CN115491429A - Detection primer and detection kit for rice wx gene and application of detection kit - Google Patents

Detection primer and detection kit for rice wx gene and application of detection kit Download PDF

Info

Publication number
CN115491429A
CN115491429A CN202210443780.1A CN202210443780A CN115491429A CN 115491429 A CN115491429 A CN 115491429A CN 202210443780 A CN202210443780 A CN 202210443780A CN 115491429 A CN115491429 A CN 115491429A
Authority
CN
China
Prior art keywords
primer
rice
gene
genes
detection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210443780.1A
Other languages
Chinese (zh)
Inventor
倪大虎
周燃
宋丰顺
甘泉
林翠香
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rice Research Institute of Anhui Academy of Agricultural Sciences
Original Assignee
Rice Research Institute of Anhui Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rice Research Institute of Anhui Academy of Agricultural Sciences filed Critical Rice Research Institute of Anhui Academy of Agricultural Sciences
Priority to CN202210443780.1A priority Critical patent/CN115491429A/en
Publication of CN115491429A publication Critical patent/CN115491429A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a detection primer and a detection kit for rice wx gene and application thereof. Aiming at the rice wx gene functional region, sequence difference of waxy rice and non-waxy rice wx genes is found out through sanger dideoxy sequencing technology to determine a waxy allelic specific molecular marker, two pairs of wx gene detection primers with nucleotide sequences shown as SEQ ID No.1-SEQ ID No.4 are designed, the number and the size of fragments of PCR products are detected through electrophoresis, whether the detected rice sample contains wx genes or not is judged, the contained wx genes are heterozygous or homozygous allelic types is judged, and then auxiliary breeding of the wx genes, quick judgment of wx gene homozygotes and heterozygotes and identification of the purity of the waxy rice varieties are realized. The invention obviously reduces the detection cost and provides technical support for the cultivation of new glutinous rice varieties, the purification and rejuvenation of core germplasm and the purity identification of production seeds.

