CN105671042A - Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker - Google Patents

Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker Download PDF

Info

Publication number
CN105671042A
CN105671042A CN201610180607.1A CN201610180607A CN105671042A CN 105671042 A CN105671042 A CN 105671042A CN 201610180607 A CN201610180607 A CN 201610180607A CN 105671042 A CN105671042 A CN 105671042A
Authority
CN
China
Prior art keywords
ssiii
rice
molecular marker
amylose content
fine
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
CN201610180607.1A
Other languages
Chinese (zh)
Inventor
饶玉春
徐江民
钱前
曾大力
马路
肖飒清
胡瑚倩
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.)
Zhejiang Normal University CJNU
Original Assignee
Zhejiang Normal University CJNU
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 Zhejiang Normal University CJNU filed Critical Zhejiang Normal University CJNU
Priority to CN201610180607.1A priority Critical patent/CN105671042A/en
Publication of CN105671042A publication Critical patent/CN105671042A/en
Pending legal-status Critical Current

Links

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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

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 discloses a molecular marker SSIII-1-m for rice amylose content micro-control genes SSIII-1. The molecular marker is characterized in that paddy rice is used as a species, primers of the molecular marker are selectively primer pairs, nucleotide sequences are 5'->3', the molecular marker SSIII-1-m in a forward direction is shown as AAGAAGGGAAGGGAGTCAGC, and the molecular marker SSIII-1-m in a reverse direction is shown as GCCATCTCCATTGCCAGC. The invention further discloses application of the molecular marker SSIII-1-m. The molecular marker SSIII-1-m can be used for identifying rice amylose contents of the paddy rice and/or assisting in selectively breeding progenies of the paddy rice. Single plants, with banding patterns consistent with Nipponbare banding patterns, of the progenies are selected for breeding when the progenies are progenies of Nipponbare indica rice.

