CN114292947B - CAPS mark for identifying number of corn aleurone layers and detection method thereof - Google Patents
CAPS mark for identifying number of corn aleurone layers and detection method thereof Download PDFInfo
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Abstract
The invention discloses a CAPS mark for identifying the number of corn gluten layers and a detection method thereof, belonging to the technical field of molecular biology. SNP locus for identifying the number of layers of corn aleurone layer, wherein the nucleotide at the 491 th locus of CDS sequence of DEK48 gene of corn is G or A; CAPS markers were developed based on SNP sites. The invention provides a rapid, high-accuracy and high-throughput CAPS marker for identifying a mutation site of dek, so as to assist the application of the site in improving the aleurone layer of corn kernels, and lay a molecular foundation for improving the nutrition quality of the corn kernels.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a CAPS mark for identifying the number of layers of corn steep liquor powder and a detection method thereof.
Background
Corn is the first large grain crop in our country, and is also an important animal feed and industrial raw material. The kernel endosperm is the main nutrient storage organ of corn and accounts for more than 80% of the weight of the kernel. Mature corn endosperm mainly comprises four parts of starchy endosperm, aleurone layer, transfer layer and periembryo layer. The aleurone layer is located at the outermost layer of cereal grain endosperm and is a cubic thick-wall cell containing a large amount of aleurone particles. When seeds germinate, the aleurone layer receives hormonal signals to produce amylase and other hydrolytic enzymes, which break down nutrients in the endosperm to provide energy for germination. In addition, the aleurone layer of cereal crops is accumulated with rich high-quality protein, B vitamins and minerals, which are the part of the very rich nutrition components in the grains. The aleurone layer of wheat and corn is only one cell layer, the rice endosperm contains one to three cell layers, and in wild type barley is three cell layers. Research on the regulatory mechanisms of aleurone layer development suggests that multiple levels of genetic regulation control the fate, differentiation and organization of aleurone layer cells, and that many genes are involved in these processes. For example, DEK1 (deffectkernel 1) is a key gene for aleurone layer development, and its encoded protein has 21 transmembrane regions, an extracellular polypeptide loop and a cysteine protease domain, and participates in aleurone layer cell specification and its fate maintenance, and DEK1 mutant shows aleurone layer cell deletion. Another gene in maize, crinkly4 (CR 4), encodes a receptor-like kinase that specifically receives signals related to aleurone layer localization, and participates in aleurone layer cell fate specification through a protein phosphorylation cascade. Mutants CR4 and DEK1 are very similar phenotypically, and immunobiological studies have found that both the DEK1 and CR4 proteins are localized on endocytic vesicles on the plasma membrane, with CR4 possibly downstream of DEK 1. Unlike the two mutants above, the SAL1 (supernumerary aleurone layers 1) mutant had multiple layers of aleurone layer cells, indicating that the SAL1 gene is a negative regulator of aleurone layer cell development. SAL1 gene codes E type vacuole sorting protein, and is involved in membrane vesicle transport, and research shows that the protein is also positioned on endocytic membrane vesicles and is positioned at upstream to negatively regulate CR4 and DEK1 genes. In addition, THK1 (thin aleurone 1) is also involved in negative regulation of the number of aleurone layer cells, downstream of DEK 1. Numerous studies have proposed a possible model for interpreting aleurone layer cell fate specification, namely that the DEK1 gene accepts and conducts a localization signal to downstream CR4, which CR4 transmits to aleurone layer precursor cells, promoting aleurone layer cell specification. The upstream SAL1 gene then negatively regulates the concentration of DEK1 and CR4 proteins in the plasma membrane through endosome-mediated recycling/degradation. The negative regulatory gene THK1 located downstream of DEK1 is inhibited by DEK1, limiting the differentiation of the aleurone layer to the outermost cells of the endosperm. In summary, research on development of the aleurone layer of corn kernels has been advanced at home and abroad, however, the regulation and control mechanism of differentiation and development of the aleurone layer is complex, and the gene capable of being applied to thickening the aleurone layer of corn to improve the nutritional value of corn is still lacking at present. Based on the gene, the method has important significance in further excavating the aleurone layer development regulating gene.
Accordingly, it would be desirable to provide CAPS markers and methods for identifying the number of layers of corn meal and to provide methods for detecting the same.
Disclosure of Invention
In view of the above, the present invention provides a CAPS tag for identifying the number of layers of corn steep liquor and a detection method thereof.
In a prior study, a mutant dek defective in maize kernel development by EMS mutagenesis was found, which exhibited a phenotype of retarded kernel development, diminished mature kernel embryos and endosperm (FIG. 1).
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a SNP locus for identifying the number of layers of corn aleurone, wherein the SNP locus is positioned at 491 of CDS sequence of DEK48 gene of corn; the nucleotide at the SNP site is G or A.
Further, a CAPS marker for identifying the number of layers of corn steep liquor, which CAPS marker was developed based on the SNP site.
Further, CAPS markers for identifying the layer number of the corn steep liquor powder are obtained by amplifying specific primers CAPS491-F and CAPS 491-R;
the primer sequences of CAPS491-F and CAPS491-R are as follows:
CAPS491-F：5’-GGGGGGTAGTTGGACAAGAGC-3’；SEQ ID NO.6；
CAPS491-R：5’-CTGGCTTGATGCTCGGGTC-3’；SEQ ID NO.7。
further, a detection method of CAPS marks for identifying the number of layers of corn steep liquor powder comprises the following specific steps:
(1) Extracting DNA of corn kernels to be detected;
(2) Taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by utilizing the specific primer;
(3) Performing BceAI digestion on the PCR product, and performing agarose gel electrophoresis after digestion;
(4) And (3) judging an electrophoresis result: if an 417bp electrophoresis band is obtained, the SNP locus is A, which is a maize homozygous dek mutant, and the layer number of the maize aleurone layer is increased; if two electrophoresis bands of 302bp and 115bp are obtained, the SNP locus is G, the wild type is corn homozygous, and the layers of corn aleurone are normal.