Description

Detection primer and detection kit for rice wx gene and application of detection kit
Technical Field
The invention relates to a detection primer and a detection kit for a rice wx gene, and further relates to application of the detection primer for the rice wx gene in the aspects of auxiliary breeding of the rice wx gene, rapid judgment of a wx gene homozygote and a heterozygote, identification of the purity of a glutinous rice variety and the like, belonging to the fields of molecular markers, detection primers and application of the rice wx gene.
Background
The rice is one of the important grain crops in China, the planting area of the rice in China accounts for about 30% of the area of the grain crops, and the yield is close to half of the total grain yield. Because of its good eating quality, glutinous rice is widely used in health-care nourishing food, wine brewing, cake making, cold drink and medicine, etc., and is popular among people.
Because the waxy character is recessive, the factors such as mixing and powder stringing in production easily cause waxy character loss or degeneration, and the waxy character is fatal to the seed property maintenance. In order to ensure the specificity, consistency and stability of the conventional variety, the variety needs to be purified and rejuvenated to maintain the excellent variety of the variety. The common methods are a strain cycle method and a three-year three-nursery method, and the core of the two methods is to select a typical single plant or a typical single spike, and the selection is based on an empirical phenotype, so that certain blindness exists. In the process of breeding glutinous rice, the separation generation is mostly in a heterozygous state due to the glutinosity, the amylose content cannot accurately reflect whether the glutinous character is homozygous, accurate analysis can be carried out only after the strain is stable, and the breeding workload is greatly increased.
Starch synthesis in rice is controlled by the waxy gene (Wx) encoding starch synthase (GBSS I) bound to starch granules. Allelic variation of the Wx gene results in differences in amylose content among different rice varieties. The amylose content in the waxy rice variety is generally lower than 2 percent, and belongs to recessive mutation (Wx) of the Wx gene. Previous researches show that in the currently reported glutinous rice, a 23bp insertion exists in the second exon region of the Wx gene, so that frame shift mutation occurs in the expression of the Wx gene. The scholars Sun Huaqin, tian Zhixi, etc. designed functional molecular markers for this point difference to distinguish waxy/non waxy varieties. Meanwhile, sun Huaqin and the like indicate that, as the base sequence of the wx gene functional region has higher GC content, GC Buffer and high fidelity enzyme (LA Taq) suitable for GC rich structure amplification should be selected during PCR reaction, and higher annealing temperature needs to be set, so that the detection cost is higher. Tian Zhixi and other markers Wx M1 designed for Wx gene functional regions select an annealing temperature of 58 ℃ during PCR reaction, and have more miscellaneous bands during electrophoresis detection, so that the result judgment is difficult.
Therefore, designing a detection primer with strong specificity, high sensitivity and low detection cost aiming at the rice wx gene functional region is a technical problem to be solved urgently.
Disclosure of Invention
One of the purposes of the invention is to provide a rice wx gene detection primer;
the invention also aims to provide a detection kit containing the rice wx gene detection primer.
The third purpose of the invention is to apply the detection primer of the rice wx gene to the aspects of the auxiliary breeding of the rice wx gene, the rapid judgment of the pure and hybrid of the wx gene or the identification of the purity of the glutinous rice variety, and the like.
The above object of the present invention is achieved by the following technical solutions:
the invention firstly provides a rice wx gene detection primer, which is selected from any one of a primer pair (1) or a primer pair (2), wherein the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.
The invention further provides a rice wx gene PCR detection kit, which comprises: taq enzyme, dNTP, mg 2+ Deionized water and a detection primer; wherein the detection primer is designed by taking a rice wx gene functional region as a targetDetecting a primer; as a preferred embodiment, the detection primer is selected from any one of a primer pair (1) or a primer pair (2), wherein the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.
The invention also comprises the application of the rice wx gene detection primer in the aspects of auxiliary breeding of the rice wx gene, rapid judgment of wx gene homozygote and heterozygote, identification of glutinous rice variety purity and the like.
