CN108676796B - Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice - Google Patents

Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice Download PDF

Info

Publication number
CN108676796B
CN108676796B CN201810042202.0A CN201810042202A CN108676796B CN 108676796 B CN108676796 B CN 108676796B CN 201810042202 A CN201810042202 A CN 201810042202A CN 108676796 B CN108676796 B CN 108676796B
Authority
CN
China
Prior art keywords
ossnb
rice
grain
molecular marker
indel
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.)
Active
Application number
CN201810042202.0A
Other languages
Chinese (zh)
Other versions
CN108676796A (en
Inventor
马孝松
余舜武
刘鸿艳
罗利军
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.)
SHANGHAI AGROBIOLOGICAL GENE CENTER
Original Assignee
SHANGHAI AGROBIOLOGICAL GENE CENTER
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 SHANGHAI AGROBIOLOGICAL GENE CENTER filed Critical SHANGHAI AGROBIOLOGICAL GENE CENTER
Priority to CN201810042202.0A priority Critical patent/CN108676796B/en
Publication of CN108676796A publication Critical patent/CN108676796A/en
Application granted granted Critical
Publication of CN108676796B publication Critical patent/CN108676796B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Landscapes

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

Abstract

The invention relates to a molecular marker of a rice grain type gene OsSNB and application thereof, wherein the method for breeding large-grain rice by applying the molecular marker OsSNB _ indel in an auxiliary way is to take rice genome DNA as a template and OsSNB _ indel as a primer for PCR amplification, detect an amplification product by 1% agarose gel electrophoresis, and if a single band of 441bp exists, indicate that a detection sample contains the allele type of a large-grain variety IRAT 109; if a single band of 215bp exists, the genotype of the detection sample is Nipponbare allele; if the two bands of 215bp and 441bp exist, the detection sample is indicated to be the OsSNB heterozygous genotype. The marker has no genetic exchange through sequence difference design, and has high accuracy; the kit is directly used for agarose gel electrophoresis detection, and is simpler and more convenient; the marker is a codominant marker, homozygous and heterozygous individuals can be detected, the breeding period is shortened, and the efficiency is improved.