Further, the SNP locus is applied to auxiliary corn kernel aleurone layer improvement.
Further, the CAPS mark is applied to auxiliary corn kernel aleurone layer improvement.
Further, the SNP locus is applied to identification of dek mutation.
Further, the CAPS markers are used in the identification of dek mutations.
The DEK48 gene was mapped using map-based cloning techniques. The DEK mutant caused an increased aleurone layer number phenotype due to the occurrence of a SNP mutation with "G" to "A" in the DEK48 gene CDS 491. In order to further apply the dek gene to the layer-by-layer improvement of the corn aleurone, a simple and high-throughput co-dominant molecular marker needs to be developed for distinguishing different genotypes and assisting the layer-by-layer selection of the aleurone. In the SNP variation detection technology, CAPS markers can be used for efficiently and rapidly realizing SNP locus detection through simple PCR amplification, restriction endonuclease digestion and product gel electrophoresis, and become an important molecular marker technology in biological research, and are widely applied to the fields of genetic map construction, germplasm identification, auxiliary breeding and the like. The invention provides a rapid, high-accuracy and high-throughput CAPS marker for identifying a mutation site of dek, so as to assist the application of the site in improving a corn kernel aleurone layer.
Compared with the prior art, the CAPS mark for identifying the number of layers of corn aleurone and the detection method thereof are provided, and a molecular basis is laid for improving the nutrition quality of corn kernels.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of dek compared to the phenotype of wild type Zheng58 grain;
wherein the outer circle is wild seed (WT), the seed is full, and embryo and endosperm develop normally; the inner circle is dek seed mutant, the seed is smaller, and embryo and endosperm are abnormal in development; scale = 1cm;
FIG. 2 is a graph showing a comparison of dek to wild type Zheng58 seed aleurone layer number 20 days after pollination;
wherein, the left graph shows dek mutant grains 20 days after pollination, and the number of aleurone layers is multiple; the right side is wild grains 20 days after pollination, and the number of aleurone layers is 1; scale = 100 μm; a aleurone layer is arranged in the rectangular frame;
FIG. 3 is a diagram showing cloning of the DEK48 gene of the present invention;
wherein A: DEK48 Gene Structure, DEK mutant a mutation of "G" to "A" on the second exon; b: the DEK48 codes for a protein structure, and amino acid 164 of DEK mutein is changed from Gly to Asp; c: mutation site analysis, sequencing at dek mutation site "a", sequencing of other inbred lines "G";
FIG. 4 is a drawing of CAPS491 marker development of the present invention; developing CAPS markers at dek mutation sites, wherein the wild type contains a cleavage site for BceAI restriction enzyme near the mutation sites, and the dek mutant cannot be cleaved by BceAI due to the mutation from "G" to "A";
FIG. 5 of the accompanying drawingsWild Type (WT), dek mutant and F of the invention 1 CAPS491 primer amplification and cleavage; wild type can be cut into two bands of 302 and 115bp, dek48 cannot be cut into 417bp, heterozygous F 1 The three bands of 417, 302 and 115bp are not obvious after enzyme digestion due to the smaller 115bp fragment;
FIG. 6 is a drawing of the invention F 2 A group construction process; b73 as female parent and heterozygous mutant DEK48 (+/-) hybrid to obtain F 1 ，F 1 Selfing to obtain F 2 Locating group, F 2 The ratio of wild-type (+/+), heterozygous (+/-) and mutant (-/-) in the population was 1:2:1, a step of;
FIG. 7 is a drawing of the invention F 2 Restriction enzyme strips of the population; wild type can be cut into two bands of 302 and 115 bp; dek48 cannot be cut, is 417bp; the heterozygote is 417, 302 and 115bp three bands, and the band is not obvious after enzyme digestion due to the smaller 115bp fragment;
wherein F is 2 -5、F 2 -6、F 2 -10、F 2 -11、F 2 -16、F 2 -20、F 2 -22、F 2 -26 total 8 parts of material band type is A type (417 bp); f (F) 2 -2、F 2 -8、F 2 -15、F 2 -23、F 2 -28、F 2 -30 total 6 parts of material bands are B type (302+115 bp); f (F) 2 -1、F 2 -3、F 2 -4、F 2 -7、F 2 -9、F 2 -12、F 2 -13、F 2 -14、F 2 -17、F 2 -18、F 2 -19、F 2 -21、F 2 -24、F 2 -25、F 2 -27、F 2 -29, 16 total material bands are H-shaped (417+302+115 bp); blank is to use ddH 2 O as a negative control for the template;
FIG. 8 is a diagram showing the enzymatic cleavage strips of the mutants and breeding inbred lines of the invention; the inbred line can be digested into two bands of 302bp and 115bp, and dek cannot be cut.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The dek mutant is described in "phenotypic identification and Gene localization of maize kernel mutant dek, dan Huimin, crop theory, 2020,46 (9): 1359-1367".
Example 1
Paraffin section observation of grains of different days after pollination of the DEK mutant revealed that there was different degrees of thickening of the aleurone layer cells of the mutant (FIG. 2), indicating that the DEK48 gene was involved in fate specification of the maize aleurone layer cells.