The invention provides a method for determining whether wx genes or homozygote and heterozygote of wx genes exist in a rice sample by applying a rice wx gene detection primer, which comprises the following steps:
(1) Extracting DNA of a rice sample to be detected;
(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer;
(3) Amplifying by using the primer (1), and if the amplification result is only a 140bp strip, determining that the rice sample to be detected is a wx gene homozygote; if the amplification result is only 117bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 140bp and 117bp at the same time, the rice sample to be detected is a hybrid strain containing wx genes;
amplifying by using the primer pair (2), wherein if the amplification result only has 199bp bands, the rice sample to be detected is a wx gene homozygote; if the amplification result is only 176bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 199bp and 176bp, the rice sample to be detected is a hybrid strain containing wx genes.
In order to transfer the wx gene into rice material with excellent characters, the invention also provides a method for carrying out rice wx gene auxiliary breeding by applying the rice wx gene detection primer, which comprises the following steps:
hybridizing and backcrossing the excellent rice strain and the glutinous rice material containing wx genes, amplifying wx gene sites of each hybridization (backcross) generation single plant by using a primer pair (1) (wx-1) or a primer pair (2) (wx-2), and performing polyacrylamide gel electrophoresis on the amplified PCR product; (2) Amplifying by using the primer (1), if the amplification result contains a 140bp strip, judging that wx genes exist in the sample, amplifying by using the primer (2), and if the amplification result contains a 199bp strip, judging that wx genes exist in the sample; thereby assisting in breeding the individual plants which contain the waxy gene (wx) and have excellent agronomic characters.
The invention also provides a method for rapidly judging wx gene homozygote of a selfing segregation population single plant by applying the rice wx gene detection primer, which comprises the following steps:
(1) Extracting DNA of a single plant of a self-separation group to be detected; (2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer; (3) Amplifying by using a primer pair (1), if the amplification result only has 140bp bands, determining that the single detection sample plant is a homozygous single plant carrying wx genes, and if the amplification result simultaneously contains two bands of 140bp and 117bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes; amplifying by using a primer pair (2) wx-2, if the amplification result only has 199bp bands, determining that the single detection sample plant is a homozygous plant carrying wx genes, and if the amplification result simultaneously contains two bands of 199bp and 176bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes.
The invention also provides a method for identifying the purity of a glutinous rice variety by applying the rice wx gene detection primer, which comprises the following steps:
(1) Extracting DNA of a glutinous rice variety to be detected;
(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer;
(3) Amplifying by using a primer pair (1), if the electrophoresis result has only 140bp bands which are glutinous rice single plants carrying homozygous wx genes, and simultaneously contains two bands of 140bp and 117bp which are glutinous rice single plants carrying heterozygous wx genes, and only contains 117bp bands which are non-glutinous rice single plants mixed;
amplifying by using a primer pair (2), if the electrophoresis result has only 199bp bands which are glutinous rice single plants carrying homozygous wx genes, single plants containing two bands of 199bp and 176bp are glutinous rice single plants carrying heterozygous wx genes, and single plants containing only 176bp bands which are non-glutinous rice single plants are mixed;
(4) The purity of the rice sample is calculated according to the following formula:
Figure BDA0003615707940000051
in the formula:
p-sample purity;
N T the number of seeds (number of seedlings, number of plants) to be tested;
N D the number of seeds (number of seedlings, number of plants) determined as a mixed plant.
As a preferred embodiment of the present invention, the reaction system for PCR amplification comprises: taq enzyme 0.2. Mu.l, dNTP 2. Mu.l, mg 2+ 2 ul, 14 ul DNA template, 1 ul upstream primer, 1 ul downstream primer, 5.8 ul deionized water, 4 ul 5 XBuffer.
As a preferred embodiment of the present invention, the amplification procedure of the PCR amplification comprises: pre-denaturation at 95 deg.C for 5min, circulation at 94 deg.C for 1min,67 deg.C for 30s,72 deg.C for 40s, extension at 72 deg.C for 10min, and storage at 12 deg.C.
