CN105734056B - The molecular labeling of rice ear sprouting period main effect QTL and its application - Google Patents

The molecular labeling of rice ear sprouting period main effect QTL and its application Download PDF

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CN105734056B
CN105734056B CN201610203246.8A CN201610203246A CN105734056B CN 105734056 B CN105734056 B CN 105734056B CN 201610203246 A CN201610203246 A CN 201610203246A CN 105734056 B CN105734056 B CN 105734056B
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CN105734056A (en
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占小登
孙滨
程式华
曹立勇
张迎信
沈希宏
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China National Rice Research Institute
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Abstract

The invention discloses the molecular labeling of rice ear sprouting period main effect QTL and its application.The molecular labeling is H70 and 301K, wherein, H70 primer pairs sequence described in SEQ ID No.1 and SEQ ID No.2 forms；301K primer pairs sequence shown in SEQ ID No.3 and SEQ ID No.4 forms；The two molecular labelings and heading stage QTL close linkage, available for identification and breeding rice heading stage kind.The invention also discloses utilize above-mentioned molecular markers for identification, the method at breeding rice heading stage.The main effect QTL at present invention control heading stage of the finely positioning on the 5th the short arm of a chromosome first, and obtain the InDel marks H70 and 301K of close linkage.The amplified band characteristic of these marks need to only be detected, it can be determined that the variation presence or absence of identification heading stage main effect QTL, to predict the heading stage phenotype of rice, for the breeding work for instructing rice ear sprouting period to improve.