EXAMPLE 2 cloning of the DEK48 Gene
DEK48 gene was located precisely within 130kb interval of maize chromosome 3, and 6 protein-encoding genes were found in the interval using Gramene (http:// www.gramene.org /) website. Including WRKY family transcription factors (Zm 00001d 039532), cysteine oxidases (Zm 00001d 039533), F-box family proteins (Zm 00001d 039534), acetyltransferases (Zm 00001d 039535), synaptic fusion proteins (Zm 00001d 039536) and ALWAYS EARLY protein 3 (Zm 00001d 039537). Sequencing analysis of the exons of the 6 candidate genes showed that the 65 th base of the second exon of Zm00001d039535 gene was mutated from G to a in dek mutant (mutation of G to a at 491 of CDS sequence) resulting in mutation of the glycine at the original site to aspartic acid (fig. 3A). Sequencing of the segments of 5 maize inbred lines (Zheng 58, chang7-2, zong31, Q319, CL 11) containing this mutation site, these maize inbred lines were found to be G at this site (fig. 3C), further indicating that the mutation of this SNP may be responsible for the dek grain development defective phenotype. The results of the Gramene database on the structural and functional annotation of the DEK48 gene show that the DEK48 has a total length of 2854bp (SEQ ID NO. 1), codes for acetyl transferase (FIG. 3B), belongs to the BAHD family and comprises two conserved regions of HXXDG and DYGFG. Primers are designed at the 5 'and 3' ends of the gene to amplify the full length of cDNA, and the result shows that the CDS full length 1254bp (SEQ ID NO. 2) of the DEK48 gene has two exons. The coded amino acid sequence of the DEK48 gene is shown as SEQ ID NO. 3.
The DEK48 genomic sequence is as follows:
ATGGCGGGGTTCAAGGTGACGCGGATCTCGGAGGGCCCCGTGAAGCCGGCGTCGGCGACGCCCGAGGAGACGCTGCCGCTGGCTTGGGTGGACCGGTACCCGACGCACCGTGGCCTGGTGGAGTCGATGCACATCTTCCGGTCGGGCGCGGGCGAGGCCCCGGCCGTGATCCGGGCGGCGCTGGCCAAGGCGCTGGCCTTCTTCTACCCGCTGGCGGGCCGCATCGTGGAGGGGGAGCAGCCGGGGCGCCCCGCCATCCGCTGCACCGCCGACGGCGTCTACTTCGCGGAGGCCGAGGCGGACTGCAGCCTGGAGGACGTGCGCTTCCTGGAGCGGCCCCTGCTGCTGCCCAAGGAGGACCTCGTGCCTTACCCCGGCGACGACCGCTGGCCCGTCGAGCCGCACAACACCATCATGATGATGCAGGTATCTTAGACCACACCACACCTCGCCTCACCTCATCCCGTCAGTCAATTCCGCAGCGAGCTTTGTTTGGACCATACGACCCGCCGACGGCCGACGAGCTCAGCTCAGCGCGCGCGCTTTGTTTGGAGAGGGATCCGTTGTTCGTCGGGCCAGATCCGGGCACGCGCGCGCGGTAGATCTGGGGAGCTGCAGCCTGGCGAGGGGTCAAAAGCCCAGGGTCAGAGAAGGCGCGCTCACGGCCACCTAGGGAGGTTGACCGTGTCGGTTAATCAGGATGGCAAGTGGGACCAACGCTGCCAGATTCCCGCCGTCGCATAATCACGCACATCGTGCTTCCATAGTACTGTTTTTTTTTTATTATTTTCTTCTTGCATTTGAATCGAAAAAAATAATGCCTAAAACATAGTTTTAGTTTCCGAAATAAAATAGTGAGACAAAATCCTAGAAACCAAACGGTCAGGTAAAAAGCAGGGAGGGACTACCGGACAGGCAGCGAGAACGATTAATGGGTTAACGGCTACTAGTAGTACTAGCAGCGGCTGTAGGGAGATTCCCTCGACGCCGCGGTGGCGGTGGGAGCGGATGGCCACCGTACAGTACTGAATGCGGGCGGTGAGGCTGATCCGCGCCGATCTGGATATGCGTGAGCGTCCGTGCTCGCGTCCGTGCTCCGGCAGACGAGCCCAGGTTGGTCGTCTGACACGCATGGCGTCTGAGGTCAGCAGTGGAGAACTTGCGGCCAATTCTCAATTCCGCCGACAGAAGCCGCTACGTCACACTCCAGCGTGACCTTGCGTCGACTATCCAACGTGACCTTGGGTGGTGGGTGGGTGGGTAGGGTGGGCGGCTGGTAGGTAGGTCCGCGCTGGCGTAGACGGCTGCCGCGAACTCGGTGCCCGAAAGCGGTGCCAGCGCCACGATGTGAACCCCGGACAAAGAAGCGGGGAGAAGAGGCGTGCTGAACTGGGCTTGCTCTGTCCACGACGCTCCAATTTGCCATTTGGTAGTAGTACATGTTGGTAGTGGTCATCTTGCCGTATATATTGCCCTGCCCTTATAAAGGCACGACGAACTGTACCCGTGGTCATCTTGCCATGTCCTAGTTTTCAATATATTTATGACAGTAGTAGATGCTGGTAGTGGTCATCTTGCCATACACCCATATTTAGCCACAAAATTGTACCCGTGGTGTTGGTGCCGAGTGCCGACCGGGCATGACCAGTAGCTGACCGGTTGTTGTCGTTGTCGTTGCCGGTAGTTTACTCGATCGTACGTGGATGTTGCCTTTGCGATCAGGGTTCAGGGATCAGCCATGGCTTTAGCTTCTTTTCCTGCTTTCGTCCACGGCCCACCGACATCTTTGCGGGGGGTAGTTGGACAAGAGCCGTGTCAGTATAGATCACAACTTTCGCAACTACTGCACCTTTCATACTGCTGCCAGTGGTTGCCACCGACTGCACACCCTTTCCTTTCTTCACGGTTGCGTGCGACTGATTTGCCACCGAGAGACGATTAATTAAGACGCACGCTTCGGATCTGAACAAAATAAACCTGTGTAGAAAGAAAGAAAAAAAAAATAACGTCAATTGCATGCTCTCAGATCACCAAGTTCACCTGCGGCGGCTTCGTGATGGGCCTGCGGTTCAACCACGCGTCGGCGGACGGCATGGGCGCGGCGCAGTTCATCAACGCGGTGGGGGACATGGCGCGGGGGCTGGCGGAGCCGAGGGTGCTGCCCGTGTGGCACCGGGAGAAGTTCCCGGACCCGAG