Aiming at wx gene function regions, sequence differences of wx genes of waxy rice and non-waxy rice are found out through a sanger dideoxy sequencing technology, wx gene specific function markers wx-1 and wx-2 are developed, a PCR technology is utilized to amplify the marker sites, the number and the size of fragments of PCR products are detected through electrophoresis, whether the detected rice sample contains wx genes or not and whether the contained wx genes are heterozygous or homozygous allelic types are judged, and then auxiliary breeding of the wx genes, quick judgment of wx gene homozygotes and heterozygotes and identification of waxy rice variety purity are realized.
The invention develops the waxy allelic specific molecular marker for the sequence difference on the second exon of wx gene of waxy rice/non-waxy rice variety, realizes accurate differentiation of the genotype of the waxy rice and the non-waxy rice through the optimization of a reaction system and a reaction program, obviously reduces the detection cost, and provides technical support for the cultivation of new waxy rice varieties, the purification and rejuvenation of core germplasm and the purity identification of production seeds.
The present invention relates to key term definitions and abbreviations
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.
The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Cassol et al (1992); rossolini et al, mol cell. Probes 8.
Gene molecular marker: an effective gene marker closely linked with a trait is developed based on sequence difference of a certain gene.
Drawings
FIG. 1 shows the base sequence of the second exon in the Wx gene.
FIG. 2 shows the molecular assisted selective breeding process of glutinous rice gene.
FIG. 3 depicts a wx-1 electropherogram; m:20bp DNA standard molecular weight. 1-8 are glutinous rice varieties which are respectively as follows: anhui cultivated glutinous rice No.1, anhui cultivated glutinous rice No.2, anhui cultivated glutinous rice No. 5, anhui cultivated glutinous rice 1116, anhui cultivated glutinous rice No. 19, xuanjing glutinous rice No.1, guangming glutinous rice No.1 and Anhui rice No. 68;9-23 are non-waxy varieties, respectively: anhui cultivated japonica No.2, anhui cultivated japonica No.3, anhui cultivated japonica No. 8, anhui cultivated japonica No. 516, anhui cultivated japonica No. 11036, anhui cultivated Jinqing, anhui rice No. 14, anhui rice No. 15, anhui rice No. 18, anhui cultivated japonica No.2, anhui cultivated japonica No. 10, chang nong cultivated No.1, evening japonica No. 22, tian japonica No.2 and Ningjing No. 3.
FIG. 4 depicts a wx-2 electropherogram; m:20bp DNA standard molecular weight. 1-8 are glutinous rice varieties which are respectively as follows: anhui cultivated glutinous rice No.1, anhui cultivated glutinous rice No.2, anhui cultivated glutinous rice No. 5, anhui cultivated glutinous rice 1116, anhui cultivated glutinous rice No. 19, xuanjing glutinous rice No.1, guangming glutinous rice No.1 and Anhui rice No. 68;9-23 are non-waxy varieties, respectively: anhui cultivated japonica No.2, anhui cultivated japonica No.3, anhui cultivated japonica No. 8, anhui cultivated japonica No. 516, anhui cultivated japonica No. 11036, anhui cultivated Jinqing, anhui rice No. 14, anhui rice No. 15, anhui rice No. 18, anhui cultivated japonica No.2, anhui cultivated japonica No. 10, chang nong cultivated No.1, evening japonica No. 22, tian japonica No.2 and Ningjing No. 3.
FIG. 5 shows marker wx-1, wx-2 for the auxiliary breeding of wx gene selfing progeny; m:20bp DNA standard molecular weight; 1-21: detecting the single plant; 1,wx gene donor (xuanjing glutinous rice No. 7); 2, wuyujing No. 3; 9. 18, 21: a wx gene homozygote; 5. 6, 12, 13, 15, 20: a wx gene hybrid; the rest is single plants without wx gene.
FIG. 6 shows the purity of two glutinous rice varieties tested by wx-1 and wx-2 markers;
a tangle-solidup: hybrid strain: does not contain WX gene hybrid strains.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 comparison of functional regions of wx Gene of Rice and design and verification of wx Gene detection primers
Extraction of rice genome DNA by CTAB method
The materials include 8 glutinous rice varieties such as Anhui reclamation glutinous rice No.1 and Zhen glutinous rice No. 19 and 15 non-glutinous rice varieties such as Anhui reclamation japonica No.2 and late japonica No. 22. Shearing about 0.1g of young leaves of a test material for each variety, transferring the young leaves into a 2.0mL centrifuge tube, adding 2 grinding beads into the centrifuge tube, freezing the young leaves by using liquid nitrogen, fully grinding the young leaves into powder by using a tissue grinder, adding 800 mu L of CTAB extraction buffer solution preheated at 65 ℃ (81.7 g of NaCl and 20g of CTAB are fully dissolved in a proper amount of water, then adding 1mol/L of Tris-HCl 100mL and 0.5mol/L of EDTA 40mL, fixing the volume to 1000mL, storing the young leaves at 4 ℃), incubating the young leaves at 65 ℃ for 1h, and reversing the young leaves and the buffer solution uniformly every 15 min; adding equal volume of chloroform/isoamyl alcohol (24); centrifuging