Description

Rice amylose content micro-control gene SSIII-1 molecular marker and application
Technical field
The invention belongs to agricultural biotechnology engineering, particularly to the molecular marker relevant with rice grain amylose content controlling gene SSIII-1 and application thereof.
Background technology
High yield, high-quality are always up the target of rice breeding long-sought. Through long-term effort, the especially utilization of hybrid paddy rice technology, China achieves universally acknowledged achievement in Rice Production. But owing to the past places the problem of food and clothing how solving people above the other things always, so the emphasis of rice breeding work is largely focused on the cultivation of new high-yielding rice varieties, thus causing that the breeding of fine quality rice is seriously delayed, the particularly general deviation of hybrid paddy rice quality of some high yields.
Causing that grain quality improving is in progress another slower main cause is complexity and the limitation of traditional breeding method means of rice quality heredity. The quality trait of rice includes all many-sides such as exterior quality, processing quality, cooking quality, nutritional quality and Cooking Quality, and Rice Cooking is the most important index evaluating rice quality. Cooking quality refers to the characteristic that rice shows in digestion process, mainly by amylose content (AmyloseCOntent, AC), gel consistence (GelCOnsistency, GC), gelatinization point (GelatinizationTEmperature, GT) three physical and chemical indexs evaluate, and wherein AC affects the topmost physical and chemical index of rice quality. Breeding scholar and geneticist have done substantial amounts of work to seek the genetic base of rice AC, but the result of different experiments room has bigger difference. The research of early stage shows that rice AC is controlled by a major gene resistance, and is subject to the regulation and control [1,2] of other minor effects QTL. Research subsequently find Waxy (Wx) for rice AC number there is conclusive effect, it is possible to control the major gene resistance [3-5] of AC exactly. Except contaminating and 1 main effect QTL detected the 6th on body, minor effect QTL number and position that different researchs detect are very inconsistent. Such as, Tan etc. detects two minor effect QTLs [6] the 1st, on 2 chromosomes, and He etc. detects a minor effect QTL [6] at the 5th chromosome, and Aluko etc. detects minor effect QTLs [7] respectively on the 3rd and 8 chromosomes. The research such as Huang Zuliu finds except the 6th chromosomal Wx gene loci, also detects that a main effect QTL controlling rice AC at the 3rd chromosome;Other 5 minor effect QTLs lay respectively on the 4th, 4,6,9,11 chromosomal different seats [8]. Other laboratorys the 4th, 6,7 chromosomes also detected that the QTLs [9] controlling AC. Wu Changming etc. do not find the QTL site relevant with AC on the 6th chromosome, simply find 5 QTL site [10] on the 1st, 7,8,9,12 chromosome.
Cause that grain quality improving another slower main cause of progress is the limitation in traditional breeding method means. Favo(u)rable target character is mainly oriented selection and fixing by traditional breeding way, cultivates improved Varieties, and this tool bears the character of much blindness and unpredictability [11]. Further, the method for individual selection is that the economical character meeting breeding objective is directly selected, and what namely select is individual Phenotype rather than genotype. Owing to having one between gene because of multiple-effect, how because of the effect of an effect, controlling gene and modifying gene etc., often there is larger difference in individual Phenotype and genotype, the accuracy carrying out individual selection thereby through field phenotypic character is poor. Molecular marker assisted selection (Marker-assistedSElection, MAS) technology provides new approach to rice breeding, and combining with traditional breeding technology is greatly improved breeding efficiency, shortens breeding cycle. The core of MAS is the Phenotypic Selection in conventional breeding to be converted into genotype select, it directly reflects the sequence difference of DNA, not by the impact of gene expression, result reliability is strong, and not by the impact [12] of the growth and development stage of plant and environmental condition.
The list of references above related to is as follows:
1.BollichC.W., BD.Inheritanceofamyloseintwohybridpopulationsofrice.Cere alChem.1973,50,631-636 (BollichC.W., BD. the heredity at two hybrid Populations of amylose content. cereal chemistry .1973,50:631-636);
2.McKenzieK.R., JN.Geneticanalysisofamylosecontent, alkalispreadingscore, andgraindimensionsinrice.CropSci.1983,23,306-311 (genetic analysis of McKenzieK.R., JN. content of amylose in rice, caustic SCC and Grain Morphology. crop science .1983,23:306-311);
3.SanoY.Differentialregulationofwaxygeneexpressioninrice endosperm.Theor.Appl.Genet.1984,68,467-473 (the difference regulation and control that SanoY. paddy endosperm waxy gene is expressed. theoretical and application heredity .1984,68:467-473);
4.KumarI.K., GS.Juliano, BO.Geneticanalysisofwaxylocusinrice (OryzasativaL.) .Theor.Appl.Genet.1987,73,481-488 (KumarI.K., the genetic analysis in GS.Juliano, BO. paddy endosperm waxy gene site. theoretical and application heredity .1987,73:481-488);
5.KumarI.K., GS.Inheritanceofamylosecontentinrice (OryzasativaL.) .Euphytica1988,38,261-269 (KumarI.K., GS. the heredity of content of amylose in rice. European plant .1988,38:261-269);
6.TanY.F., LiJ.X., YuS.B., etal.Thethreeimportanttraitsforcookingandeatingqualityof ricegrainsarecontrolledbyasinglelocusinanelitericehybrid, Shanyou63.Theor.Appl.Genet.1999,99,642-648 (TanY.F., LiJ.X., YuS.B., Deng. three important indicators of excellent hybrid paddy rice Shanyou 63 rice cooking and eating quality are controlled by unit point. theoretical and application heredity .1999,99:642-648);
7.AlukoG., MartinezC., TohmeJ., etal.QTLmappingofgrainqualitytraitsfromtheinterspecificc rossOryzasativaxO.glaberrima.Theor.Appl.Genet.2004,109,6 30-639 (AlukoG., MartinezC., TohmeJ., etc. Rice Kernel character QTL location in Asian Cultivated Rice and Oryza glaberrima Steud intervarietal hybridization colony. theoretical and application heredity .2004,109:630-639.);