Description

Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice
Technical Field
The invention relates to the field of molecular genetics, in particular to a molecular marker of a rice grain type gene OsSNB, a molecular marker applied to the molecular marker and an auxiliary selective breeding method for large-grain rice.
Background
The rice is the most important grain crop, more than half of the population in the world takes the rice as staple food, the rice yield factors comprise four basic factors of spike number per unit area, grain number per spike (spike number of flowers per spike), seed setting rate and grain weight, and the grain weight is mainly determined by the factors of grain length, grain width, grain thickness and the like. Therefore, breeding and planting large-grain rice varieties to increase the grain weight of rice is one of the important ways to increase the yield of rice. Therefore, digging and utilizing the large grain gene to improve the rice grain type is an important way for improving the rice yield.
Rice grain type is a typical quantitative trait, and is controlled by multiple genes. Molecular marker assisted selection is a modern breeding technology which utilizes markers closely linked with grain type genes or functional markers in genes to select target traits by combining genotype and phenotype identification in later generations. The method not only can greatly shorten the breeding period and improve the breeding efficiency, but also saves a large amount of labor and material cost. The OsSNB large grain allelic gene type is derived from a few japonica rice varieties and is located on No. 7 chromosome of rice, and the varieties used in large area in the current production are all small grain variety Nipponbare allelic gene types, so that the OsSNB large grain allelic gene type has great application value in the aspects of improving the rice grain type and increasing the rice yield, and the development of the functional marker of the OsSNB gene has important significance for fully utilizing the gene, further improving the rice grain type and increasing the rice yield.
Disclosure of Invention
The invention aims to solve the technical problem of providing a molecular marker of a rice grain type gene OsSNB, and a positive primer and a negative primer and application thereof.
The technical scheme provided for solving the technical problems is to provide a molecular marker of a rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice, wherein the molecular marker is numbered as OsSNB _ indel, and the forward and reverse primer sequences of the molecular marker OsSNB _ indel are as follows:
OsSNB_indel F：5’-TAATACACCCTACATACACAAAACCA-3’；
OsSNB_indel R：5’-GTGAGGGAGACGAAGGGTAT-3’。
in a further scheme, the molecular marker OsSNB _ indel is used for identifying the rice grain type gene OsSNB haplotype.
In a further scheme, the molecular marker assisted selective breeding for the rice grain type gene OsSNB comprises the following experimental steps:
i. extracting a genome of a test rice sample;
ii. Performing PCR amplification on the genome extracted in the step i by using a positive primer and a negative primer combination of the molecular marker OsSNB _ indel to obtain an amplification product;
iii, detecting the amplification product through 1% agarose gel electrophoresis, wherein if a single band of 215bp exists, the sample is of a Nipponbare allelic type; when a single band of 441bp exists, the sample contains the allele type of the large-particle variety IRAT 109; when the two bands of 215bp and 441bp exist, the sample is heterozygous for the OsSNB gene.
In a further scheme, paired primers are synthesized within 300bp of the upstream and downstream of the corresponding genome position, and the amplified sequence segment contains the molecular marker of the rice grain type gene OsSNB.
The invention has the beneficial effects that:
the invention designs the functional molecular marker of the OsSNB gene by utilizing the nucleotide sequence difference, has no genetic exchange and high accuracy; the amplified fragments with larger difference can be directly used for agarose gel electrophoresis detection, so that the method is simpler and more convenient; the marker is a codominant marker, homozygous and heterozygous individuals can be detected, the breeding period is shortened, and the efficiency is improved.
Drawings
FIG. 1 is an electrophoretic map of the molecular OsSNB _ indel marker of the present invention on 8 rice varieties. Note: wherein 1 to 8 respectively represent rice varieties of nippon fine (grain length is 7.29mm, grain width is 3.36mm), zhenshan 97B (grain length is 7.82mm, grain width is 3.36mm), IRAT109 (grain length is 8.49mm, grain width is 3.73mm), CICA4 (grain length is 8.09mm, grain width is 4.11mm), yunnan 8 (grain length is 9.85mm, grain width is 4.11mm), mikanka (grain length is 10.09mm, grain width is 4.45mm), late upland rice (grain length is 7.87mm, grain width is 4.12mm), millenna grain (grain length is 9.13mm, grain width is 4.06 mm); and M represents a D2000 Marker.
Detailed Description
The molecular marker of rice grain type gene OsSNB and its application are further explained below with reference to the accompanying drawings.
Example 1: development of rice grain type gene OsSNB gene molecular marker
(1) And test materials: large grain variety: IRAT 109; granule rice variety: nipponbare.
(2) The method for extracting the rice genome DNA refers to the method in Loujiujun (2006). In a mortar, 20mg of leaf were taken, and 400. mu.l of 1.5 XCTAB (1.5% CTAB, 75mmol/L Tris-HCl, 15mmol/L EDTA, 1.05mol/L NaCl, pH 8.0) was added thereto and ground into a slurry, and then 400. mu.l of 1.5 XCTAB was added thereto, and the slurry was sucked into a 1.5ml centrifugal tube, followed by addition of550 μ l of chloroform: isoamyl alcohol (24:1), mixing, centrifuging at 12000r/min for 6min, taking the supernatant to another centrifuge tube, adding precooled isopropanol with the same volume, centrifuging at 12000r/min for 8min, taking the supernatant, drying the precipitate, and finally adding 400 mu l ddH2Dissolving O to obtain the final product. (Compare of three rice genome DNA rapid extraction methods of Loujun, Chengliang, Roulijun [. J ]]Molecular plant breeding, 2006,3(5):749-
(3) And development of a gene molecular marker OsSNB _ indel: the OsSNB is located on the 7 th chromosome of rice, the accession number of the reference genome of Nipponbare is AP014963.1, and the 231bp is inserted in the upstream-357 site of the initiation codon of the OsSNB in the gene sequence of the large-grain variety IRAT 109.
Table 1: base comparison of Nipponbare with IRAT109
Figure GDA0003145175010000041
Figure GDA0003145175010000051
(4) And designing a Primer OsSNB _ indel by using Primer Premier 5.0 software according to the nucleotide sequence difference, wherein the sequence of a positive Primer and a reverse Primer is as follows:
OsSNB_indel F：5’-TAATACACCCTACATACACAAAACCA-3’
OsSNB_indel R：5’-GTGAGGGAGACGAAGGGTAT-3’。
example 2: parent polymorphism detection of OsSNB-indel gene molecular marker
(1) And extracting the genome of the tested rice variety by a conventional CTAB method. The varieties of the tested rice are respectively as follows: nipponbare, Zhenshan 97B, IRAT109, CICA4, Yunlong No. 8, Mirabka, late upland rice and millettia.
(2) And performing PCR amplification on the rice variety to be tested by using the functional marker OsSNB _ indel, wherein the PCR amplification system is as follows: 20ng of rice genome DNA template, 10uL of Taq PCR Mastermix (Tiangen Biochemical technology (Beijing) Co., Ltd.), 1uL of each of 10uM primers and 10uM primers, and dd H supplement2O to 20 uL. The PCR reaction program is:pre-denaturation at 95 ℃ for 5min, followed by 32 cycles at 95 ℃ for 30s, 54 ℃ for 30s, 72 ℃ for 1min, and final extension at 72 ℃ for 5 min.
(3) And taking 8uL of the PCR product to load the PCR product on a 1% agarose gel for electrophoresis detection. The results are shown in figure 1, a single strip of 215bp is amplified from 97B of Nipponbare and Zhenshan of the small-grain rice variety, a single strip of 441bp is amplified from other large-grain rice varieties, the electrophoresis strips are clear, and the difference is obvious.
Example 3: verification and application of OsSNB gene molecular marker
Hybrid F of rice maintainer line Zhenshan 97B and large-grain variety IRAT109 by using OsSNB gene molecular marker OsSNB _ indel1Carrying out PCR amplification on the genome DNA of the generation individual plant, carrying out electrophoresis typing on the product in 1% agarose gel, detecting two bands of 215bp and 441bp in the hybrid F1 individual plant, and indicating that the detection samples are OsSNB gene heterozygosis and the grain type is obviously larger than that of Zhenshan 97B.
Using OsSNB gene molecular marker OsSNB _ indel to perform F detection on 188 IRAT109 and Zhenshan 97B10PCR amplification is carried out on the genome DNA of a single generation recombinant inbred line, electrophoresis detection is carried out on products in 1% agarose gel, 117 strains respectively contain the Zhenshan 97B allele type, 71 strains contain the allele type consistent with the IRAT109, and the grain type identification result shows that the grain type of the strains containing the Zhenshan 97B allele type is extremely obviously smaller than that of the strains containing the IRAT109 allele type.
226 parts of rice germplasm resources, namely 128 parts of indica rice and 98 parts of japonica rice, are subjected to PCR amplification by using OsSNB gene molecular marker OsSNB _ indel, and products are subjected to electrophoresis detection in 1% agarose gel, so that 215bp bands are found in 204 parts of germplasm resources, 22 parts of japonica type germplasm resources are 441bp bands, and the grain width of the germplasm resources containing 441bp is remarkably larger than that of the germplasm resources containing 215bp (P germplasm resources containing 441 bp)<10-6) The grain length has no statistical difference, and the domestic main cultivar parents (Minghui 63, Zhenshan 97B, Baohe 7B, 9311, Hanhui No. 3, Shanghaihong 7B, C418, Zhong413, etc.) are all 215bp band types.
Therefore, the marker can be used for identifying the rice grain type gene OsSNB and/or assisting in selective breeding of large-grain rice.
The present invention is not limited to the above embodiments, and the technical solutions of the above embodiments of the present invention may be combined with each other in a crossing manner to form a new technical solution, and all technical solutions formed by using equivalent substitutions fall within the scope of the present invention.
Sequence listing
<120> molecular marker of rice grain type gene OsSNB and application thereof
<140> 201810042202.0
<141> 2018-01-17
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> sequence of SNB in IRAT109
<212> RNA
<213> Rice Genes
<400> 1
<210> 2
<211> SNB sequence in Nipponbare
<212> RNA
<213> Rice Genes
<400> 2

