcgcatctg 1800
aagcaagggg gggaagggcc aagggggatc ggtcttgttc tcggagaggg ttgagctgct 1860
tgacggtttt gactcggatc atggtgtgca gctctatcag atagagagct gacgtgaggt 1920
gtgagacgcg gatcgtggag tactactcag taggagaggt ggggaggggg aagaaggcga 1980
tagccgggga ggatggtgct tgtgcttgcg ctgggggatc tgcacatccc gcaccgggcg 2040
cccgacctcc ccgccaaatt caagtccatg ctcgtgcccg gcaagatcca acacatcatc 2100
tgcactggca atctctgcat caaggtaacc ttaccttccg cgcctggccg gccggccggg 2160
taaccggcgt ccctcggcga tcgattttac aatcctttcc tctcgcctca tgtgtccaat 2220
ttttgcgctg gctaggtcgt actgccgcgg atagttgcat cacgtgagaa atcgttatgg 2280
ttcttaggtc gtgctgggat cagccgcatc gctgtattag tctactgcag ttgtttgttt 2340
ggttcggtcg g 2351
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<221>
<400> 5
ctcaccccca caagtcaaga 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<221>
<400> 6
ccgaccgaac caaacaaaca 20
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<221>
<400> 7
gagagagaga gagagaga 18
Claims (1)
1, a1) or a2) or A3):
A1) a method for identifying the grain length of corn kernels comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapKernel length of corn of II>HaplotypeHapIV, haplotypeHapIII or haplotypeHapGrain length of the corn of I;
A2) a method for identifying the grain width of corn grains comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapSeed grain width of corn of III<HaplotypeHapI. HaplotypeHapII or haplotypeHapIV, the grain width of the corn;
A3) a method for identifying the grain shape of corn kernels comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapIII or haplotypeHapKernel shape of corn of II>HaplotypeHapIV or haplotypeHapKernel shape of the corn of I; the grain shape is grain length/grain width;
the haplotypeHapThe corn of I is based on the SNP S-1830, the genotype of which is AA homozygous and has 'deletion type II of 2bp sequence with 9 tandem repeats';
the haplotypeHapThe II corn is the corn which is based on the SNP S-1830 and has the genotype of GG homozygous type and 2bp sequence of 9 tandem repeats;
the haplotypeHapThe maize of III is the maize based on SNP S-1830, the genotype of which is GG homozygous and has 'deletion type I of 2bp sequence with 9 tandem repeats';
the haplotypeHapThe IV corn is the corn which is based on the SNP S-1830, has the genotype of GG homozygous type and has 'deletion type II of 2bp sequence with 9 tandem repeats';
the SNP S-1830 is 189 th nucleotide from the 5' end of a target sequence of a primer pair consisting of a primer F and a primer R in a maize genome;
the primer F is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;
the '2 bp sequences with 9 tandem repeats' are shown as a sequence 7 in a sequence table;
the deletion type I of the 2bp sequence of the 9 tandem repeats is that the 1 st and the 2 nd nucleotides of the 2bp sequence of the 9 tandem repeats are deleted;
the deletion type II of the 2bp sequence of the 9 tandem repeats is that the 1 st to 4 th nucleotides are deleted from the 2bp sequence of the 9 tandem repeats.