8. yellow ancestral six, and the Molecular mapping of Tan Xuelin, TragoonrungS., etal. rice grain amylose content gene locus. Acta Agronomica Sinica 2000,26,777-782;
9.LancerasJ.C., HuangZ.L., NaivikulO., etal.MappingofgenesforcookingandeatingqualitiesinThaijas minerice (KDML105) .DNARes.2000,7,93-101 (LancerasJ.C., HuangZ.L., NaivikulO., etc. the gene mapping .DNA of THAI fragrant rice cooking and eating quality studies .2000,7:93-101);
10. Wu is kept burning day and night, Sun Chuanqing, Chen Liang, etc. the QTL of content of amylose in rice and indica japonicadifferentiation and interrelationship study thereof. China Agricultural University journal 2000,5,6-11;
11. Li Zhi health. the strategy of China's Rice molecular breeding plan. Molecular Plant Breeding 2005,3,603-608;
12. Feng builds up. Molecular Marker Assisted Selection Technology application on rice breeding. China agronomy circular 2006,22,43-47.
Separately, the invention " Molecular marker of rice amylose content micro-control gene GBSSII and application " of the patent No. 201310586095.5, 201310585846.1 invention " molecular marker of rice amylose content micro-control gene SSIVb and application ", 201310581339.0 invention " rice amylose content micro-control gene AGPS2a molecular marker and application " give some molecular markers relevant with rice starch synthetic gene, above-mentioned it is labeled as GBSSII-m, SSIVb-m and AGPS2a-m, these labellings are minor gene, reality finds, minor gene polymerization is more many, choice accuracy will be more high, so developing more amylose content minor gene or the bigger gene of contribution rate, it is particularly important.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of molecular marker relevant with rice starch synthetic gene SSIII-1 and application thereof, the genetic marker that molecular marker SSIII-1-m is rice amylose content micro-control gene SSIII-1 of gained of the present invention, identifies and/or its offspring's assisted selection for amylose content of rice.
In order to solve above-mentioned technical problem, the present invention provides a kind of genetic marker with rice amylose content micro-control gene SSIII-1, and using Oryza sativa L. as species, this molecular marker primer is selected from following primer pair, and nucleotides sequence therein is classified as 5 ' → 3 ',
SSIII-1-m forward: AAGAAGGGAAGGGAGTCAGC
Reverse: GCCATCTCCATTGCCAGC.
The present invention also provides the purposes of above-mentioned molecular marker simultaneously: identify and/or its offspring's assisted selection for amylose content of rice.
The improvement of purposes as the molecular marker SSIII-1-m of the present invention: when for the offspring of Japan fine (japonica rice) and long-grained nonglutinous rice (be such as special blue or green, 93-11), the individual plant that in selection offspring, banding pattern is consistent with Japan fine banding pattern is used for breeding.
Remarks illustrate:
Primer SSIII-1-mPCR expands the molecular marker SSIII-1-m of gained sequence in Japan is fine: AAGAAGGGAAGGGAGTCAGCTCTGAGGAGGGAGAGGGAGAGGGAGGGAGGAGGAGG GGATCAGGGGGAGAGGAGGGAGGTTTTGGCGCTGGCAATGGAGATGGC;
Primer SSIII-1-mPCR expands the molecular marker SSIII-1-m of gained sequence in special green grass or young crops: AAGAAGGGAAGGGAGTCAGCTCTGAGGAGGGAGAGGGAGAGGGAGGAGGGAGGAGG AGGGGATCAGGGGGAGAGGAGGGAGGTTTTGGCGCTGGCAATGGAGATGGC.
The development approach of the molecular marker of the present invention, comprises the following steps:
1), carry out hybridizing, backcrossing and selfing with the special green grass or young crops as high amylose starches so that japonica rice variety Japan is fine as low amylose content gene donor parents, thus obtaining the individual plant as offspring's rice low amylose content;
2), parental rice seedling and filial generation seedling genomic DNA are extracted by CTAB (cetyltriethylammonium bromide, HexadecyltrimethylammoniumBromide) method;
3), Indel (insertion/deletion fragment, insertion/deletions) molecule labelling method is adopted to carry out the screening of rice low amylose content gene labelling;
4) an Indel molecular marker SSIII-1-m, is developed.
The molecular marker SSIII-1-m relevant with rice low amylose content, specifically obtains by following method:
1) nucleotide sequence, according to gene SSIII-1, design, development Indel molecular marker, the polymorphism blue or green for detecting the warm and fine high amylose content spy of low amylose content Japan; By checking order to further determine that the sequence interval for primer SSIII-1-m difference between the special green grass or young crops of the warm and fine high amylose content of Japan of low amylose content; By hybridizing, backcrossing and selfing incorporation of markings assisted Selection, it is thus achieved that the Oryza sativa L. new germ plasm of the low amylose content of special blue or green background;
2), parental rice seedling and filial generation seedling genomic DNA are extracted by CTAB method;
3) genetic marker that Indel molecule labelling method carries out rice low amylose content, is adopted to screen the Oryza sativa L. new germ plasm of low amylose content;
4), an Indel molecular marker SSIII-1-m is identified, through polymorphic detection, it has been found that it is associated with rice grain amylose content.
Indel molecular marker SSIII-1-m is adopted to carry out the method for rice grain amylose content screening specifically:
(1), Indel is marked at the DNA polymorphism analysis between the warm and fine special green grass or young crops of high and low rice grain amylose content kind Japan:
Nucleotide sequence according to gene SSIII-1, design, development Indel molecular marker SSIII-1-m, it is used for the polymorphism detecting between the special green grass or young crops of the warm and fine high amylose content of Japan of low amylose content. Primer entrusts the synthesis of Shanghai Shen Neng betting office, and in the enterprising performing PCR amplification of PTC-225PCR instrument, PCR reaction system is: 20ng/ul oryza sativa genomic dna 1ul, 10 × PCRBuffer2.0ul, 25mMMgCl22.0ul, 2mMdNTP2.0ul, 10uM primer 2 .0ul, 5U/ulTaqDNA polymerase 0.2ul, ddH2O10.8ul, total system 20ul. Response procedures: 95 DEG C of degeneration 5 minutes; 94 DEG C of degeneration 1 minute, anneal 1 minute for 55 DEG C, and 72 DEG C extend 1 minute, 40 circulations; 72 DEG C of polishings 10 minutes; Product detect: containing 0.5%ug/ulEB 4.0% agarose gel electrophoresis, under uviol lamp observe and film recording result.