Claims (4)

1. A molecular marker of a rice grain type gene OsSNB is used for auxiliary selective breeding of large-grain rice, and is numbered as OsSNB _ indel, and is characterized in that forward and reverse primer sequences of the molecular marker OsSNB _ indel are as follows:
OsSNB_indel F：5’-TAATACACCCTACATACACAAAACCA-3’；
OsSNB_indel R：5’-GTGAGGGAGACGAAGGGTAT-3’。
2. the molecular marker of the rice grain type gene OsSNB as claimed in claim 1 is used for auxiliary selective breeding of large-grain rice, and is characterized in that: the molecular marker OsSNB _ indel is used for identifying the rice grain type gene OsSNB haplotype.
3. The molecular marker of the rice grain type gene OsSNB as claimed in claim 2 is used for the auxiliary selective breeding of large-grain rice, and is characterized in that the molecular marker auxiliary selective breeding of the rice grain type gene OsSNB comprises the following experimental steps:
i. extracting a genome of a test rice sample;
ii. Performing PCR amplification on the genome extracted in the step i by using a positive primer and a negative primer combination of the molecular marker OsSNB _ indel to obtain an amplification product;
iii, detecting the amplification product through 1% agarose gel electrophoresis, wherein if a single band of 215bp exists, the sample is of a Nipponbare allelic type; when a single band of 441bp exists, the sample contains the allele type of the large-particle variety IRAT 109; when the two bands of 215bp and 441bp exist, the sample is heterozygous for the OsSNB gene.
4. The molecular marker of the rice grain type gene OsSNB as claimed in claim 1 is used for auxiliary selective breeding of large-grain rice, and is characterized in that: paired primers are synthesized in 300bp upstream and downstream of the corresponding genome position, and the amplified sequence segment contains the molecular marker of the rice grain type gene OsSNB.
CN201810042202.0A 2018-01-17 2018-01-17 Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice Active CN108676796B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810042202.0A CN108676796B (en) 2018-01-17 2018-01-17 Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810042202.0A CN108676796B (en) 2018-01-17 2018-01-17 Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice

Publications (2)

Publication Number Publication Date
CN108676796A CN108676796A (en) 2018-10-19
CN108676796B true CN108676796B (en) 2021-08-20

Family

ID=63799286

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810042202.0A Active CN108676796B (en) 2018-01-17 2018-01-17 Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice

Country Status (1)

Country Link
CN (1) CN108676796B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864266B (en) * 2018-07-11 2020-08-18 中国农业大学 Protein SSH1 related to rice graininess and grain type as well as encoding gene and application thereof
CN113736898A (en) * 2021-08-09 2021-12-03 上海市农业生物基因中心 Molecular marker of rice heading stage regulation gene OsGI and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174462A1 (en) * 2011-06-16 2012-12-20 Ceres, Inc. Sorghum with increased sucrose purity
CN106701778A (en) * 2015-11-16 2017-05-24 华中农业大学 Method for increasing grain number per ear and reducing plant height by use of rice SNB genes
CN106754957A (en) * 2016-12-05 2017-05-31 上海市农业生物基因中心 The application of OsSCAMP13 genes and encoding proteins with resistance and acquisition methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174462A1 (en) * 2011-06-16 2012-12-20 Ceres, Inc. Sorghum with increased sucrose purity
CN106701778A (en) * 2015-11-16 2017-05-24 华中农业大学 Method for increasing grain number per ear and reducing plant height by use of rice SNB genes
CN106754957A (en) * 2016-12-05 2017-05-31 上海市农业生物基因中心 The application of OsSCAMP13 genes and encoding proteins with resistance and acquisition methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Expression character of rice SUPERNUMERARY BRACT (OsSNB) in response to abiotic stress and hormone;Zhang shaoxuan等;《Journal of Agricultural Science and Technology (Beijing)》;20151231;第17卷(第1期);第42-48页 *
利用GS3基因功能性分子标记改良水稻粒型的研究;李扬等;《上海农业学报》;20160101;第32卷(第1期);第1-5页 *

Also Published As

Publication number Publication date
CN108676796A (en) 2018-10-19

Similar Documents

Publication Publication Date Title
BRPI0812744B1 (en) METHODS FOR SEQUENCE-TARGETED MOLECULAR IMPROVEMENT
CN107058516A (en) The molecular labeling of wide gene GW2 of rice grain a kind of and its application
CN110684858B (en) Molecular marker of rice slender grain type gene and application thereof
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
CN108676796B (en) Molecular marker of rice grain type gene OsSNB for auxiliary selective breeding of large-grain rice
KR102461815B1 (en) TaqMan molecular marker for identifying HMW-GS and use thereof
WO2017139544A9 (en) Qtls associated with and methods for identifying lodging resistance in soybean
CN111471790B (en) Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-7D.1 and application thereof
CN114717352B (en) Molecular marker of rice high temperature resistant regulatory gene Hsp70 and application thereof
CN112609018B (en) SNP molecular marker of rice grain type related gene GLW2 and application thereof
CN115786564A (en) Rice Pi-ta and Ptr dominant functional molecular marker and application thereof
CN112143830B (en) Molecular marker of rice sword leaf width regulation gene NAL1 and application thereof
CN111118192B (en) KASP molecular marker of wheat ear base small ear fruition main effect QTL and application thereof
JP4101493B2 (en) Novel primer set for detecting nucleic acid markers derived from barley chromosomes in the background of wheat and use thereof
CN112980985B (en) PCR primer group for identifying or screening cabbage hybrid lethal parent type 1 and application thereof
CN116855592B (en) Method for identifying, screening or controlling thousand seed weight of wheat based on A583G SNP locus
KR102198575B1 (en) Marker for predicting leaf rolling in rice using QTL associated with leaf rolling and uses thereof
CN116640878B (en) Molecular marker of wheat floret number QTL per spike developed based on novel mode of multi-single-combined-standard-evidence and application thereof
CN111826461B (en) Development and application of molecular marker of sorghum miscanthus
CN116814841B (en) Primer group for identifying rice black brown glume gene HK4, and method and application thereof
CN116397040B (en) Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same
RU2740404C1 (en) Dna-marker for selection of spring barley varieties, resistant to spotting atrobrunneis hordeum
Wu et al. Genome mapping, markers and QTLs
CN113736898A (en) Molecular marker of rice heading stage regulation gene OsGI and application thereof

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
GR01 Patent grant
GR01 Patent grant