CATCAAGCCAGGCCCGCTCCCGGAGCTGCCCGTGCTGGCGCTGGACTACGTCGTGCTCGACTTCCCCACGGCCTACATCGACGGGCTCAAGCGGGAGTACAAGGCGCACAGCGGCAGGTTCTGCTCCGGCTTCGACGTGCTCACGGCCAAGCTCTGGCAGTGCCGCACCCGGGCGCTGGCCCTGGACCCGGCCGCCGAGGTCAAGCTCTGCTTCTTCGCCAGCGTCCGCCACCTGCTCAAGCTCGACCGGGGGTACTACGGCAACTCCATCTTCCCCGTCAAGATGTCCGCGCCGGCCGACAAGGTGCTGGCCTCCTCGCTCGTGGAGGTGGTCGACATCATCCGGGAGGCCAAGGACAGGATGGCCGTCGAGTTCTCCCGCTTCGCTGGGGAGGAGACGGACCAGGACCCGTTCCAGATGACCTTCAACTACGAGTCCATCTACGTCTCCGACTGGAGCAAGCTCGGCTTCTCCGAGGTCGACTACGGCTTCGGCCCGCCCATCTTCGCCGGCCCGCTCGTCAACAACGACTTCATCGCCTCCGTCGTCTTCCTCAAGGCGCCGCTCCCGCTCGACGGCACCAGGATGCTCGCCAGCTGCGTCACCAAGGAACACTCCGAGGAGTTCGCCCGTGGCATGAAGGAAGACCTGCCCTGA；SEQ ID NO.1。
the DEK48 CDS sequence is as follows:
ATGGCGGGGTTCAAGGTGACGCGGATCTCGGAGGGCCCCGTGAAGCCGGCGTCGGCGACGCCCGAGGAGACGCTGCCGCTGGCTTGGGTGGACCGGTACCCGACGCACCGTGGCCTGGTGGAGTCGATGCACATCTTCCGGTCGGGCGCGGGCGAGGCCCCGGCCGTGATCCGGGCGGCGCTGGCCAAGGCGCTGGCCTTCTTCTACCCGCTGGCGGGCCGCATCGTGGAGGGGGAGCAGCCGGGGCGCCCCGCCATCCGCTGCACCGCCGACGGCGTCTACTTCGCGGAGGCCGAGGCGGACTGCAGCCTGGAGGACGTGCGCTTCCTGGAGCGGCCCCTGCTGCTGCCCAAGGAGGACCTCGTGCCTTACCCCGGCGACGACCGCTGGCCCGTCGAGCCGCACAACACCATCATGATGATGCAGATCACCAAGTTCACCTGCGGCGGCTTCGTGATGGGCCTGCGGTTCAACCACGCGTCGGCGGACGGCATGGGCGCGGCGCAGTTCATCAACGCGGTGGGGGACATGGCGCGGGGGCTGGCGGAGCCGAGGGTGCTGCCCGTGTGGCACCGGGAGAAGTTCCCGGACCCGAGCATCAAGCCAGGCCCGCTCCCGGAGCTGCCCGTGCTGGCGCTGGACTACGTCGTGCTCGACTTCCCCACGGCCTACATCGACGGGCTCAAGCGGGAGTACAAGGCGCACAGCGGCAGGTTCTGCTCCGGCTTCGACGTGCTCACGGCCAAGCTCTGGCAGTGCCGCACCCGGGCGCTGGCCCTGGACCCGGCCGCCGAGGTCAAGCTCTGCTTCTTCGCCAGCGTCCGCCACCTGCTCAAGCTCGACCGGGGGTACTACGGCAACTCCATCTTCCCCGTCAAGATGTCCGCGCCGGCCGACAAGGTGCTGGCCTCCTCGCTCGTGGAGGTGGTCGACATCATCCGGGAGGCCAAGGACAGGATGGCCGTCGAGTTCTCCCGCTTCGCTGGGGAGGAGACGGACCAGGACCCGTTCCAGATGACCTTCAACTACGAGTCCATCTACGTCTCCGACTGGAGCAAGCTCGGCTTCTCCGAGGTCGACTACGGCTTCGGCCCGCCCATCTTCGCCGGCCCGCTCGTCAACAACGACTTCATCGCCTCCGTCGTCTTCCTCAAGGCGCCGCTCCCGCTCGACGGCACCAGGATGCTCGCCAGCTGCGTCACCAAGGAACACTCCGAGGAGTTCGCCCGTGGCATGAAGGAAGACCTGCCCTGA；SEQ ID NO.2。
the amino acid sequence encoded by the DEK48 gene is as follows:
MAGFKVTRISEGPVKPASATPEETLPLAWVDRYPTHRGLVESMHIFRSGAGEAPAVIRAALAKALAFFYPLAGRIVEGEQPGRPAIRCTADGVYFAEAEADCSLEDVRFLERPLLLPKEDLVPYPGDDRWPVEPHNTIMMMQITKFTCGGFVMGLRFNHASADGMGAAQFINAVGDMARGLAEPRVLPVWHREKFPDPSIKPGPLPELPVLALDYVVLDFPTAYIDGLKREYKAHSGRFCSGFDVLTAKLWQCRTRALALDPAAEVKLCFFASVRHLLKLDRGYYGNSIFPVKMSAPADKVLASSLVEVVDIIREAKDRMAVEFSRFAGEETDQDPFQMTFNYESIYVSDWSKLGFSEVDYGFGPPIFAGPLVNNDFIASVVFLKAPLPLDGTRMLASCVTKEHSEEFARGMKEDLP；SEQ ID NO.3。
the gene amplification primer sequences were as follows:
DEK48-F：5’-ATGGCGGGGTTCAAGGTGAC-3’；SEQ ID NO.4；
DEK48-R：5’-GGGCAGGTCTTCCTTCATGCC-3’；SEQ ID NO.5。
EXAMPLE 3CAPS marker development
In maize homozygous wild type SNP491 (or position 2091 of genomic sequence, SNP 2091) is G, which is recognized and cleaved by the restriction enzyme BceAI, whereas in homozygous dek grain mutant this site is a, which is not recognized by BceAI, and CAPS491 marker development is performed at this site (fig. 4). Theoretically, the wild type DNA amplification product contains 1 restriction site, can be cut into 302 and 115bp by BceAI restriction enzyme, and the DNA amplification product of the mutant dek has no restriction site, and the strip length is 417bp.