(2), the sequence of interval of Indel labelling SSIII-1-m genome sequence difference between the warm and fine special green grass or young crops of rice grain amylose content kind low, high Japan:
According to the Indel molecular marker SSIII-1-m obtained, for the genome sequence that the warm and fine high amylose content kind spy of pcr amplification low amylose content kind Japan is blue or green, pcr amplification product entrusts Shanghai Ying Jun Bioisystech Co., Ltd to carry out sequencing analysis.Pcr amplification carries out with reference to above-mentioned (1), and reclaiming of PCR primer selects the PCR primer of Beijing hundred Tyke Bioisystech Co., Ltd exploitation to reclaim test kit (centrifugal column type, catalog number (Cat.No.): DP1403).
(3) Indel labelling SSIII-1-m, is utilized to carry out the assisted selection of low amylose content
The genetic donor parent Japan of low amylose content is fine, hybridize with the rice variety spy green grass or young crops of high amylose content, by backcrossing, selfing incorporation of markings assisted Selection, the fine low amylose content gene SSIII-1 of Japan is imported in the special green grass or young crops of high amylose content, select the individual plant that the fine banding pattern of banding pattern and Japan is consistent in segregating population for breeding improvement, it is thus achieved that the material with Japanese fine SSIII-1 gene of some parts of special blue or green backgrounds; Gather in the crops the seed tied on these plant, detect its amylose content, it has been found that its amylose content significantly reduces.
Amylose content height is the main factor that rice quality is inferior. The present invention adopts molecular biology method fine for material with the Japan of low amylose content, develops and screen new and the stable molecular marker that can reduce rice grain amylose content and method thereof, for the assisted selection of fine quality rice; Owing to the material of institute can effectively reduce the amylose content of general rice, the improvement of China's rice quality is had universality by it.
The invention Indel labelling SSIII-1-m of rice starch synthesis related gene SSIII-1. Profit is in this way, not only overcome the shortcomings such as conventional breeding methods required time cycle length, targetedly the fine low amylose content gene SSIII-1 of Japan can be selected to obtain and on purpose carry out the polymerization of multiple high-quality in laboratory, thus cultivating the new rice variety with high-quality. In the present invention, when gained plant occurs Japanese fine band after testing, we judge that it belongs to the Oryza sativa L. of low amylose content; When gained plant occur after testing special blue or green band or occur simultaneously special blue or green+Japan's fine band time, we judge that it belongs to the Oryza sativa L. of high amylose content.
Labelling between the applicable most long-grained nonglutinous rice of the present invention (such as special blue or green, 93-11) and japonica rice (as fine in Japan) selects.
Therefore, result of the present invention is significant in Oryza sativa L. quality breeding is put into practice; Its advantage is specifically summarized as follows:
(1) molecular marker that rice grain amylose content can be regulated and controled of the present invention, be by the fine hybridization blue or green with the long-grained nonglutinous rice spy of high amylose content of the japonica rice of low amylose content Japan, backcross, in selfing screening obtain, rice grain amylose content can be significantly reduced, and stable existence, can be used for the assisted selection of fine quality rice;
(2) present invention is based on the nucleotide sequence development of rice starch synthetic gene SSIII-1 and the Indel molecular marker that obtains, compared with amylose content molecular marker GBSSII-m, SSIVb-m and AGPS2a-m relevant with other, the labelling of the present invention is positioned on the chromosome that Oryza sativa L. is different, and the effect that labelling of the present invention screens is more preferably, and greatly improve the efficiency of assisted Selection, that is, the labelling of the present invention can make the effect of the assisted selection of rice low amylose content more preferably.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the Indel labelling SSIII-1-m electrophoretic band figure the long-grained nonglutinous rice spy green grass or young crops of the warm and fine high amylose content of japonica rice Japan of low amylose content;
Fig. 2 is the pcr amplification product of primer SSIII-1-m sequence difference between the warm and fine special green grass or young crops of Japan;
Fig. 3 is the Indel labelling SSIII-1-m special blue or green electrophoretic band figure identifying the 12 parts of low amylose contents obtained;
Fig. 4 is the amylose content of 12 parts of materials of labelling SSIII-1-m assist-breeding;
Symbol in above-mentioned Fig. 1, Fig. 3～4 is explained as follows respectively:
1 represents: the long-grained nonglutinous rice spy of high amylose content is blue or green;
2 represent: the japonica rice Japan of low amylose content is fine;
3 represent: fine/special blue or green F1 plant of Japan;
4,5,6,7,8,9,10,11,12,13,14,15 all represent: the warm and fine special blue or green offspring of Japan, obtain after the screening of Indel labelling SSIII-1-m.
Detailed description of the invention
Embodiment 1, use Indel labelling SSIII-1-m identify the polymorphism that the long-grained nonglutinous rice spy of the warm and fine high amylose content of japonica rice Japan of low amylose content is blue or green
Specific practice is: choose rice material Japan warm and fine special blue or green, obtains its F1 with the warm and fine special blue or green hybridization of Japan, utilizes primer SSIII-1-m to identify its polymorphism (Fig. 1).
One, DNA is extracted
1), preparation DNA extraction buffer:
Add the DNA extraction solution (0.35Msorbitol of 1 volume in order successively; 0.1MTris, pH8.2; 0.005MEDTA; All the other are water), karyorhexis liquid (0.2MTris, the pH7.5 of 1 volume; 0.05MEDTA; 2MNaCl; 0.055MCTAB; All the other are water) and 5% (mass concentration) sarkosyl solution (i.e. the aqueous solution of lauroyl-sarcosine sodium) of 0.4 volume; It is eventually adding sodium sulfite, is configured to DNA extraction buffer; The sodium sulfite final concentration of 0.02M in DNA extraction buffer.
