CN105734056A - Molecular markers of major QTL for rice heading period and application of molecular marker - Google Patents
Molecular markers of major QTL for rice heading period and application of molecular marker Download PDFInfo
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- 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
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- 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
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- 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
Abstract
The invention discloses molecular markers of major QTL for the rice heading period and application of the molecular marker.The molecular markers include an H70 primer pair and a 301K primer pair, wherein the H70 primer pair is composed of a sequence shown as SEQ ID No.1 and a sequence shown as SEQ ID No.2; the 301K primer pair is composed of a sequence shown as SEQ ID No.3 and a sequence shown as SEQ ID No.4; the two molecular markers are closely linked with QTL for the heading period and can be used for authenticating and breeding varieties for the rice heading period.The invention further discloses a method for authenticating and breeding the varieties for the rice heading period through the molecular markers.The major QTL which are located on the galianconism of the fifth chromosome and used for controlling the heading period are precisely positioned for the first time, and the InDel markers H70 and 301K which are closely linked are obtained.Whether variation of the major QTL for the heading period exists or not can be judged and authenticated just by detecting the characteristics of amplified bands of the markers, the phenotype of rice in the heading period can be predicted, and the molecular markers are used for guiding breeding work for an improvement in the rice heading period.
Description
Technical field
The invention belongs to molecular genetic breeding field, in particular it relates to the molecular marker of rice ear sprouting period main effect QTL and application thereof.
Background technology
Oryza sativa L. is one of most important cereal crops in the world, and there is more than 110 countries and regions rice cultivation in the whole world, and there is more than half population nearly in China with rice for staple food, and Rice Production is for ensureing that China's grain security has played important function.But, development along with modern agricultural production and society, the Planting characteristic particularly Regional suitability of Oryza sativa L. is had higher requirement by cultivation technique intensive, modern, tradition rice varieties can not meet production needs, cultivates high yield, target that stable yields, high-quality, the eurytopic new varieties person that become rice breeding pursues jointly.Heading stage is one of Oryza sativa L. Main Agronomic Characters, and length at heading stage (from sprouting to beginning fringe) directly reflects Different Rice Varieties During Growth Duration length.Rice ear sprouting period length not only directly determines Local Adaptation and the seasonal adaptation of rice varieties, and the high yield of rice varieties, stable yields are played an important role.After experienced by long-term nature and artificial selection, Oryza sativa L. defines rich and varied Genetic characteristics type, the envirment factors such as different light, temperature and farming custom make different rice workspace that Oryza sativa L. Genetic characteristics type have particular/special requirement, and suitable period of duration plays pivotal player in maintaining rice high yield stable yields.Therefore, discover and use new Heading date gene and be always up the focus of research.
According to the data that Gramene website (http://www.gramene.org/qtl/) announces, QTL relevant with rice ear sprouting period at present has 711, is distributed on each bar chromosome of Oryza sativa L..But, in these researchs, most of QTLs only Primary Location, their high-accuracy collection of illustrative plates is still unknown, limits and it is carried out further map based cloning.CSSL population (chromosomesegmentsubstitutionlines, CSSLs) and NIL (nearlyisogeniclines, NILs) is primarily now utilized to carry out QTL detection and separation.By use NIL strategy, at least 13 heading stage QTLs (Hd1, Hd2, Hd3a, Hd3b, Hd6, Hd8, Hd9, Ehd1, DTH8, Ghd7, DTH2, DTH12, Hd17) finely positioned, wherein 10 QTLs (Hd1, Hd3a, Hd6, Ehd1, DTH8, Hd17, Hd16, DTH2, Ghd7, DTH7) by successful clone.But be cloned heading stage number very little, significantly limit rice ear sprouting period application in production practices, therefore, be badly in need of strengthen rice ear sprouting period quantitative trait locus mask work.
Rice ear sprouting period is important economical character, directly related with yield traits, therefore excavate new rice ear sprouting period QTL site and carry out fine positional cloning research, can for being further elucidated with rice ear sprouting period genetic mechanism and regulated and control network and high yield, precocity, the offer theory and practice support of eurytopicity rice breeding.