2, B1) or B2) or B3):
B1) a method for identifying the grain length of corn kernels comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapKernel length of corn of II>HaplotypeHapIV, haplotypeHapIII or haplotypeHapGrain length of the corn of I;
B2) a method for identifying the grain width of corn grains comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapSeed grain width of corn of III<HaplotypeHapI. HaplotypeHapII or haplotypeHapIV, the grain width of the corn;
B3) a method for identifying the grain shape of corn kernels comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapIII or haplotypeHapKernel shape of corn of II>HaplotypeHapIV or haplotypeHapKernel shape of the corn of I; the grain shape is grain length/grain width;
the haplotype of the corn to be detected is detected as haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapThe method of IV' comprises the following steps: detecting whether the genome DNA of the corn to be detected contains a sequence 1 in a sequence table, a sequence 2 in the sequence table and a sequence tableA DNA fragment shown in a sequence 3 or a sequence 4 of a sequence table, and then carrying out the following judgment:
if the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 1 in the sequence table and does not contain the DNA segments shown in the sequence 2 in the sequence table, the sequence 3 in the sequence table and the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotypeHapI；
If the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 2 in the sequence table and does not contain the DNA segments shown in the sequence 1 in the sequence table, the sequence 3 in the sequence table and the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotypeHapII；
If the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 3 in the sequence table and does not contain the DNA segments shown in the sequence 1 in the sequence table, the sequence 2 in the sequence table and the sequence 4 in the sequence table, the haplotype of the corn to be detected is haplotypeHapⅢ；
If the genome DNA of the corn to be detected contains the DNA segment shown in the sequence 4 in the sequence table and does not contain the DNA segments shown in the sequence 1 in the sequence table, the sequence 2 in the sequence table and the sequence 3 in the sequence table, the haplotype of the corn to be detected is haplotypeHapⅣ。
3, C1) or C2) or C3):
C1) a method for identifying the grain length of corn kernels comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapKernel length of corn of II>HaplotypeHapIV, haplotypeHapIII or haplotypeHapGrain length of the corn of I;
C2) a method for identifying the grain width of corn grains comprises the following steps: detecting the haplotype of the corn to be detected as the haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapSeed grain width of corn of III<HaplotypeHapI. HaplotypeHapII or haplotypeHapIV, the grain width of the corn; C3) a method for identifying the grain shape of corn kernels comprises the following steps: detecting corn to be detectedThe haplotype of (A) is a haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapIV, haplotypeHapIII or haplotypeHapKernel shape of corn of II>HaplotypeHapIV or haplotypeHapKernel shape of the corn of I; the grain shape is grain length/grain width;
the haplotype of the corn to be detected is detected as haplotypeHapI. HaplotypeHapII. HaplotypeHapIII is also haplotypeHapThe method of IV' comprises the following steps in sequence:
(1) taking the genome DNA of the corn 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, and then judging as follows:
if the nucleotide sequence of the PCR amplification product is shown as sequence 1 in the sequence table, the haplotype of the corn to be detected is haplotypeHapI; if the nucleotide sequence of the PCR amplification product is shown as the sequence 2 in the sequence table, the haplotype of the corn to be detected is the haplotypeHapII, performing phase-change reaction; if the nucleotide sequence of the PCR amplification product is shown as the sequence 3 in the sequence table, the haplotype of the corn to be detected is the haplotypeHapIII; if the nucleotide sequence of the PCR amplification product is shown as the sequence 4 in the sequence table, the haplotype of the corn to be detected is the haplotypeHapⅣ；
The primer F is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 6 of a sequence table.
4. The application of a molecular marker A shown as a sequence 1 in a sequence table, a molecular marker B shown as a sequence 2 in the sequence table, a molecular marker C shown as a sequence 3 in the sequence table or a molecular marker D shown as a sequence 4 in the sequence table in the identification of the corn grain traits; the grain trait is f1) and/or f2) and/or f 3): f1) the grains are long; f2) the grain width is wide; f3) kernel length/kernel width.
5. The application of a molecular marker A shown as a sequence 1 in a sequence table, a molecular marker B shown as a sequence 2 in the sequence table, a molecular marker C shown as a sequence 3 in the sequence table or a molecular marker D shown as a sequence 4 in the sequence table in the preparation of a product for identifying the corn grain properties; the grain trait is f1) and/or f2) and/or f 3): f1) the grains are long; f2) the grain width is wide; f3) kernel length/kernel width.
6. The application of a molecular marker A shown as a sequence 1 in a sequence table, a molecular marker B shown as a sequence 2 in the sequence table, a molecular marker C shown as a sequence 3 in the sequence table or a molecular marker D shown as a sequence 4 in the sequence table in corn breeding.
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| CN101687898A (en) * | 2006-08-14 | 2010-03-31 | 孟山都技术有限责任公司 | maize polymorphisms and methods of genotyping |
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| CN101687898A (en) * | 2006-08-14 | 2010-03-31 | 孟山都技术有限责任公司 | maize polymorphisms and methods of genotyping |
| WO2010120727A1 (en) * | 2009-04-13 | 2010-10-21 | E. I. Du Pont De Nemours And Company | Genetic loci associated with fusarium ear mold resistance in maize |
| CN105219858A (en) * | 2015-10-15 | 2016-01-06 | 中国农业科学院作物科学研究所 | Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof |
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