The preparation method of above-mentioned DNA extraction solution is: the sorbitol (Sorbitol) of 0.35mol, the Tris (trishydroxymethylaminomethane, pH8.2) of 0.1mol, 0.005mol EDTA (ethylenediaminetetraacetic acid) add water and be settled to 1L.
The preparation method of above-mentioned karyorhexis liquid is: the Tris (trishydroxymethylaminomethane, pH7.5) of 0.2mol, the EDTA (ethylenediaminetetraacetic acid) of 0.05mol, the NaCl (sodium chloride) of 2mol, 0.055mol CTAB (cetyl trimethylammonium bromide) add water and be settled to 1L.
2), above-mentioned Japan is fine, special green grass or young crops, F1 rice leaf be handled as follows respectively:
1., weigh the rice leaf liquid nitrogen grinding powdering of 0.1g, be subsequently adding the above-mentioned steps 1 of 700 μ l) the DNA extraction buffer prepared, 65 DEG C of water-baths 40 minutes. Add the chloroform of 700 μ l again: isoamyl alcohol (volume ratio of 24:1), and mix. 10,000rpm are centrifuged 5 minutes, are transferred in new centrifuge tube by supernatant.
2., after above-mentioned steps is 1. centrifugal, the supernatant of gained adds 2/3～isopropanol of 1 times of volume pre-cooling (to 4 DEG C), mix gently to DNA and precipitate. 13,000rpm are centrifuged 8 minutes, pour out supernatant.
3. the DNA precipitate of above-mentioned steps 2. gained, is washed again with the alcohol 200 μ l of 70% (volumetric concentration).
4., the DNA after above-mentioned washing is dried and is dissolved in 100 μ lTE buffer or pure water.
5., the concentration of DNA sample of ultraviolet spectrophotometry detection above-mentioned steps 4. gained, the integrity of the agarose gel electrophoresis detection DNA of 0.7%. Completely suitable DNA is used for pcr amplification, and incomplete DNA then extracts again, until obtaining complete DNA.
Two, pcr amplification
1), reaction system:
Oryza sativa genomic dna 20ng/ul1ul, 10 × PCRBuffer2.0ul, 25mMMgCl22.0ul, 2mMdNTP2.0ul, 10uM primer each 1.0ul, 5U/ulTaqDNA polymerase 0.2ul, ddH2O10.8ul, total system 20ul.
Described primer is: SSIII-1-m forward: AAGAAGGGAAGGGAGTCAGC
Reverse: GCCATCTCCATTGCCAGC.
2), response procedures:
95 DEG C of degeneration 5 minutes; 94 DEG C of degeneration 1 minute, anneal 1 minute for 55 DEG C, and 72 DEG C extend 1 minute, 40 circulations; 72 DEG C of polishings 10 minutes.
Three, electrophoresis detection
Take amplified production 20ul, agarose gel (containing the 0.5%ug/ulEB) electrophoresis with 4.0%, observe and film recording result under uviol lamp. As shown in Figure 1.
In FIG, the fine band for 104bp of Japan, special green grass or young crops is the band of 107bp, the band that " F1 " is 104bp+107bp.
According to Fig. 1, we can draw following conclusion: Indel molecular marker SSIII-1-m can detect special blue or green and Japan fine between polymorphism, and the fine pcr amplification product fragment of Japan is less than special blue or green, be indicated above SSIII-1-m can be used for special blue or green and Japan fine between Molecular Detection and the marker assisted selection of offspring.
Embodiment 2, use Indel molecular marker SSIII-1-m identify the sequence difference that the long-grained nonglutinous rice spy of the warm and fine high amylose content of japonica rice Japan of low amylose content is blue or green
Specific practice is: the warm and fine special blue or green genomic DNA of Japan is carried out pcr amplification by application Indel molecular marker SSIII-1-m, and amplified production entrusts Shanghai Ying Jun Bioisystech Co., Ltd to check order, and compares the difference (Fig. 2) of its sequence.
One, DNA is extracted
1), preparation DNA extraction buffer:
With embodiment 1.
2), the warm and fine special blue or green rice leaf of above-mentioned Japan is handled as follows respectively:
With embodiment 1.
3) pcr amplification
With embodiment 1.
4) recovery of PCR primer
Reclaiming of PCR primer selects the PCR primer of Beijing hundred Tyke Bioisystech Co., Ltd exploitation to reclaim test kit (centrifugal column type, catalog number (Cat.No.): DP1403), requiring to carry out with reference to the description of product, the PCR primer of recovery entrusts Shanghai Ying Jun Bioisystech Co., Ltd to check order.
According to Fig. 2, we are concluded that the warm and fine special blue or green SSIII-1-m amplified production of Japan exists the base difference (shown in the underscore in Fig. 2) of 3bp, this is that we can use Indel molecular marker SSIII-1-m to detect the reason place of its polymorphism, is also that we can be used for the hereditary basis of its offspring's marker assisted selection.
Embodiment 3, Indel labelling SSIII-1-m is utilized to carry out the assisted selection of low amylose content
Specific practice is: the genetic donor parent Japan of low amylose content is fine, it is sequentially carried out hybridization with the rice variety spy green grass or young crops of high amylose content, backcrosses and selfing, assisted Selection to gained offspring binding molecule labelling SSIII-1-m, selects the individual plant that the fine banding pattern of banding pattern and Japan is consistent in segregating population to be used for breeding improvement.
One, DNA is extracted
1), preparation DNA extraction buffer:
With embodiment 1.
2), above-mentioned Japan is fine, special green grass or young crops, gained offspring rice leaf be handled as follows respectively:
With embodiment 1.
Two, Indel marker detection
1), pcr amplification
With embodiment 1.
2), electrophoresis detection
With embodiment 1.
Three, Indel molecular marker SSIII-1-m carries out the assisted selection of low amylose content
The fine common rice variety spy green grass or young crops with high amylose content of genetic donor japonica rice variety Japan of low amylose content hybridizes, backcross and selfing, the assisted Selection of binding molecule labelling SSIII-1-m, the individual plant that the fine banding pattern of banding pattern and Japan is consistent in segregating population is selected to be further used for breeding improvement (Fig. 3), eliminate the blue or green banding pattern one of banding pattern and high amylose content spy to make peace the individuality of heterozygosis banding pattern (there is the warm and fine special leukorrhagia with greenish discharge type of Japan simultaneously), the rice grain amylose content of breeding material adopts national standard (GB/T15683-2008) to detect.Analysis shows, selected 12 individual plants with the fine banding pattern of Japan are all blue or green than recurrent parent spy substantially reduces (Fig. 4). This test result indicate that: Indel labelling SSIII-1-m may be used for the assisted selection of rice grain amylose content.
Remarks illustrate:
" 4～15 " in Fig. 3 and Fig. 4 are Japan warm and fine special green grass or young crops and are sequentially carried out hybridization, backcross and selfing acquisition, and are the individual plants that have selected " banding pattern with Japan fine banding pattern consistent ".
Test 1, utilize Indel labelling SSIII-1-m to differentiate rice grain amylose content
Specific practice is: the individuality that heterozygosis banding pattern of being made peace by banding pattern one blue or green with high amylose content spy for the banding pattern being eliminated in the step 3 of embodiment 3 (has the warm and fine special leukorrhagia with greenish discharge type of Japan) simultaneously proceeds plantation, by the mensuration to its offspring's Rice Kernel amylose content, the reliability of further analyzing molecules labelling SSIII-1-m assisted Selection.
One, DNA is extracted
1), preparation DNA extraction buffer:
With embodiment 1.
2), above-mentioned rice leaf is handled as follows respectively:
With embodiment 1.
Two, Indel marker detection
1), pcr amplification
With embodiment 1.
2), electrophoresis detection
With embodiment 1.
Three, Indel labelling SSIII-1-m differentiates rice grain amylose content
Randomly choose 4 banding patterns that are eliminated in the step 3 of embodiment 3 and continue plantation with the blue or green consistent individual plant of spy, detect through Indel molecular marker SSIII-1-m, its offspring all shows consistent banding pattern blue or green with spy, gathers in the crops these individual plant seeds respectively and measures its amylose content. Additionally, the individuality randomly choosing wherein 1 heterozygosis banding pattern (simultaneously having the warm and fine special leukorrhagia with greenish discharge type of Japan) is used for continuing plantation, behind in generation, randomly select 16 individual plants, Indel molecular marker SSIII-1-m detection shows, these 16 individual plants have 4 special leukorrhagia with greenish discharge types of individual plant performance, 8 individual plant performance heterozygosis banding patterns, 4 show Japanese fine banding pattern, meet the separation relation of 1:2:1; After plant to be planted maturation, gather in the crops these individual plants respectively and measure the amylose content of its seed. Table 1 is the amylose content of the Rice Kernel of these 20 individual plants (strain), and 4 individual plants consistent with special leukorrhagia with greenish discharge type all show similar high amylose content blue or green with spy; And in 16 offspring's individual plants of banding pattern heterozygosis, have 12 individual plants to behave like the high amylose content (wherein, the banding pattern of 4 individual plants show special leukorrhagia with greenish discharge type, the banding pattern performance heterozygosis banding pattern of 8 individual plants) blue or green with spy; 4 individual plants in Japanese fine banding pattern, its amylose content is substantially less than special blue or green. This test result indicate that: Indel molecular marker SSIII-1-m may be used for differentiating rice grain amylose content.
The genotype of table 1. rice grain amylose content and correspondence thereof
The plant selected and genotype thereof Offspring's individual plant and genotype thereof Amylose content (%)
XT-1(T) XT-1-1(T) 25.52±0.111
XT-2(T) XT-2-2(T) 25.28±0.123
XT-3(T) XT-3-5(T) 24.95±0.120
XT-4(T) XT-4-2(T) 25.90±0.121
XH-2(H) XH-2-4(T) 26.01±0.120
XH-2-7(T) 25.80±0.135
XH-2-5(T) 24.21±0.123
XH-2-7(T) 25.48±0.113
XH-2-9(H) 24.90±0.110
XH-2-4(H) 26.00±0.121
XH-2-1(H) 24.59±0.115
XH-2-11(H) 25.10±0.125
XH-2-8(H) 25.60±0.113
XH-2-10(H) 27.00±0.111
XH-2-12(H) 26.50±0.123
XH-2-6(H) 25.95±0.126
XH-2-8(N) 22.10±0.113 8 -->
XH-2-10(N) 21.30±0.116
XH-2-12(N) 21.95±0.123
XH-2-6(N) 21.21±0.125
Note: in bracket, letter represents the genotype of this individual plant, T is special leukorrhagia with greenish discharge type, and H is heterozygosis banding pattern, and N is Japanese fine banding pattern.
Comparative example 1 and significantly high labelling SSIII-1-m-1 and SSIII-1-m-2 (such as table 2, nucleotides sequence therein is classified as 5 ' → 3 ') of the forward and reverse primer homology of SSIII-1-m; Detect according to the said method of the present invention, find that the two labelling expanding effect is very bad, it does not have band occurs or non-characteristic band is a lot, it is impossible to be used for differentiating rice grain amylose content, namely, it is impossible to become rice amylose content micro-control gene molecular marker.
Table 2
Forward Reversely
SSIII-1-m-1 AGGGAAGGGAGTCAGCTCTG CCATCTCCATTGCCAGCGCC
SSIII-1-m-2 GGAGTCAGCTCTGAGGAGG TCTCCATTGCCAGCGCCAAA
Comparative example 2,4 kinds of molecular marker AGPS2a-m, SSIVb-m, GBSSII-m, SSIII-1-m (present invention) methods described in embodiment 3 as described in Table 3 are tested, now, the genetic donor parent of low amylose content is still that Japan is fine, and the rice variety of high amylose content has been made into 93-11 by special green grass or young crops.
Table 3
Forward Reversely
AGPS2a-m TCTTCTTTTAGATCTTAAATTTCAGA CACATCAAAGTTGTAGTTTTGTGA
SSIVb-m ATTTTCCTCAGTAGTAAGCAAGAGTT AAAACATTGCTCCAAAACAGC
GBSSII-m TGTCAGTCGCTGTCCTCGTA GATCTCATCCCATGCTAAGTTACT
SSIII-1-m AAGAAGGGAAGGGAGTCAGC GCCATCTCCATTGCCAGC
Selecting the individual plant that the fine banding pattern of banding pattern and Japan is consistent in segregating population, rice grain amylose content employing national standard (GB/T15683-2008) of breeding material detects.
The selected result such as table 4 below with the amylose content in the fine banding pattern individual plant of Japan compared with recurrent parent 93-11:
Table 4
This experiment has carried out the repetition of several times, described in result all substantially as above table 4.
Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above. It is clear that the invention is not restricted to above example, it is also possible to there are many deformation. All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.

Claims (3)

1. the molecular marker SSIII-1-m of rice amylose content micro-control gene SSIII-1, using Oryza sativa L. as species, is characterized in that: described molecular marker primer is selected from following primer pair, and nucleotides sequence therein is classified as 5 ' → 3 ',
SSIII-1-m forward: AAGAAGGGAAGGGAGTCAGC
Reverse: GCCATCTCCATTGCCAGC.
2. the purposes of molecular marker SSIII-1-m as claimed in claim 1, is characterized in that: identify and/or its offspring's assisted selection for amylose content of rice.
3. the purposes of molecular marker SSIII-1-m according to claim 2, is characterized in that: when for the offspring of Japanese warm and fine long-grained nonglutinous rice, selects the individual plant that in offspring, the fine banding pattern of banding pattern and Japan is consistent to be used for breeding.
CN201610180607.1A 2016-03-28 2016-03-28 Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker Pending CN105671042A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610180607.1A CN105671042A (en) 2016-03-28 2016-03-28 Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610180607.1A CN105671042A (en) 2016-03-28 2016-03-28 Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker

Publications (1)

Publication Number Publication Date
CN105671042A true CN105671042A (en) 2016-06-15

Family

ID=56224227

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610180607.1A Pending CN105671042A (en) 2016-03-28 2016-03-28 Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker

Country Status (1)

Country Link
CN (1) CN105671042A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106591435A (en) * 2016-11-22 2017-04-26 南京农业大学 Molecular marker method for site of low-amylose-content gene of dark endosperm mutant w54 of japonica rice variety Koshihikari
CN107619873A (en) * 2017-08-30 2018-01-23 上海交通大学 Based on molecular labeling in association analysis and KASP exploitation waxy1 genes
CN108424974A (en) * 2017-11-10 2018-08-21 浙江省农业科学院 The Genetic identification and molecular mark method of rice grain amylose content QTL site qSAC3
CN114480720A (en) * 2022-03-16 2022-05-13 四川省农业科学院作物研究所 Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066050A1 (en) * 1998-06-15 1999-12-23 National Starch And Chemical Investment Holding Corporation Improvements in or relating to plants and plant products
CN1726282A (en) * 2002-12-19 2006-01-25 拜尔作物科学有限公司 Plant cells and plants which synthesize a starch with an increased final viscosity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066050A1 (en) * 1998-06-15 1999-12-23 National Starch And Chemical Investment Holding Corporation Improvements in or relating to plants and plant products
CN1726282A (en) * 2002-12-19 2006-01-25 拜尔作物科学有限公司 Plant cells and plants which synthesize a starch with an increased final viscosity

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
TIAN ZHIXI等: "Development of gene-tagged molecular markers for starch synthesis-related genes in rice", 《CHINESE SCIENCE BULLETIN》 *
中华人民共和国国家质量监督检验检疫总局: "大米 质量淀粉含量的测定", 《中华人民共和国国家标准》 *
康翠芳等: "水稻淀粉合成相关基因SSⅠ、SSⅢ-1和PUL对稻米品质的影响", 《农业生物技术学报》 *
柳发财: "利用近等基因系研究淀粉合酶相关基因对稻米品质的影响", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
许顺菊等: "回交重组自交系中SSⅢ-1、SBE3和PUL基因对稻米蒸煮食味品质的影响", 《西北植物学报》 *
陈秀兰等: "《核技术农业应用的理论与实践》", 30 November 2009 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106591435A (en) * 2016-11-22 2017-04-26 南京农业大学 Molecular marker method for site of low-amylose-content gene of dark endosperm mutant w54 of japonica rice variety Koshihikari
CN107619873A (en) * 2017-08-30 2018-01-23 上海交通大学 Based on molecular labeling in association analysis and KASP exploitation waxy1 genes
CN108424974A (en) * 2017-11-10 2018-08-21 浙江省农业科学院 The Genetic identification and molecular mark method of rice grain amylose content QTL site qSAC3
CN108424974B (en) * 2017-11-10 2021-09-03 浙江省农业科学院 Genetic identification and molecular marker assisted breeding method for rice amylose content QTL locus qSAC3
CN114480720A (en) * 2022-03-16 2022-05-13 四川省农业科学院作物研究所 Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof
CN114480720B (en) * 2022-03-16 2024-06-04 四川省农业科学院作物研究所 Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof

Similar Documents

Publication Publication Date Title
CN103602673B (en) Rice amylose content micro-control gene AGPS2a molecule marker and application
CA2697860C (en) Method and compositions for breeding for preferred traits associated with goss` wilt resistance in plants
CN103602675B (en) Molecular marker of rice amylose content micro-control gene GBSSII and application
CN103602674B (en) The molecule marker of rice amylose content micro-control gene SSIVb and application
CN106011287B (en) The molecular labeling in the site rice bacterial leaf streak major gene resistance BLS1 and its application
CN106929574A (en) No. 4 chromosomes of upland cotton SNP marker related to fibre strength
CN103993011B (en) Semen Sesami dominant genic male sterility gene molecule marker and preparation method and application
CN105671042A (en) Molecular marker for rice amylose content micro-control genes SSIII-1 and application of molecular marker
CN108588272A (en) A kind of molecular labeling and its application with Plant Height in Wheat and spike length character main effect QTL compact linkage
CN109929945A (en) The molecular labeling BrSF2604 primer and its application in cabbage type rape florescence and maturity period main effect QTL site
US20170022574A1 (en) Molecular markers associated with haploid induction in zea mays
CN105671183B (en) Rice amylose content micro-control gene AGPL3 molecular labeling and application
AU2020103461A4 (en) Molecular marker of rice amylose content micro-control gene SSIIIb and application thereof
CN105838819A (en) Molecular marker for rice grain amylose content micro-control gene ISA and application thereof
CN105861725B (en) The molecular labeling of rice amylose content micro-control gene AGPL1 and application
CN106048011A (en) Molecular marker of rice grain AC (amylose content) micro-control gene SSIIIb and application
CN110004242A (en) The molecular labeling BrSF0239 primer and its application in cabbage type rape florescence and maturity period main effect QTL site
WO2015174825A1 (en) Method of predicting or determining plant phenotypes in oil palm
CN111172317B (en) Molecular marker HSRC3911 closely linked with major QTL site in flowering phase of sesame and application thereof
CN103667484B (en) Oil content character main gene locus of rape 6F313 and applications thereof
CN105671182A (en) Molecular marker for rice gel consistency control genes PUL and application of molecular marker
CN105671043A (en) Molecular marker for rice gel consistency control genes SBE1 and application of molecular marker
CN108546778A (en) A kind of SNP marker and its application for detecting anti-cucumber powdery mildew character
CN113151563B (en) Method, primer pair and kit for identifying watermelon pulp color and application of kit
Kumar et al. Development of mapping populations

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20160615