Account for little step on etc. with long-grained nonglutinous rice BG1 be donor parents, japonica rice XLJ constructs the RIL containing 269 familys for receptor parent hybridization and detects for QTL, and main effect QTL at heading stage, called after qHD5 detected at the 5th chromosome.Process backcrosses and molecule assisted Selection constructs BC4F2Segregating population, (accounts for little stepping on, within 2015, be published in GENE) by its Primary Location within the scope of 309kb.It is carried out fine Position Research, finds molecular marker closely linked with major gene loci at heading stage, rice ear sprouting period will be greatly improved and identify Breeding Efficiency, accelerate Advances in Breeding.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of rice ear sprouting period that can improve and identifies Breeding Efficiency, accelerates the molecular marker of the rice ear sprouting period main effect QTL of Advances in Breeding.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that
The present invention, on the basis of qHD5 Primary Location, expands target group further, adopts BC4F2The method that in colony, heterozygosis individual plant carries out selfing, obtains BC4F3Colony is for finely positioning qHD5；And be located in the region of 80kb, have found two molecular marker H70 and 301Ks closely linked with it.This interval did not have any report of Heading date gene, and we temporary designations this main effect QTL at heading stage is DTH5.Wherein, H70 primer pair sequence described in SEQIDNo.1 and SEQIDNo.2 forms；301K primer pair sequence shown in SEQIDNo.3 and SEQIDNo.4 forms.
Present invention also offers the method utilizing above-mentioned molecular markers for identification, breeding rice heading stage, comprise the steps:
(1) foundation of PCR amplification system: adopt in H70,301K primer pair arbitrary to or two pairs, with Oryza sativa L. individual plant complete genome DNA to be measured for template, carry out pcr amplification；
(2) amplification of DNA fragments detection is analyzed: if H70 primer pair can amplify 171bp fragment, then this individual plant genotype is DTH5, and its phenotype is late heading；If 165bp fragment can be amplified, then this individual plant genotype is dth5, and its phenotype is early heading；If heterozygosis banding pattern can be amplified, then this individual plant genotype is DTH5dth5, and heading stage falls between；If 301K primer pair can amplify 178bp fragment, then this individual plant genotype is DTH5, and its phenotype is late heading；If 169bp fragment can be amplified, then this individual plant genotype is dth5, and its phenotype is early heading；If heterozygosis banding pattern can be amplified, then this individual plant genotype is DTH5dth5, and heading stage falls between.
Present invention also offers the application in rice ear sprouting period qualification, selection-breeding of the molecular marker of above-mentioned rice ear sprouting period main effect QTL.
The present invention finely located control main effect QTL at heading stage (DTH5) novel site being positioned on Chromosome 5 of Rice galianconism first, and obtains closely linked InDel labelling H70 and 301K.Have only to detect the amplified band characteristic of these labellings, it can be determined that whether the potentiation variation in main effect at heading stage site exists, and predicts the phenotype at heading stage of Oryza sativa L., for the breeding work instructing rice ear sprouting period to improve.Molecular marker not only just can distinguish the genotype of rice varieties in seedling stage, and can convenient, fast, directly realize target gene qualification in Rice Germplasm Resources and breeding progeny, greatly reduce labour cost, saving time and be not subject to the impact of environment and anthropic factor.
Accompanying drawing explanation
Above-mentioned is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, below in conjunction with accompanying drawing, the present invention is described in further detail with detailed description of the invention.
Fig. 1 is the Oryza sativa L. phenotype comparison diagram containing DTH5 and dth5；
Fig. 2 is scattergram at heading stage；
Fig. 3 is the positioning analysis process of rice ear sprouting period QTL site, the wherein region of the 80kb that DTH5 is positioned between H70 and 301K；
Fig. 4 is that molecular marker 301K is to segregating population BC4F3Part effect for colony's individual plant amplification.
Detailed description of the invention
In the segregating population related in following embodiment, following embodiment, method therefor is conventional method if no special instructions.
The acquisition of embodiment 1 and the closely linked molecular marker of major gene resistance at heading stage
(1) target group and test method
1, target group
Account for little stepping on qHD5 Primary Location etc. between molecular marker RM17788 and RM5374.On this basis, the present invention expands target group further, adopts BC4F2The method that in colony, heterozygosis individual plant carries out selfing, obtains BC4F3Advanced lines segregating population.
The present invention utilizes BC4F3First qHD5 is carried out genetic analysis by segregating population, as in figure 2 it is shown, early heading meets 1:3 (χ with heading in evening segregation ratio2=1.0159, P=0.3135 > 0.05) segregation ratio, illustrate that this gene is subject to single Mendelian factor and controls, and heading in evening to early heading for dominant.It it is the Oryza sativa L. phenotype comparison diagram containing DTH5 and dth5 shown in Fig. 1.
The present invention utilizes BC4F3Extreme recessive individual plant more than in advanced lines segregating population 2000 will be located in the 5th chromosomal heading stage main effect QTL finely position.