at 12,000rpm for 10min, sucking supernatant, transferring to another 1.5mL centrifuge tube, adding equal volume of isopropanol, mixing, centrifuging at-20 deg.C for 10min at 4 deg.C and 12,000rpm, discarding supernatant, and adding 500 μ L75% ethanol to wash precipitate; after centrifugation at 12,000rpm for 10min, ethanol was discarded, and after drying at room temperature, 50. Mu.L of sterile water was added and sufficiently dissolved. The concentration of each sample was measured by an apple Nanophotometer ultramicro ultraviolet spectrophotometer to detect OD 260 /OD 280 The ratio, after being qualified, the concentration is uniformly diluted into 50 ng/mu L by sterile water and stored at the low temperature of minus 20 ℃ for later use.
2. Determination of the functional region sequence of wx Gene
2.1 wx gene function region DNA sequencing primer sequence
TABLE 1Wx Gene sequencing primer information
Figure BDA0003615707940000081
2.2 PCR amplification
The sequencing primer was used to amplify the genomic DNA of rice, and the distribution of each component in the amplification system is shown in Table 2.
TABLE 2 PCR reaction System
Figure BDA0003615707940000091
Note: the reaction volume can be selected between 10-50. Mu.L, and the concentration of template DNA source can be between 20-200 ng/. Mu.L.
The PCR reaction program is: after pre-denaturation at 95 ℃ for 5min, at 94 ℃ for 1min, at 55/58 ℃ for 30s, at 72 ℃ for 40s, the cycle is repeated for 35 times, at 72 ℃ for 10min, and the product is preserved at 12 ℃. The PCR amplification products were detected by 1.5% agarose electrophoresis.
Comparison of the functional region sequences of wx genes
SeqMan in DNAstar software is used for completing the assembly of a plurality of fragments in the sequencing result, clustal Omega software is used for carrying out multi-sequence alignment, and the specific differential site of wx gene is found out.
4.Wx Gene detection primer design and verification
4.1wx Gene detection primer design
According to the specific difference of wx gene functional regions, PCR detection primers wx-1 and wx-2 are designed by using Primer Premier 5 software, and selected Primer sequences are compared on NCBI-blast to check the specificity of the primers.
TABLE 3 functional marker information for the waxy genes wx
Figure BDA0003615707940000092
Figure BDA0003615707940000101
4.2wx Gene detection verification
The primers wx-1 and wx-2 were used to amplify waxy and non-waxy rice varieties, respectively, and the PCR reaction system is shown in Table 2.
The reaction procedure is as follows: pre-denaturation at 95 deg.C for 5min, circulation at 94 deg.C for 1min,67 deg.C for 30s,72 deg.C for 40s, extension at 72 deg.C for 10min, and storage at 12 deg.C. The product is separated by 10 percent polyacrylamide gel electrophoresis, which not only can accurately judge whether wx gene exists, but also can judge homozygote and heterozygote of wx gene.
As a result of amplification using the primer wx-1, a wx gene homozygote was found in which only the 140bp band was present, a wx gene-free homozygote was found in which only the 117bp band was present, and a wx gene-containing heterozygote was found in which the single strain was found to contain both the 140bp and 117bp bands.
The primer wx-2 is used for amplification, and as a result, only 199bp bands are homozygotes containing wx genes, only 176bp bands are not containing wx genes, and single strains containing 199bp bands and 176bp bands are hybrid strains containing wx genes.
4.WX Gene assisted selection
In order to transfer wx gene into rice material with excellent character, excellent rice strain is hybridized and backcrossed with glutinous rice material containing wx gene, each hybridization (backcross) generation individual plant wx gene site is amplified by using primer wx-1 or wx-2, and polyacrylamide gel electrophoresis is carried out on the amplified PCR product to judge whether wx gene exists or not, thereby assisting in breeding individual plant containing glutinous gene (wx) and having excellent agronomic character.
Rapid determination of wx Gene homozygote
And judging whether the detected individual plant contains wx genes and whether the wx genes are in a homozygous type according to the difference of enzyme cutting bands of amplified products of the individual plants of the selfing segregation population by the markers wx-1 and wx-2.
The primer wx-1 is used for amplification, and as a result, only a 140bp strip is a homozygous single plant carrying wx genes, and a single plant containing two strips of 140bp and 117bp is a heterozygous plant carrying wx genes. The primer wx-2 is used for amplification, and as a result, only 199bp bands are homozygous single plants carrying wx genes, and single plants containing 199bp and 176bp bands are hybrid plants carrying wx genes.