EXAMPLE 4 CAPS-labeled primer design and Synthesis
CAPS mark specific primer is designed according to the genome sequence of DEK48 gene, CAPS491-F primer (1791-1811 bp of genome sequence) is designed on the first intron of DEK48 gene, CAPS491-R primer (2189-2207 bp of genome sequence) is designed on the second exon, and the length of amplified fragment is 417bp. Primers were synthesized by the division of biological engineering (Shanghai).
The specific primer sequences are as follows:
CAPS491-F：5’-GGGGGGTAGTTGGACAAGAGC-3’；SEQ ID NO.6；
CAPS491-R：5’-CTGGCTTGATGCTCGGGTC-3’；SEQ ID NO.7。
example 5
A detection method for CAPS marks for identifying the number of corn gluten layers comprises the following specific steps:
(1) Extracting DNA of corn kernels to be detected;
the first step: about 0.1g of kernel endosperm was cut and placed into a 2mL centrifuge tube, and the steel beads were milled with Geno/Grinder and shaken at 1450rpm for 45s.
And a second step of: 600. Mu.L of the preheated 2% CTAB extract was added to each centrifuge tube with a lance, and the mixture was rapidly inverted and shaken well, and then placed into a 65℃water bath for 30min, and slightly shaken well once every 10 min.
And a third step of: 600 μl phenol was added: chloroform: isoamyl alcohol (25:24:1) extract was continuously extracted for 20min and centrifuged at 8000rpm for 15min. The supernatant was transferred to a new centrifuge tube. 2. Mu.LRNaseA (10 mg/mL) was added to each centrifuge tube and the tube was water-bath at 37℃for 30min.
Fourth step: the supernatant was added with equal volumes of chloroform to isoamyl alcohol (24:1), shaken upside down, centrifuged at 8000rpm for 15min and transferred to a new centrifuge tube. Adding 0.7 times of pre-cooled isopropanol, shaking thoroughly, mixing for 15s, and placing in a refrigerator at-20deg.C for 20min.
Fifth step: centrifuging at 8000rpm for 15min, pouring out supernatant, adding 700 μl of 70% alcohol, centrifuging at 3000rpm for 5min, and repeating one time, wherein one time soaking for more than 1 hr. The alcohol was decanted and left at room temperature for several minutes.
Sixth step: mu.L ddH was added to each centrifuge tube 2 O, mixing was reversed to dissolve the DNA sufficiently.
Seventh step: the quality of the DNA was checked with a 1% agarose gel and the concentration was checked with nanodrop 2000.
(2) Performing PCR amplification reaction by using the DNA extracted in the step (1) as a template and utilizing specific primers CAPS491-F and CAPS 491-R;
PCR amplification System (the enzyme used for PCR amplification is MF-002, beijing polymerase Biotechnology Co., ltd.): template DNA<1μg，2×M5 Taq HiFi PCRMix 10μl，CAPS491-F(10μM)0.5μl，CAPS491-R(10μM)0.5μl，ddH 2 O was made up to 20. Mu.l.
PCR reaction conditions: 94 ℃ for 3min;94℃30sec,60℃25sec,72℃35sec,30-35cycles;72 ℃ for 5min; forever at 4 ℃.
(3) Performing BceAI enzyme digestion on the PCR product; performing agarose gel electrophoresis after enzyme digestion;
BceAI restriction enzyme (NEB, R0623S), recognition site ACGGC (12/14);
and (3) enzyme cutting system: PCR products<1μg，10x Buffer 2μl，BceAI 1μl，ddH 2 O was made up to 20. Mu.l.
And (3) enzyme cutting: storing at 37 deg.C for 1 hr and at 4 deg.C.
(4) And (3) judging an electrophoresis result: if a 417bp electrophoresis band is obtained, the SNP locus is A, the SNP locus is a maize homozygous dek mutant, and the number of layers of the maize aleurone is multiple; if two electrophoresis bands of 302bp and 115bp are obtained, the SNP locus is G, the SNP locus is a corn homozygous wild type, and the number of layers of corn paste powder is 1.
Wild type inbred line B73 and F thereof using primers CAPS491-F and CAPS491-R pair dek48 1 The hybrid was PCR amplified and electrophoresed on a 1% agarose gel with a single amplified band and clear (FIG. 5) and the Sanger sequencing was correct. All PCR amplified products were digested with BceAI restriction enzyme, reacted at 37℃for 1 hour, and then electrophoresed on a 3% agarose gel. The experimental result shows that 3 types of bands A (417 bp), B (302+115 bp) and H (417+302+115 bp) are obtained after enzyme digestion. dek48 mutant band is A type band, B73 band is B type band, F 1 The hybrid band is H-shaped (FIG. 5). The CAPS marker has strong specificity and is separated from the character co-dominance, and can be used for detecting mutation sites.