2, test method (DNA extraction and pcr amplification)
Individual plant takes young leaflet tablet, extracts individual DNA by CTAB method.PCR reacts: 1 μ LDNA (100ng), 1 μ L primer (10mmol, primer after primer and 0.5 μ L before 0.5 μ L), 1 μ L10 × Taqbuffer (20mMMg2+), 0.2 μ LdNTPs (10mM), 0.2 μ LTaqpolymerase (2U/ μ L) and 6.6 μ LddH2O.PCR response procedures is: 95 DEG C of denaturations 4 minutes (72 DEG C extend 30s, totally 32 circulations for 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s), last 72 DEG C extend 10 minutes.In the enterprising performing PCR amplification of PCR instrument, amplified production is separated by electrophoresis on 8% polyacrylamide gel.
(2) molecular markers development (at the InDel labelling that the design of the target area of Primary Location is new)
Whole genome sequence according to the warm and fine indica rice 93-11 of japonica rice Japan announced in Gramene (http://www.gramene.org), use the genome sequence of the warm and fine 93-11 of BLAST (http://blast.ncbi.nlm.nih.gov) comparison Japan, find insertion/deletion site (InDel), utilize PrimerPremiers5.0 software design InDel molecular marker (primer is synthesized) by Shanghai Ying Weijie base trade Co., Ltd, and carry out polymorphism screening, PCR reaction system and response procedures are with above-mentioned PCR amplification system, obtain 7 to polymorphism mark, fine location for gene.
(3) positioning result
Positioning result is as it is shown on figure 3, finally by gene mapping between H70 and 301K, H70 and 301K all has 1 exchange individual plant.H70 and 301K primer sequence is in Table 1.Result according to RAP-DBPrimer-Blast, between H70 and 301K, on japonica rice variety Japan is fine, physical distance is 80kb, and the genetic distance between qHD5 is close, can be used for molecular marker assisted selection breeding.With molecular marker 301K to segregating population BC4F3Expanding for population segment individual plant, expanding effect is as shown in Figure 4.
Table 1
(4) result and analysis
Having screened 20 heterozygosis individual plants with molecular marker H70 and 301K, after planting into family, each strain all occurs in that heading stage separates, and its screening accuracy reaches 100%.
Embodiment 2 (method utilizing closely linked molecular marker H70 and 301K qualification, breeding rice kind at suitable heading stage)
Specifically comprise the following steps that
(1) extraction of DNA: water intaking rice young leaflet tablet, CTAB method extracts genomic DNA；
(2) foundation of standard PCR amplification system: with any pair in H70 and 301K or two pairs of labeled primers, is template to Oryza sativa L. individual plant complete genome DNA to be measured, carries out pcr amplification；
(3) amplification of DNA fragments detection is analyzed: PCR primer detects through the polyacrylamide gel electrophoresis of 8%, if labelling H70 can amplify 171bp fragment, illustrates that this individual plant genotype is DTH5, and its phenotype is late heading；If 165bp fragment can be amplified, illustrating that this individual plant genotype is dth5, its phenotype is early heading；If heterozygosis banding pattern can be amplified, illustrate that this individual plant genotype is DTH5dth5；Heading stage between.Amplifying 178bp fragment with 301K, illustrate that this individual plant genotype is DTH5, if its phenotype is late heading can amplify 169bp fragment, illustrate that this individual plant genotype is dth5, its phenotype is early heading；If heterozygosis banding pattern can be amplified, illustrate that this individual plant genotype is DTH5dth5, heading stage between.
Above-mentioned molecular marker and method, mainly in the utilization of the molecular marker auxiliary improvement at rice varieties heading stage, the breeding of molecular marker auxiliary and germ plasm resource.By detecting the molecular marker chain with major gene loci at heading stage, may determine that the major gene loci with or without controlling heading stage imports in breeding lines, improve purposiveness and the effectiveness of the improvement of rice ear sprouting period adaptability, improve the efficiency of selection of this kind, accelerate Breeding progress.
The above; it it is only presently preferred embodiments of the present invention; not the present invention being done any pro forma restriction, those skilled in the art utilize the technology contents of the disclosure above to make a little simple modification, equivalent variations or modification, all fall within protection scope of the present invention.
。
Claims (3)
1. the molecular marker of rice ear sprouting period main effect QTL, it is characterised in that adopt H70 primer pair or 301K primer pair；
Described H70 primer pair sequence described in SEQIDNo.1 and SEQIDNo.2 forms；Described 301K primer pair sequence shown in SEQIDNo.3 and SEQIDNo.4 forms.
2. the method utilizing molecular markers for identification described in claim 1, breeding rice heading stage, it is characterised in that comprise the steps:
(1) foundation of PCR amplification system:
Adopt in H70,301K primer pair arbitrary to or two pairs, with Oryza sativa L. individual plant complete genome DNA to be measured for template, carry out pcr amplification；
(2) amplification of DNA fragments detection is analyzed:
If H70 primer pair can amplify 171bp fragment, then this individual plant genotype is DTH5, and its phenotype is late heading；If 165bp fragment can be amplified, then this individual plant genotype is dth5, and its phenotype is early heading；If heterozygosis banding pattern can be amplified, then this individual plant genotype is DTH5dth5, and heading stage falls between；
If 301K primer pair can amplify 178bp fragment, then this individual plant genotype is DTH5, and its phenotype is late heading；If 169bp fragment can be amplified, then this individual plant genotype is dth5, and its phenotype is early heading；If heterozygosis banding pattern can be amplified, then this individual plant genotype is DTH5dth5, and heading stage falls between.
3. the molecular marker of the rice ear sprouting period main effect QTL described in claim 1 application in rice ear sprouting period qualification, selection-breeding.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636184A (en) * | 2016-11-17 | 2017-05-10 | 中国科学院东北地理与农业生态研究所 | Application of rice heading-date gene vector |
| CN110157835A (en) * | 2019-07-09 | 2019-08-23 | 湖南省水稻研究所 | One kind InDel molecular marker and primer thereof relevant to Rice Heading blooming stage heat resistance and application |
| CN111206113A (en) * | 2020-02-12 | 2020-05-29 | 广西壮族自治区农业科学院 | InDel molecular marker for assisting selection of early heading genes of rice and application of InDel molecular marker |
| CN111304355A (en) * | 2020-04-10 | 2020-06-19 | 山东省农业科学院生物技术研究中心 | InDel molecular marker closely linked with rice heading stage gene and application |
| CN112501341A (en) * | 2020-12-09 | 2021-03-16 | 浙江师范大学 | Major QTL for regulating heading stage of rice, molecular marker and application |
| CN113981130A (en) * | 2021-11-30 | 2022-01-28 | 扬州大学 | Method for screening rice growth period |
| CN114854893A (en) * | 2021-12-23 | 2022-08-05 | 山西农业大学农业基因资源研究中心 | SNPs (single nucleotide polymorphism) marker associated with millet heading stage character and identification method thereof |
| WO2022188287A1 (en) * | 2021-03-10 | 2022-09-15 | 中国农业科学院作物科学研究所 | Protein for shortening heading stage of rice, and encoding gene and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636184A (en) * | 2016-11-17 | 2017-05-10 | 中国科学院东北地理与农业生态研究所 | Application of rice heading-date gene vector |
| CN110157835A (en) * | 2019-07-09 | 2019-08-23 | 湖南省水稻研究所 | One kind InDel molecular marker and primer thereof relevant to Rice Heading blooming stage heat resistance and application |
| CN110157835B (en) * | 2019-07-09 | 2023-03-28 | 湖南省水稻研究所 | InDel molecular marker related to heat resistance of rice at heading and flowering stage as well as primer and application thereof |
| CN111206113A (en) * | 2020-02-12 | 2020-05-29 | 广西壮族自治区农业科学院 | InDel molecular marker for assisting selection of early heading genes of rice and application of InDel molecular marker |
| CN111206113B (en) * | 2020-02-12 | 2021-07-02 | 广西壮族自治区农业科学院 | InDel molecular marker for assisting selection of early heading genes of rice and application of InDel molecular marker |
| CN111304355A (en) * | 2020-04-10 | 2020-06-19 | 山东省农业科学院生物技术研究中心 | InDel molecular marker closely linked with rice heading stage gene and application |
| CN111304355B (en) * | 2020-04-10 | 2022-06-03 | 山东省农业科学院生物技术研究中心 | InDel molecular marker closely linked with rice heading stage gene and application |
| CN112501341A (en) * | 2020-12-09 | 2021-03-16 | 浙江师范大学 | Major QTL for regulating heading stage of rice, molecular marker and application |
| WO2022188287A1 (en) * | 2021-03-10 | 2022-09-15 | 中国农业科学院作物科学研究所 | Protein for shortening heading stage of rice, and encoding gene and application thereof |
| CN113981130A (en) * | 2021-11-30 | 2022-01-28 | 扬州大学 | Method for screening rice growth period |
| CN113981130B (en) * | 2021-11-30 | 2024-04-26 | 扬州大学 | Method for screening rice growth period |
| CN114854893A (en) * | 2021-12-23 | 2022-08-05 | 山西农业大学农业基因资源研究中心 | SNPs (single nucleotide polymorphism) marker associated with millet heading stage character and identification method thereof |
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