Thus, when identifying the selfed segregating population: the electrophoresis result shows that the single plant only has a 140bp band when the marker wx-1 is used for amplification; the marked wx-2 is used for amplification, and electrophoresis results show that single plants with 199bp bands are single plants with homozygous wx genes.
Test example 1wx Gene-assisted selection and Breeding application test Using wx Gene detection primers
1. Extracting 8 parts of glutinous rice material and 15 parts of non-glutinous rice material genome DNA by a CTAB method:
1) About 100 seeds of each 23 varieties are taken and cultured in an illumination incubator at the temperature of 28-32 ℃ for about 1 week;
2) Cutting single seedlings, placing the single seedlings into a 2.0mL centrifuge tube, adding 2 grinding beads, freezing by liquid nitrogen, grinding the single seedlings into powder by using a tissue grinder, and processing 5 single seedlings per part of material;
3) To the centrifuge tube was added 800. Mu.L of 65 ℃ preheated CTAB extraction buffer (81.7 g NaCl and 20g CTAB dissolved well in an appropriate amount of water, followed by addition of 1mol/L Tris-HCl 100mL,0.5mol/L EDTA 40mL, volume to 1000mL, storage at 4 ℃. ) (ii) a
4) Water bath at 65 deg.C for 1h, and mixing by reversing every 15min for several times;
5) Adding equal volume of chloroform/isoamyl alcohol (24);
6) Centrifuging at 12,000rpm for 10min, sucking supernatant, transferring to another 1.5mL centrifuge tube,
7) Adding isopropanol of the same volume, mixing, and freezing at-20 deg.C for 30min.
8) The mixture was centrifuged at 12,000rpm for 10min at 4 ℃ and the supernatant was discarded.
9) Adding 500 mu L of 75% ethanol to wash the precipitate; centrifuging at 12,000rpm for 10min, and removing ethanol;
10 ) repeat step 9
11 ) drying at room temperature, adding 50. Mu.L of sterilized water, and dissolving sufficiently for use.
2.wx gene function region differential sequence amplification and detection
1) Amplification primer of wx gene functional region
The amplification primers for the wx gene functional region were as follows:
wx-1F:5’CGTCGTCGCTGCTCCGC 3’
wx-1R:5’ACCGCTGCTGCTTGGGC 3’
wx-2F:5’CGTCCCAGCTCGCCACCT 3’
wx-2R:5’CACCGACCGCTGCTGCTT 3’
2) PCR amplification and electrophoresis detection
Sequentially adding the components into a 0.2mL thin-wall tube, wherein the reaction system comprises the following components:
TABLE 4 PCR reaction System
Figure BDA0003615707940000121
And (3) amplification procedure: pre-denaturation at 95 deg.C for 5min, circulation at 94 deg.C for 1min,67 deg.C for 30s,72 deg.C for 40s, extension at 72 deg.C for 10min, and storage at 12 deg.C.
3.Wx gene auxiliary breeding by using markers wx-1 and wx-2
1) The breeding process of wx gene-containing materials (figure 2).
2) Extraction of rice genome DNA
The rice material used: and the BC2F4 single plant is bred by hybridization of Xuanjing glutinous No. 7 and Wuyujing No. 3. Shearing about 0.1g of young leaves of the rice at the adult stage, and extracting genome DNA by a CTAB method;
3) PCR amplification electrophoresis detection of rice genome DNA
Amplifying the genome DNA of the individual strains of the Xuanjing glutinous rice No. 7, wuyujing glutinous No.3 and BC2F4 groups by using primers wx-1 and wx-2, and detecting the PCR product by using 10% polyacrylamide gel electrophoresis.
4) Selection of progeny containing wx gene
The wx gene homozygous single plant is marked with wx-1 amplification only containing 140bp bands, and marked with wx-2 amplification only containing 199bp bands; the wx gene heterozygous single strain has two bands of 140bp and 117bp in wx-1 amplification and 199bp and 176bp in wx-2 amplification. In the backcross progeny, wx-gene-heterozygous individuals were selected, and in the selfed progeny, wx-gene-homozygous individuals were selected (fig. 5).
Test example 2 determination of purities of hybrid between glutinous and non-glutinous rice Using wX Gene detection primers
According to the difference of electrophoresis bands of amplification products of the markers wx-1 and wx-2 between the waxy rice and the non-waxy rice, the purity of the waxy rice can be identified.
The primer wx-1 is used for amplification, and the electrophoresis result shows that only 140bp bands are glutinous rice single plants carrying homozygous wx genes, two bands of 140bp and 117bp are glutinous rice single plants carrying heterozygous wx genes, and only 117bp bands are non-glutinous rice single plants mixed. The primer wx-2 is used for amplification, and the single glutinous rice plant carrying the homozygous wx gene only has 199bp bands, the single glutinous rice plant carrying the heterozygous wx gene only has 199bp and 176bp bands, and the single non-glutinous rice plant only having 176bp bands is mixed as shown in figure 6.
The purity test result of the rice sample is calculated according to the following formula:
Figure BDA0003615707940000131
in the formula:
p-sample purity;
N T the number of seeds to be tested (number of seedlings, number of plants);
N D the number of seeds (number of seedlings, number of plants) determined as a mixed plant.