EXAMPLE 6CAPS Label at F 2 Detection in isolated populations
30 parts of F are marked with CAPS491 2 The isolated population (figure 6) was subjected to genotyping,the results are shown in FIG. 7. As can be seen from FIG. 7, at F 2 After 22 parts of wild type materials with normal grain development are subjected to enzyme digestion, 6 parts of bands are B,16 parts of bands are H, 8 parts of mutant grains are subjected to enzyme digestion, the bands are A, and the detection result of the molecular marker and the accuracy rate of the phenotype reach 100.0%. F (F) 2 Genotype (wild type: heterozygous: mutant) distribution in the segregating population meets 1:2:1 (χ2)<3.84 Conforming to mendelian genetic rules, again verifying that the maize dek grain shape is controlled by a recessive single gene.
Example 7 detection of CAPS markers in maize diaphysis inbred lines
Further, CAPS491 was used to examine the backbone inbred lines (Zheng 58, chang7-2, B73, mo17, fu 31, huangzao four, huangC, zizan 319, 178, ye 478) in 10 maize breeds, and the bands after cleavage were all B-type, indicating that none of the backbone inbred lines contained mutant SNP sites (FIG. 8). Therefore, the corn kernel mutant dek can be used for improving the corn kernel aleurone layer, and the CAPS491 mark is developed, so that the corn kernel aleurone layer has an important effect on auxiliary site selection.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> institute of crop science at national academy of agricultural sciences
<120> CAPS marker for identifying the number of layers of corn steep liquor and detection method thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2854
<212> DNA
<213> Artificial Sequence
<400> 1
atggcggggt tcaaggtgac gcggatctcg gagggccccg tgaagccggc gtcggcgacg 60
cccgaggaga cgctgccgct ggcttgggtg gaccggtacc cgacgcaccg tggcctggtg 120
gagtcgatgc acatcttccg gtcgggcgcg ggcgaggccc cggccgtgat ccgggcggcg 180
ctggccaagg cgctggcctt cttctacccg ctggcgggcc gcatcgtgga gggggagcag 240
ccggggcgcc ccgccatccg ctgcaccgcc gacggcgtct acttcgcgga ggccgaggcg 300
gactgcagcc tggaggacgt gcgcttcctg gagcggcccc tgctgctgcc caaggaggac 360
ctcgtgcctt accccggcga cgaccgctgg cccgtcgagc cgcacaacac catcatgatg 420
atgcaggtat cttagaccac accacacctc gcctcacctc atcccgtcag tcaattccgc 480
agcgagcttt gtttggacca tacgacccgc cgacggccga cgagctcagc tcagcgcgcg 540
cgctttgttt ggagagggat ccgttgttcg tcgggccaga tccgggcacg cgcgcgcggt 600
agatctgggg agctgcagcc tggcgagggg tcaaaagccc agggtcagag aaggcgcgct 660
cacggccacc tagggaggtt gaccgtgtcg gttaatcagg atggcaagtg ggaccaacgc 720
tgccagattc ccgccgtcgc ataatcacgc acatcgtgct tccatagtac tgtttttttt 780
ttattatttt cttcttgcat ttgaatcgaa aaaaataatg cctaaaacat agttttagtt 840
tccgaaataa aatagtgaga caaaatccta gaaaccaaac ggtcaggtaa aaagcaggga 900
gggactaccg gacaggcagc gagaacgatt aatgggttaa cggctactag tagtactagc 960
agcggctgta gggagattcc ctcgacgccg cggtggcggt gggagcggat ggccaccgta 1020
cagtactgaa tgcgggcggt gaggctgatc cgcgccgatc tggatatgcg tgagcgtccg 1080
tgctcgcgtc cgtgctccgg cagacgagcc caggttggtc gtctgacacg catggcgtct 1140
gaggtcagca gtggagaact tgcggccaat tctcaattcc gccgacagaa gccgctacgt 1200
cacactccag cgtgaccttg cgtcgactat ccaacgtgac cttgggtggt gggtgggtgg 1260
gtagggtggg cggctggtag gtaggtccgc gctggcgtag acggctgccg cgaactcggt 1320
gcccgaaagc ggtgccagcg ccacgatgtg aaccccggac aaagaagcgg ggagaagagg 1380
cgtgctgaac tgggcttgct ctgtccacga cgctccaatt tgccatttgg tagtagtaca 1440
tgttggtagt ggtcatcttg ccgtatatat tgccctgccc ttataaaggc acgacgaact 1500
gtacccgtgg tcatcttgcc atgtcctagt tttcaatata tttatgacag tagtagatgc 1560
tggtagtggt catcttgcca tacacccata tttagccaca aaattgtacc cgtggtgttg 1620
gtgccgagtg ccgaccgggc atgaccagta gctgaccggt tgttgtcgtt gtcgttgccg 1680
gtagtttact cgatcgtacg tggatgttgc ctttgcgatc agggttcagg gatcagccat 1740
ggctttagct tcttttcctg ctttcgtcca cggcccaccg acatctttgc ggggggtagt 1800
tggacaagag ccgtgtcagt atagatcaca actttcgcaa ctactgcacc tttcatactg 1860
ctgccagtgg ttgccaccga ctgcacaccc tttcctttct tcacggttgc gtgcgactga 1920
tttgccaccg agagacgatt aattaagacg cacgcttcgg atctgaacaa aataaacctg 1980