Description

The molecular labeling of rice ear sprouting period main effect QTL and its application
Technical field
The invention belongs to molecular genetic breeding field, in particular it relates to the molecular labeling of rice ear sprouting period main effect QTL and its Using.
Background technology
Rice is that there are individual countries and regions rice cultivation, China more than 110 in one of most important cereal crops, the whole world in the world There is the nearly population of more than half using rice as staple food, Rice Production has played important function for guarantee China's grain security.But With the development of modern agricultural production and society, intensive, modernization cultivation technique is particularly to the Planting characteristic of rice Regional suitability proposes higher requirement, and traditional rice varieties can not meet to produce needs, cultivate high yield, stable yields, it is high-quality, Eurytopic new varieties turn into the target that rice breeding person pursues jointly.Heading stage is one of rice Main Agronomic Characters, is taken out Ear period length directly reflects Different Rice Varieties During Growth Duration length (from sprouting beginning fringe).Rice ear sprouting period length not only directly determines The Local Adaptation and seasonal adaptation of rice varieties, and high yield to rice varieties, stable yields play an important role.It experienced After long-term nature and artificial selection, rice forms the rich and varied envirment factor such as Genetic characteristics type, different light, temperature And farming is accustomed to causing different rice workspaces to have rice Genetic characteristics type particular/special requirement, suitable breeding time is maintaining rice high Pivotal player is play in production stable yields.Therefore, it is always the focus studied to discover and use new Heading date gene.
According to Gramene websites (http://www.gramene.org/qtl/) announce data, at present and Rice Heading Phase, the QTL of correlation had 711, was distributed on each bar chromosome of rice.However, in these researchs, most of QTLs are only preliminary Positioning, their high-accuracy collection of illustrative plates is still unknown, limits and carries out further map based cloning to it.Primarily now utilize chromosome Fragment substitution line (chromosome segment substitution lines, CSSLs) and NIL (nearly Isogenic lines, NILs) carry out QTL detections and separation.By using NIL strategy, at least 13 heading stage QTLs (Hd1, Hd2, Hd3a, Hd3b, Hd6, Hd8, Hd9, Ehd1, DTH8, Ghd7, DTH2, DTH12, Hd17) by finely positioning, Wherein 10 QTLs (Hd1, Hd3a, Hd6, Ehd1, DTH8, Hd17, Hd16, DTH2, Ghd7, DTH7) are by successful clone.But by The heading stage number of clone very little, significantly limit application of the rice ear sprouting period in production practices, therefore, be badly in need of strengthening water The mask work of rice heading stage quantitative trait locus.
Rice ear sprouting period is important economical character, directly related with yield traits, therefore excavates new rice ear sprouting period QTL site simultaneously carries out finely positioning clone's research, can be to further elucidate rice ear sprouting period genetic mechanism and regulated and control network and height Production, precocious, eurytopicity rice breeding provide theory and practice and supported.
It is that receptor parent hybridization is constructed containing 269 familys to account for small step on etc. using long-grained nonglutinous rice BG1 as donor parents, japonica rice XLJ RIL detects for QTL, and detects main effect heading stage QTL in the 5th chromosome, is named as qHD5.By backcrossing and Molecule assisted Selection constructs BC4F2Segregating population, by its Primary Location in the range of 309kb (account for it is small step on, deliver within 2015 In GENE).Finely positioning research is carried out to it, finds molecular labeling with heading stage major gene loci close linkage, will be big It is big to improve rice ear sprouting period identification Breeding Efficiency, accelerate Advances in Breeding.
The content of the invention
Rice ear sprouting period identification Breeding Efficiency can be improved the technical problem to be solved in the present invention is to provide one kind, accelerate to educate The molecular labeling of the rice ear sprouting period main effect QTL of kind progress.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
The present invention further expands target group, using to BC on the basis of qHD5 Primary Locations4F2Heterozygosis in colony The method that individual plant is selfed, has obtained BC4F3Colony is used for finely positioning qHD5；And be located in 80kb region, It has found two molecular labeling H70 and 301K with its close linkage.This section did not had any report of Heading date gene, I Temporary designations main effect heading stage QTL be DTH5.Wherein, H70 primer pairs are as described in SEQ ID No.1 and SEQ ID No.2 Sequence forms；301K primer pairs sequence shown in SEQ ID No.3 and SEQ ID No.4 forms.
Present invention also offers using above-mentioned molecular markers for identification, the method at breeding rice heading stage, comprise the following steps：
(1) foundation of PCR amplification system：Using any pair in H70,301K primer pair or two pairs, with rice list to be measured Strain complete genome DNA is template, enters performing PCR amplification；
(2) amplification of DNA fragments tests and analyzes：If H70 primer pairs can amplify 171bp fragments, the individual plant genotype For DTH5, its phenotype is late heading；If 165bp fragments can be amplified, the individual plant genotype is dth5, and its phenotype is early Heading；If heterozygosis banding pattern can be amplified, the individual plant genotype is DTH5dth5, and heading stage falls between；If 301K primer pairs can amplify 178bp fragments, then the individual plant genotype is DTH5, and its phenotype is late heading；If it can expand Go out 169bp fragments, then the individual plant genotype is dth5, and its phenotype is early eared；If heterozygosis banding pattern can be amplified, the list Pnca gene type is DTH5dth5, and heading stage falls between.
Present invention also offers the molecular labeling of above-mentioned rice ear sprouting period main effect QTL in rice ear sprouting period identification, seed selection Application.
Present invention control heading stage main effect QTL (DTH5) of the finely positioning on Chromosome 5 of Rice galianconism first Novel site, and obtain the InDel marks H70 and 301K of close linkage.Only need to detect these amplified band characteristics marked, It may determine that the synergy variation in heading stage main effect site whether there is, to predict the heading stage phenotype of rice, for instructing rice The breeding work of heading stage improvement.Molecular labeling not only just can distinguish the genotype of rice varieties in seedling stage, and can facilitate, Fast, directly realize identification of the target gene in Rice Germplasm Resources and breeding progeny, greatly reduce work into Originally, save the time and do not influenceed by environment and human factor.
Brief description of the drawings
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 With reference to accompanying drawing, the present invention is described in further detail with embodiment.
Fig. 1 is the rice phenotype comparison diagram containing DTH5 and dth5；
Fig. 2 is heading stage distribution map；
Fig. 3 is the positioning analysis process of rice ear sprouting period QTL site, and wherein DTH5 is positioned between H70 and 301K 80kb region；
Fig. 4 is molecular labeling 301K to segregating population BC4F3For the part effect of colony's individual plant amplification.
Embodiment
Method therefor is conventional side unless otherwise instructed in the segregating population that is related in following embodiments, following embodiments Method.
Embodiment 1 and the acquisition of the molecular labeling of heading stage major gene resistance close linkage
(1) target group and test method
1st, target group
Account for small step on qHD5 Primary Locations etc. between molecular labeling RM17788 and RM5374.On this basis, it is of the invention Further expand target group, using to BC4F2The method that heterozygosis individual plant is selfed in colony, has obtained BC4F3Advanced lines point Peel off body.
The present invention utilizes BC4F3Segregating population carries out genetic analysis to qHD5 first, as shown in Fig. 2 early heading is eared with evening Segregation ratio meets 1:3(χ2=1.0159, P=0.3135>0.05) segregation ratio, illustrate the gene by single Mendelian factor control System, and evening heading is dominant to early heading.It is the rice phenotype comparison diagram containing DTH5 and dth5 shown in Fig. 1.
The present invention utilizes BC4F3More than 2000 extremely recessive individual plants in advanced lines segregating population are by taking out positioned at the 5th chromosome Ear period main effect QTL finely positioning.
2nd, test method (DNA extracts to be expanded with PCR)
Individual plant takes young leaflet tablet, and individual DNA is extracted with CTAB methods.PCR reacts：1 μ L DNA (100ng), 1 μ L primers (primer after primer and 0.5 μ L before 10mmol, 0.5 μ L), 1 μ 10 × Taq of L buffer (20mM Mg2+),0.2μL dNTPs (10mM), 0.2 μ L Taq polymerase (2U/ μ L) and 6.6 μ L ddH2O.PCR response procedures are：95 DEG C of pre-degenerations 4 minutes (95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 32 circulate), last 72 DEG C extend 10 minutes.In PCR instrument Enter performing PCR amplification, amplified production is separated by electrophoresis on 8% polyacrylamide gel.
(2) molecular markers development (new InDel marks are designed in the target area of Primary Location)
According to Gramene (http://www.gramene.org) in announce japonica rice Nipponbare and indica rice 93-11 it is complete Genome sequence, use BLAST (http://blast.ncbi.nlm.nih.gov) compare Nipponbare and 93-11 genome Sequence, find insertion/deletion site (InDel), (drawn using the Software for Design InDel molecular labelings of Primer Premiers 5.0 Thing is synthesized by Shanghai Invitrogen trade Co., Ltd), and polymorphism screening is carried out, PCR reaction systems and response procedures are same as above The PCR amplification system stated, obtain 7 pairs of polymorphism marks, the finely positioning for gene.
(3) positioning result
For positioning result as shown in figure 3, finally by the assignment of genes gene mapping between H70 and 301K, H70 and 301K have 1 exchange Individual plant.H70 and 301K primer sequences are shown in Table 1.According to RAP-DB Primer-Blast result, in japonica rice between H70 and 301K Genetic distance of the physical distance between 80kb, with qHD5 is close in kind Nipponbare, is educated available for molecular marker assisted selection Kind.With molecular labeling 301K to segregating population BC4F3Expanded for population segment individual plant, expanding effect is as shown in Figure 4.
Table 1
(4) result and analysis
20 heterozygosis individual plants are screened with molecular labeling H70 and 301K, after planting into family, heading occurs in each strain Phase separates, and it screens the degree of accuracy and reaches 100%.
Embodiment 2 (utilizes molecular labeling H70 and the 301K identification of close linkage, breeding rice suitable heading stage kind Method)
Comprise the following steps that：
(1) DNA extraction：Water intaking rice young leaflet tablet, CTAB methods extraction genomic DNA；
(2) foundation of standard PCR amplification system：With any pair in H70 and 301K or two pairs of labeled primers, to be measured Rice individual plant complete genome DNA is template, enters performing PCR amplification；
(3) amplification of DNA fragments tests and analyzes：PCR primer detects by 8% polyacrylamide gel electrophoresis, if mark Note H70 can amplify 171bp fragments, and it is DTH5 to illustrate the individual plant genotype, and its phenotype is late heading；If it can amplify 165bp fragments, it is dth5 to illustrate the individual plant genotype, and its phenotype is early eared；If heterozygosis banding pattern can be amplified, illustrate this Individual plant genotype is DTH5dth5；Heading stage is between.178bp fragments are amplified with 301K, illustrate that the individual plant genotype is DTH5, if its phenotype, which is late heading, can amplify 169bp fragments, it is dth5 to illustrate the individual plant genotype, and its phenotype is early Heading；If heterozygosis banding pattern can be amplified, it is DTH5dth5 to illustrate the individual plant genotype, and heading stage is between.
Above-mentioned molecular labeling and method, mainly the molecular labeling auxiliary improvement in rice varieties heading stage, molecular labeling are auxiliary The breeding and the utilization of germ plasm resource helped.Pass through detection and the chain molecular labeling of heading stage major gene loci, it may be determined that The major gene loci for whetheing there is control heading stage is imported into breeding lines, improves the purpose of rice ear sprouting period adaptability improvement Property and validity, improve the efficiency of selection of the kind, accelerate Breeding progress.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, this Art personnel make a little simple modification, equivalent variations or modification using the technology contents of the disclosure above, all fall within this hair In bright protection domain.

Claims (3)

1. the molecular labeling of rice ear sprouting period main effect QTL, it is characterised in that to use H70 primer pairs or 301K primer pairs with water Rice individual plant complete genome DNA is the nucleotide sequence that template amplification comes out；
H70 primer pairs sequence described in SEQ ID No.1 and SEQ ID No.2 forms；The 301K primer pairs are by SEQ Sequence shown in ID No.3 and SEQ ID No.4 forms.
2. utilize molecular markers for identification, the method at breeding rice heading stage described in claim 1, it is characterised in that including as follows Step：
(1) foundation of PCR amplification system：
Using any pair in H70,301K primer pair or two pairs, using rice individual plant complete genome DNA to be measured as template, carry out PCR is expanded；
(2) amplification of DNA fragments tests and analyzes：
If H70 primer pairs can amplify 171bp fragments, the individual plant genotype is DTH5, and its phenotype is late heading；If 165bp fragments can be amplified, then the individual plant genotype is dth5, and its phenotype is early eared；If heterozygosis banding pattern can be amplified, Then the individual plant genotype is DTH5dth5, and heading stage falls between；
If 301K primer pairs can amplify 178bp fragments, the individual plant genotype is DTH5, and its phenotype is late heading；Such as Fruit can amplify 169bp fragments, then the individual plant genotype is dth5, and its phenotype is early eared；If heterozygosis band can be amplified Type, then the individual plant genotype is DTH5dth5, and heading stage falls between.
3. the molecular labeling of the rice ear sprouting period main effect QTL described in claim 1 answering in rice ear sprouting period identification, seed selection With.
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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
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
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
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
CN112501341B (en) * 2020-12-09 2022-05-03 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
CN113736898B (en) * 2021-08-09 2024-07-05 上海市农业生物基因中心 Molecular marker of rice heading stage regulation gene OsGI and application thereof
CN113981130B (en) * 2021-11-30 2024-04-26 扬州大学 Method for screening rice growth period
CN114854893B (en) * 2021-12-23 2023-06-20 山西农业大学农业基因资源研究中心 SNPs (single nucleotide polymorphisms) mark associated with millet heading stage characters and identification method thereof

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