Claims (10)

1. The detection primer of the rice wx gene is characterized by being selected from any one of a primer pair (1) or a primer pair (2), wherein the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.
2. A rice wx gene PCR detection kit comprises: taq enzyme, dNTP, mg 2+ Deionized water and a detection primer; the method is characterized in that the detection primer is selected from any one of a primer pair (1) or a primer pair (2), and the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.
3. Use of the detection primer of claim 1 for determining the presence of wx gene or homozygote and heterozygote of wx gene in a rice sample.
4. The use according to claim 3, comprising:
(1) Extracting DNA of a rice sample to be detected;
(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer according to claim 1;
(3) Amplifying by using the primer (1), and if the amplification result is only a 140bp strip, determining that the rice sample to be detected is a wx gene homozygote; if the amplification result is only 117bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 140bp and 117bp at the same time, the rice sample to be detected is a hybrid strain containing wx genes;
amplifying by using the primer pair (2), and if the amplification result is only 199bp bands, determining that the rice sample to be detected is a wx gene homozygote; if the amplification result is only 176bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 199bp and 176bp, the rice sample to be detected is a hybrid strain containing wx genes.
5. The use of the detection primer of claim 1 in wx gene-assisted rice breeding.
6. The use according to claim 5, comprising:
(1) Hybridizing and backcrossing a fine rice line and a glutinous rice material containing wx genes, amplifying wx gene sites of each hybridization or backcross generation individual plant by using the primer pair (1) or the primer pair (2) as claimed in claim 1, and performing polyacrylamide gel electrophoresis on the amplified PCR product;
(2) And (2) amplifying by using the primer (1), if the amplification result contains a 140bp strip, judging that wx genes exist in the sample, and if the amplification result contains a 199bp strip, judging that wx genes exist in the sample, so as to assist in breeding single plants which contain waxy genes and have excellent agronomic characters.
7. The use of the detection primer of claim 1 in determining whether a single plant of a rice selfed segregating population is a wx gene homozygote.
8. Use according to claim 7, characterized in that it comprises:
(1) Extracting DNA of a single plant of a self-copulated segregation population to be detected;
(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer;
(3) Amplifying by using a primer pair (1), if the amplification result only has 140bp bands, determining that the single detection sample plant is a homozygous single plant carrying wx genes, and if the amplification result simultaneously contains two bands of 140bp and 117bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes; amplifying by using a primer pair (2) wx-2, if the amplification result only has 199bp bands, determining that the single detection sample plant is a homozygous plant carrying wx genes, and if the amplification result simultaneously contains two bands of 199bp and 176bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes.
9. The use of the detection primer as claimed in claim 1 in the identification of the purity of a waxy rice variety.
10. The use according to claim 9, comprising:
(1) Extracting DNA of a glutinous rice variety to be detected;
(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer according to claim 1;
(3) Amplifying by using a primer pair (1), if the electrophoresis result has only 140bp bands which are glutinous rice single plants carrying homozygous wx genes, and simultaneously contains two bands of 140bp and 117bp which are glutinous rice single plants carrying heterozygous wx genes, and only contains 117bp bands which are non-glutinous rice single plants mixed;
amplifying by using a primer pair (2), if the electrophoresis result has only 199bp bands which are glutinous rice single plants carrying homozygous wx genes, single plants containing two bands of 199bp and 176bp are glutinous rice single plants carrying heterozygous wx genes, and single plants containing only 176bp bands which are non-glutinous rice single plants are mixed;
(4) The purity of the rice sample is calculated according to the following formula:
Figure FDA0003615707930000031
in the formula:
p-sample purity;
N T the number of seeds to be tested (number of seedlings, number of plants);
N D the number of seeds (number of seedlings, number of plants) determined as a mixed plant.
CN202210443780.1A 2022-04-26 2022-04-26 Detection primer and detection kit for rice wx gene and application of detection kit Pending CN115491429A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210443780.1A CN115491429A (en) 2022-04-26 2022-04-26 Detection primer and detection kit for rice wx gene and application of detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210443780.1A CN115491429A (en) 2022-04-26 2022-04-26 Detection primer and detection kit for rice wx gene and application of detection kit