tgtagaaaga aagaaaaaaa aaataacgtc aattgcatgc tctcagatca ccaagttcac 2040
ctgcggcggc ttcgtgatgg gcctgcggtt caaccacgcg tcggcggacg gcatgggcgc 2100
ggcgcagttc atcaacgcgg tgggggacat ggcgcggggg ctggcggagc cgagggtgct 2160
gcccgtgtgg caccgggaga agttcccgga cccgagcatc aagccaggcc cgctcccgga 2220
gctgcccgtg ctggcgctgg actacgtcgt gctcgacttc cccacggcct acatcgacgg 2280
gctcaagcgg gagtacaagg cgcacagcgg caggttctgc tccggcttcg acgtgctcac 2340
ggccaagctc tggcagtgcc gcacccgggc gctggccctg gacccggccg ccgaggtcaa 2400
gctctgcttc ttcgccagcg tccgccacct gctcaagctc gaccgggggt actacggcaa 2460
ctccatcttc cccgtcaaga tgtccgcgcc ggccgacaag gtgctggcct cctcgctcgt 2520
ggaggtggtc gacatcatcc gggaggccaa ggacaggatg gccgtcgagt tctcccgctt 2580
cgctggggag gagacggacc aggacccgtt ccagatgacc ttcaactacg agtccatcta 2640
cgtctccgac tggagcaagc tcggcttctc cgaggtcgac tacggcttcg gcccgcccat 2700
cttcgccggc ccgctcgtca acaacgactt catcgcctcc gtcgtcttcc tcaaggcgcc 2760
gctcccgctc gacggcacca ggatgctcgc cagctgcgtc accaaggaac actccgagga 2820
gttcgcccgt ggcatgaagg aagacctgcc ctga 2854
<210> 2
<211> 1254
<212> DNA
<213> Artificial Sequence
<400> 2
atggcggggt tcaaggtgac gcggatctcg gagggccccg tgaagccggc gtcggcgacg 60
cccgaggaga cgctgccgct ggcttgggtg gaccggtacc cgacgcaccg tggcctggtg 120
gagtcgatgc acatcttccg gtcgggcgcg ggcgaggccc cggccgtgat ccgggcggcg 180
ctggccaagg cgctggcctt cttctacccg ctggcgggcc gcatcgtgga gggggagcag 240
ccggggcgcc ccgccatccg ctgcaccgcc gacggcgtct acttcgcgga ggccgaggcg 300
gactgcagcc tggaggacgt gcgcttcctg gagcggcccc tgctgctgcc caaggaggac 360
ctcgtgcctt accccggcga cgaccgctgg cccgtcgagc cgcacaacac catcatgatg 420
atgcagatca ccaagttcac ctgcggcggc ttcgtgatgg gcctgcggtt caaccacgcg 480
tcggcggacg gcatgggcgc ggcgcagttc atcaacgcgg tgggggacat ggcgcggggg 540
ctggcggagc cgagggtgct gcccgtgtgg caccgggaga agttcccgga cccgagcatc 600
aagccaggcc cgctcccgga gctgcccgtg ctggcgctgg actacgtcgt gctcgacttc 660
cccacggcct acatcgacgg gctcaagcgg gagtacaagg cgcacagcgg caggttctgc 720
tccggcttcg acgtgctcac ggccaagctc tggcagtgcc gcacccgggc gctggccctg 780
gacccggccg ccgaggtcaa gctctgcttc ttcgccagcg tccgccacct gctcaagctc 840
gaccgggggt actacggcaa ctccatcttc cccgtcaaga tgtccgcgcc ggccgacaag 900
gtgctggcct cctcgctcgt ggaggtggtc gacatcatcc gggaggccaa ggacaggatg 960
gccgtcgagt tctcccgctt cgctggggag gagacggacc aggacccgtt ccagatgacc 1020
ttcaactacg agtccatcta cgtctccgac tggagcaagc tcggcttctc cgaggtcgac 1080
tacggcttcg gcccgcccat cttcgccggc ccgctcgtca acaacgactt catcgcctcc 1140
gtcgtcttcc tcaaggcgcc gctcccgctc gacggcacca ggatgctcgc cagctgcgtc 1200
accaaggaac actccgagga gttcgcccgt ggcatgaagg aagacctgcc ctga 1254
<210> 3
<211> 417
<212> PRT
<213> Artificial Sequence
<400> 3
Met Ala Gly Phe Lys Val Thr Arg Ile Ser Glu Gly Pro Val Lys Pro
1 5 10 15
Ala Ser Ala Thr Pro Glu Glu Thr Leu Pro Leu Ala Trp Val Asp Arg
20 25 30
Tyr Pro Thr His Arg Gly Leu Val Glu Ser Met His Ile Phe Arg Ser
35 40 45
Gly Ala Gly Glu Ala Pro Ala Val Ile Arg Ala Ala Leu Ala Lys Ala
50 55 60
Leu Ala Phe Phe Tyr Pro Leu Ala Gly Arg Ile Val Glu Gly Glu Gln
65 70 75 80
Pro Gly Arg Pro Ala Ile Arg Cys Thr Ala Asp Gly Val Tyr Phe Ala
85 90 95
Glu Ala Glu Ala Asp Cys Ser Leu Glu Asp Val Arg Phe Leu Glu Arg
100 105 110
Pro Leu Leu Leu Pro Lys Glu Asp Leu Val Pro Tyr Pro Gly Asp Asp
115 120 125
Arg Trp Pro Val Glu Pro His Asn Thr Ile Met Met Met Gln Ile Thr
130 135 140
Lys Phe Thr Cys Gly Gly Phe Val Met Gly Leu Arg Phe Asn His Ala
145 150 155 160
Ser Ala Asp Gly Met Gly Ala Ala Gln Phe Ile Asn Ala Val Gly Asp
165 170 175
Met Ala Arg Gly Leu Ala Glu Pro Arg Val Leu Pro Val Trp His Arg
180 185 190
Glu Lys Phe Pro Asp Pro Ser Ile Lys Pro Gly Pro Leu Pro Glu Leu
195 200 205
Pro Val Leu Ala Leu Asp Tyr Val Val Leu Asp Phe Pro Thr Ala Tyr
210 215 220
Ile Asp Gly Leu Lys Arg Glu Tyr Lys Ala His Ser Gly Arg Phe Cys
225 230 235 240
Ser Gly Phe Asp Val Leu Thr Ala Lys Leu Trp Gln Cys Arg Thr Arg
245 250 255
Ala Leu Ala Leu Asp Pro Ala Ala Glu Val Lys Leu Cys Phe Phe Ala
260 265 270
Ser Val Arg His Leu Leu Lys Leu Asp Arg Gly Tyr Tyr Gly Asn Ser
275 280 285
Ile Phe Pro Val Lys Met Ser Ala Pro Ala Asp Lys Val Leu Ala Ser
290 295 300
Ser Leu Val Glu Val Val Asp Ile Ile Arg Glu Ala Lys Asp Arg Met
305 310 315 320
Ala Val Glu Phe Ser Arg Phe Ala Gly Glu Glu Thr Asp Gln Asp Pro
325 330 335
Phe Gln Met Thr Phe Asn Tyr Glu Ser Ile Tyr Val Ser Asp Trp Ser
340 345 350
Lys Leu Gly Phe Ser Glu Val Asp Tyr Gly Phe Gly Pro Pro Ile Phe
355 360 365
Ala Gly Pro Leu Val Asn Asn Asp Phe Ile Ala Ser Val Val Phe Leu
370 375 380
Lys Ala Pro Leu Pro Leu Asp Gly Thr Arg Met Leu Ala Ser Cys Val
385 390 395 400
Thr Lys Glu His Ser Glu Glu Phe Ala Arg Gly Met Lys Glu Asp Leu
405 410 415
Pro
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
atggcggggt tcaaggtgac 20
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 5
gggcaggtct tccttcatgc c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 6
ggggggtagt tggacaagag c 21
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 7
ctggcttgat gctcgggtc 19
Claims (4)
1. A CAPS marker for identifying the number of layers of corn steep liquor, which is characterized in that the CAPS marker is obtained by amplification by specific primers CAPS491-F and CAPS 491-R;
the primer sequences of CAPS491-F and CAPS491-R are as follows:
CAPS491-F：5’-GGGGGGTAGTTGGACAAGAGC-3’；SEQ ID NO.6；
CAPS491-R：5’-CTGGCTTGATGCTCGGGTC-3’；SEQ ID NO.7；
the CAPS mark is a 417bp nucleotide sequence shown by 1791bp-2207bp in SEQ ID NO. 1;
the CAPS markers are developed based on SNP sites; the SNP locus is 2091 nucleotide sequence in SEQ ID NO.1, and the nucleotide at the SNP locus is G or A.
2. The detection method of CAPS marks for identifying the number of layers of corn steep liquor according to claim 1, comprising the specific steps of:
(1) Extracting DNA of corn kernels to be detected;
(2) Performing PCR amplification reaction by using the DNA extracted in the step (1) as a template and the specific primer of claim 1;
(3) Performing BceAI digestion on the PCR product, and performing agarose gel electrophoresis after digestion;
(4) And (3) judging an electrophoresis result: if an 417bp electrophoresis band is obtained, the SNP locus is A, which is a maize homozygous dek mutant, and the layer number of the maize aleurone layer is increased; if two electrophoresis bands of 302bp and 115bp are obtained, the SNP locus is G, the wild type is corn homozygous, and the layers of corn aleurone are normal.
3. Use of CAPS markers according to claim 1 for assisting in the improvement of a aleurone layer of corn kernels, characterized in that the improvement is carried out by the method according to claim 2.
4. Use of CAPS markers according to claim 1 for the identification of dek mutants, characterized in that the identification is performed by the method according to claim 2.
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| CN202111682108.XA CN114292947B (en) | 2021-12-31 | 2021-12-31 | CAPS mark for identifying number of corn aleurone layers and detection method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034743A (en) * | 2017-12-06 | 2018-05-15 | 上海大学 | For detecting specific primer and its application of corn Dek6 genes |
| CN111961739A (en) * | 2020-06-12 | 2020-11-20 | 上海大学 | Specific primer group for detecting corn Dek41 gene and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006314535A1 (en) * | 2005-11-15 | 2007-05-24 | Biogemma | EMP4 gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034743A (en) * | 2017-12-06 | 2018-05-15 | 上海大学 | For detecting specific primer and its application of corn Dek6 genes |
| CN111961739A (en) * | 2020-06-12 | 2020-11-20 | 上海大学 | Specific primer group for detecting corn Dek41 gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CASTORINA,G.等.drought-responsive zmFDL1/MYB94 regulates cuticle biosynthesis and cuticle-dependent leaf permeability.PLANT PHYSIOL..2020,第184卷(第·期),266-282. * |
| 欧杨虹 ; 吴雯雯 ; 邵元健 ; 吴永升 ; .CAPS标记开发及其在D8基因功能标记开发中的应用.分子植物育种.2016,(第10期),全文. * |
| 石慧敏 ; 蒋成功 ; 王红武 ; 马庆 ; 李坤 ; 刘志芳 ; 吴宇锦 ; 李树强 ; 胡小娇 ; 黄长玲 ; .玉米籽粒突变体dek48的表型鉴定与基因定位.作物学报.2020,第46卷(第09期),全文. * |
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