Publications (1)

Publication Number Publication Date
CN115491429A true CN115491429A (en) 2022-12-20

Family

ID=84465049

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210443780.1A Pending CN115491429A (en) 2022-04-26 2022-04-26 Detection primer and detection kit for rice wx gene and application of detection kit

Country Status (1)

Country Link
CN (1) CN115491429A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116334290A (en) * 2023-04-12 2023-06-27 湖北省农业科学院粮食作物研究所 Primer group and kit for identifying rice functional genes and application of primer group and kit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116334290A (en) * 2023-04-12 2023-06-27 湖北省农业科学院粮食作物研究所 Primer group and kit for identifying rice functional genes and application of primer group and kit
CN116334290B (en) * 2023-04-12 2024-04-05 湖北省农业科学院粮食作物研究所 Primer group and kit for identifying rice functional genes and application of primer group and kit

Similar Documents

Publication Publication Date Title
CN102154281B (en) Molecular marker SIsv0010 closely linked with heading-date gene of millet
CN105256031B (en) Utilize the method and its primer special of high-throughput molecular labeling transformation muskmelon female series
CN106498068B (en) Co-dominant SSR marker closely linked with tobacco TMV resistance gene N and application thereof
CN111719012A (en) dCAPS molecular marker primer pair for identifying dehydration rate genotype of corn kernel and application
CN109609687B (en) KASP marker primer combination for detecting watermelon fusarium wilt resistance and application thereof
CN115491429A (en) Detection primer and detection kit for rice wx gene and application of detection kit
CN112602589B (en) Method for rapid and accurate breeding of semi-waxy japonica rice restorer
CN111334597B (en) SNP (Single nucleotide polymorphism) site and KASP (Kaempferi protein) marker for detecting powdery mildew resistance of watermelon and application thereof
CN116622876B (en) Haplotype molecular marker related to vitamin C content of papaya pulp and application thereof
CN103834647A (en) SSR marker Xgwm537 in close linkage with wheat dwarf gene RhtDC20 and application thereof
CN109593879A (en) Rice medium amylase content gene Wxg1SNP Functional marker and its application
CN112575101B (en) Molecular marker related to resistance of Cucurbita pepo PRSV-W virus disease and application thereof
CN110257553B (en) KASP molecular marker method for identifying rice blast resistance gene Pigm
CN102154284B (en) Molecular marker SIsv0408 capable of being closely linked to Setaria italica pollen color gene
CN111254216A (en) CAPS marker and method for identifying soybean RN type CMS restoring gene
CN116837137B (en) SSR molecular marker closely linked with watermelon peel and macula lutea traits, detection method and application
CN114990251B (en) Molecular marker closely linked with rape methylselenocysteine content trait QTL and application thereof
KR101298480B1 (en) Method for developing gene specific molecular markers using promoter region polymorphism
CN116397041B (en) InDel marker SiDmr6 closely linked with millet saline-alkali sensitivity, primer and application thereof
KR101892461B1 (en) Molecular marker for discrimination genetic male sterility gene and detection method using the same
CN114807425A (en) InDel molecular marker closely linked with tomato male sterility gene ms-7, primer and application thereof
CN114854901A (en) Molecular marker and primer co-separated from bitter gourd full-female shape and application thereof
KR101794699B1 (en) Primer set for Jangdaok identification and method for identification
CN106609297B (en) Molecular marker for powdery mildew of pumpkin and primer pair for identifying powdery mildew resistance traits of pumpkin
KR101700611B1 (en) Primer set for Eolrukchal 1 identification and method for identification

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination