CN107937588A - The molecule labelling method of the heat-resisting main effect QTL site qHTF 1 of Rice Heading blooming stage and application - Google Patents
The molecule labelling method of the heat-resisting main effect QTL site qHTF 1 of Rice Heading blooming stage and application Download PDFInfo
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Abstract
A kind of molecule labelling method of the heat-resisting main effect QTL site qHTF 1 of Rice Heading blooming stage, this method utilizes a pair or two pairs of primers in the primer pair of molecular labeling RM23 or the primer pair of molecular labeling RM562, PCR amplification is carried out to rice material DNA to be identified, if amplifying the DNA fragmentation of corresponding size, indicate that heat-resisting main effect QTL qHTF 1 exists.This method passes through 2 SSR markers using heat-resisting 1 close linkages of main effect QTL qHTF of heading flowering period that are screening and being navigated on heat-resisting rice varieties the 1st chromosome of special green grass or young crops, predict the thermo-tolerance of rice material, be conducive to selection and breeding of the heat-resistant against new rice variety, accelerate the selection progress of heat-resisting rice varieties.
Description
Technical field
The invention belongs to biological technical field, and in particular to the important degeneration-resistant heat-resisting main effect QTL site qHTF-1 of character of rice
Molecule labelling method, also relate to application of the molecule labelling method in selection and breeding heat-resistant against the current rice varieties.
Background technology
Global climate persistently warms, and very serious influence is brought to rice in China production.Rice is to most temperature-sensitive
Period be heading flowering period, this period such as daily mean temperature is higher than 35 DEG C of high temperature, easily induces little Hua infertility, causes to be fertilized
Obstacle, cause cannot be solid and reduce yield.Heading flowering period meets with Climatic regionalization and causes setting percentage significantly to decline and the underproduction,
Such as the summer in 2003, southern more than 38 DEG C high temperature of part rice region continued more than 20 days, and only Hubei, two province disaster area of Anhui more than 80 ten thousand are public
Hectare, more than 1,000,000,000 kilograms of lost units；2006, especially big High Temperature Disaster occurred for Sichuan-chongqing Region, causes the rice underproduction then 25%
More than；2013,2016 and 2017, the provinces and regions such as Chongqing, Hubei, Hunan, Anhui and the Zhejiang in Yangtze River in China basin were multiple
By the high temperature damage for continuing more than 38 DEG C on a large scale, the cereal crops such as rice are caused to drop in production over a large area, under serious setting percentage
Drop 50%.Therefore, how quickly, accurate selection and breeding Climatic regionalization is the main meteorological disaster for influencing the production of China's rice safety,
Heat-resisting rice varieties are the main targets of current breeder.
The content of the invention
The technical problems to be solved by the invention are：In view of the above shortcomings of the prior art, there is provided a kind of Rice Heading is raised
The molecule labelling method of florescence heat-resisting main effect QTL site qHTF-1, while this method is also provided in selection and breeding heat-resistant rice product against the current
Application in kind.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is：A kind of Rice Heading blooming stage is heat-resisting
The molecule labelling method of main effect QTL site qHTF-1, this method are the primer pair or molecular labeling using molecular labeling RM23
A pair or two pairs of primers in the primer pair of RM562, carry out PCR amplification to the DNA of rice material to be identified, such as amplify correspondence
The DNA fragmentation of size, then indicate that the heat-resisting main effect QTL qHTF-1 of Rice Heading blooming stage exists；
Wherein, the primer pair sequence of above-mentioned molecular labeling RM23 is：Sense primer 5 '-CATTGGAGTGGAGGCTGG-3 '
(SEQ ID No.1), anti-sense primer 5 '-GTCAGGCTTCTGCCATTCTC-3 ' (SEQ ID No.2), after carrying out PCR amplification,
Electrophoresis can detect the band with the special blue or green same position of rice varieties, and amplified production size is 140-150bp；
The primer pair sequence of above-mentioned molecular labeling RM562 is：5 '-CACAACCCACAAACAGCAAG of sense primer (SEQ
ID No.3), anti-sense primer 5 '-CTTCCCCCAAAGTTTTAGCC-3 ' (SEQ ID No.4), carry out PCR amplification after, electrophoresis energy
Detect the band with the special blue or green same position of rice varieties, amplified production size is 185-195bp.
It is above-mentioned refer to PCR amplification when reaction system composition：2 μm of 0.4 μ L of ol/L primers, 1.0 μ L of DNA profiling, 2 ×
Easy Taq Super Mix 5.0 μ L and ddH23.6 μ l of O, totally 10 μ L；Reaction condition is：94 DEG C of pre-degeneration 5min are laggard
35 cyclic amplifications of row, cycling condition are 94 DEG C of denaturation 30s, and 58 DEG C of renaturation 30s, 72 DEG C of extension 1min, finally prolong 72 DEG C of ends
Stretch 5min.
Present invention simultaneously provides the molecule labelling method of the heat-resisting main effect QTL site qHTF-1 of Rice Heading blooming stage
Application in selection and breeding heat-resistant against the current rice varieties.
The present invention uses the molecule labelling method of QTL site, be using screen with the special green grass or young crops the 1st of heat-resisting rice varieties
2 SSR markers of the heat-resisting main effect QTL qHTF-1 close linkages of heading flowering period navigated on chromosome, predict rice material
Thermo-tolerance, accelerate the selection progresses of heat-resisting rice varieties.
Brief description of the drawings
Fig. 1 is rice varieties of the embodiment of the present invention spy green grass or young crops heat-resisting main effect QTL qHTF-1 of heading flowering period in the 1st chromosome
Positioning, the heat-resisting main effect QTL qHTF-1 of Rice Heading blooming stage is positioned at 500kb regions between RM23 and RM562.
Fig. 2 is SSR primers RM23 in the embodiment of the present invention in parent's (special blue or green and Yuejingsimiao 2) and special green grass or young crops/Guangdong crystalline substance silk
No. 2 F of seedling10The electrophoretogram of generation is expanded in colony.
Wherein：M marks for DNA marker, and swimming lane 1-24 is No. 2 F of special green grass or young crops/Yuejingsimiao10Colony's difference strain banding pattern,
Wherein top banding pattern is heat-resisting rice varieties spy leukorrhagia with greenish discharge type, and banding pattern is thermo-responsive No. 2 banding patterns of rice varieties Yuejingsimiao on the lower.
Fig. 3 is SSR primers RM562 in the embodiment of the present invention in parent's (special blue or green and Yuejingsimiao 2) and special green grass or young crops/Guangdong crystalline substance silk
No. 2 F of seedling10The electrophoretogram of generation is expanded in colony.
Wherein：M marks for DNA marker, and swimming lane 1-24 is No. 2 F of special green grass or young crops/Yuejingsimiao10Colony's difference strain, wherein
Top banding pattern is heat-resisting rice varieties spy leukorrhagia with greenish discharge type, and banding pattern is thermo-responsive No. 2 banding patterns of rice varieties Yuejingsimiao on the lower.
Embodiment
With reference to embodiment, the embodiment of the present invention is described in further detail.Following embodiments are only used
In the explanation present invention, but not limiting the scope of the invention.
Embodiment 1
Material to be tested：It is special blue or green for female parent with heat-resisting rice variety, it is miscellaneous for male parent with thermo-responsive rice variety Yuejingsimiao 2
Hand over, No. 2 RILs F of special green grass or young crops × Yuejingsimiao containing 182 strains are constructed with single-seed descent8And F10Colony.
1. phenotypic evaluation
Special blue or green, Yuejingsimiao 2 and special green grass or young crops/Guangdong are planted respectively at Agricultural University Of Hunan proving ground within 2015,2016
Brilliant No. 2 RILs F of silk seedling8(2015), F10182 strains of (2016) colony, the sowing of point 3 phases, respectively at 2015,2016
Year April 18, May 1, sowing on May 14, each issue every 13 days, in 2015, May 13 of 2016, May 26,6
The moon 8 was transplanted, Dan Benzhi, respectively 50 plants of the plantation of each issue of each material, and 20d or so the choosings before Rice Heading of each issue of each material are given birth to
In the 3 plants of immigration basin alms bowls of phase unanimously.
Material moved into respectively when every alms bowl at least 3 tillering tassels extraction sword-like leave 1cm or so phjytotron into
[24 DEG C (06 of row thermophilic:00-8:00；19:00-23:00)/26℃(08:00-10:00；16:00-19:00)/30℃(10:00-
16:00)/22℃(23:00-06:] and [34 DEG C (06 of high temperature 00):00-8:00；19:00-23:00)/36℃(08:00-10:
00；16:00-19:00)/38℃(10:00-16:00)/32℃(23:00-06:00)] handle, intensity of illumination 29000lx, often
When its irradiation 14 is small, relative humidity 75%.The spike of rice of mark heading at the same time, wherein special No. 2 RILs colonies of green grass or young crops × Yuejingsimiao
Only 141 strains carry out thermophilic or high-temperature process similar in selection breeding time；After continuous processing 7d, processing material is moved on into outdoor
It is allowed to grow naturally.
The average setting percentage of examination mark fringe, calculates setting percentage drop-out value, thermo-responsive index after maturation.
The setting percentage under setting percentage-high-temperature process under setting percentage drop-out value=thermophilic processing；Thermo-responsive index=(knot
Real rate drop-out value ÷ thermophilics handle setting percentage) × 100%.
2. the structure of genetic linkage maps
Choose and be uniformly distributed on 12 chromosomes of rice, cover 516 pairs of heat-resisting rice varieties of SSR primer pairs of full-length genome
Special blue or green and thermo-responsive two parent materials of rice varieties Yuejingsimiao No. 2 carry out polymorphism screening.Wherein 174 pairs of primers are in amphiphilic
This shows polymorphic, and the ratio of polymorphism primer is 33.71%.
Polymorphic obvious 174 pairs of codominance SSR markers between two parents are chosen, it is brilliant with the special green grass or young crops containing 141 strains × Guangdong
No. 2 RIL F of silk seedling8Colony, builds the Molecular Markers Linkage Map of 12 chromosome of full-length genome.The total length of the linkage map is
1710.36CM, average distance is 9.83CM between mark, and mark is relatively evenly distributed on 12 chromosomes.
3. the positioning of the heat-resisting QTL of heading flowering period
Heading flowering is carried out by 2.5 positioning software Interval mappings for composite of Windows QTL Cartographer
Phase heat-resisting qtl analysis, the qHTF-1 of main effect is detected in the 1st chromosome RM23~RM5385 sections, its LOD value is 6.15, table
Type explanation rate is 17.43%, additive effect 9.38, the effect of additive effect synergy allele from heat-resisting parent it is special blue or green etc.
Position gene.The position of the 1st article of heat-resisting main effect QTL qHTF-1 of chromosome Rice Heading blooming stage of rice in the genetic map of structure
Schematic diagram is shown in Fig. 1, and part electrophoretogram is shown in Fig. 2-Fig. 3.
For the main effect QTL of this section of finely positioning, there is polymorphic SSR between this section screens 2 two parents again
RM7075 and RM562 is marked, to encrypt the reference numerals of this section.With special No. 2 RIL F of green grass or young crops × Yuejingsimiao10The temperature-sensitive of colony
Sense index further positions qHTF-1 for phenotype index, is located between molecular labeling RM23 and RM562, the two
Distance between mark is 500kb, with reference to referring to table 1 below.
The molecular labeling in the heat-resisting main effect QTL site of 1 heading flowering period of table
4. molecular labeling R23, RM562 are to the molecular marker assisted selection of the main effect QTL.Using RM23, RM562 to spy
It is blue or green, 9311 and 36 heat resistance rice varieties such as Nipponbare carry out heading flowering period Heat tolerance identifications (the results are shown in Table 2) and rice
Genomic DNA carries out PCR amplification, wherein, Heat tolerance identification is with phenotypic evaluation method above in the present embodiment, in the present embodiment
PCR reaction systems are：2 μm of 0.4 μ L of ol/L primers, 1.0 μ L of DNA profiling, 2 × Easy Taq Super Mix, 5.0 μ L and
ddH2O 3.6μl.PCR reaction conditions are：35 cyclic amplifications are carried out after 94 DEG C of pre-degeneration 5min, cycling condition is 94 DEG C of denaturation
30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 1min, finally extend 5min eventually at 72 DEG C.Amplified production carries out electrophoresis detection, as a result table
It is bright, 9311, do not fall 17 kinds such as rice and amplify DNA fragmentation with special blue or green corresponding size, that is, indicate that those rice varieties are
Rice varieties existing for the heat-resisting main effect QTL qHTF-1 of Rice Heading blooming stage, show heat-resisting.
The representative rice varieties heading flowering period Heat tolerance identification of 2 36, table
| Rice varieties | Heat-resisting rank (heat-resisting R/ sensitivity S) | Rice varieties | Heat-resisting rank (heat-resisting R/ sensitivity S) |
| It is special blue or green | R | Nipponbare | S |
| 9311 | R | 05P30 | S |
| Do not fall rice | R | Jiang Zhuan 1 | S |
| Peach blossom rice | R | Shattering meal carrys out rice | S |
| Long awns early rice | R | IRAT109 | S |
| Close positive No. 55 | R | PADI.AMPIR | S |
| ITA147 | R | ITA216 | S |
| 98P202 | R | Water original 330 | S |
| Lemont | R | Middle round-grained rice 83-D | S |
| NP980237 | R | Town 88 | S |
| Tista | R | Precious Xian 97B | S |
| NP980384 | R | 2954 | S |
| Zhejiang 174 | R | Still state 12 | S |
| Cloud peak 7 | R | Yellow cun of paddy | S |
| Long rough rice | R | Cloud peak 3 | S |
| Glutinous No. 10 of Tianjin | R | Nanyang 6 | S |
| Middle Xian 91499 | R | Old shaven head | S |
| Shattering little Bai awns | R | Distant salt 9 | S |
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Agricultural University Of Hunan
<120>The molecule labelling method of the heat-resisting main effect QTL site qHTF-1 of Rice Heading blooming stage and application
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cacaacccac aaacagcaag 20
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Claims (4)
- A kind of 1. molecule labelling method of the heat-resisting main effect QTL site qHTF-1 of Rice Heading blooming stage, it is characterised in that this method It is using a pair or two pairs of primers in the primer pair of molecular labeling RM23 or the primer pair of molecular labeling RM562, to be identified The DNA of rice material carries out PCR amplification, if amplifying the DNA fragmentation of corresponding size, indicates that Rice Heading blooming stage is heat-resisting Main effect QTL qHTF-1 exists；Wherein, the primer pair sequence of above-mentioned molecular labeling RM23 is as shown in SEQ ID No.1 and SEQ ID No.2；Amplified production Size is 140-160bp；The primer pair sequence of above-mentioned molecular labeling RM562 is as shown in SEQ ID No.3 and SEQ ID No.4, amplified production size For 185-195bp.
- 2. the molecule labelling method of the heat-resisting main effect QTL site qHTF-1 of Rice Heading blooming stage as claimed in claim 1, it is special Sign is, the composition of the PCR reaction systems：2 μm of 0.4 μ L of ol/L primers, 1.0 μ L of DNA profiling, 2 × Easy Taq Super Mix 5.0 μ L and ddH23.6 μ l of O, totally 10 μ L.
- 3. the molecule labelling method in the heat-resisting main effect QTL site of Rice Heading blooming stage as claimed in claim 1, its feature exist In the PCR reaction conditions are：35 cyclic amplifications are carried out after 94 DEG C of pre-degeneration 5min, cycling condition is 94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 1min, finally extend 5min eventually at 72 DEG C.
- 4. the molecule mark of the heat-resisting main effect QTL site qHTF-1 of Rice Heading blooming stage as any one of claim 1-4 Application of the note method in the heat-resisting rice varieties of selection and breeding.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570516A (en) * | 2018-05-15 | 2018-09-25 | 江苏高航农业科技有限公司 | The molecular labeling of rare delicacies rice storage endurance main effect QTL and its application |
| 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 |
| CN110923355A (en) * | 2019-12-26 | 2020-03-27 | 安徽农业大学 | Linkage KASP molecular marker for rice high temperature resistance character and application thereof |
| 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 |
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2017
- 2017-12-08 CN CN201711293242.4A patent/CN107937588B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570516A (en) * | 2018-05-15 | 2018-09-25 | 江苏高航农业科技有限公司 | The molecular labeling of rare delicacies rice storage endurance main effect QTL and its application |
| CN108570516B (en) * | 2018-05-15 | 2021-10-22 | 福建六三种业有限责任公司 | Molecular marker of precious rice storage-resistant main effect QTL and application thereof |
| 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 |
| CN110923355A (en) * | 2019-12-26 | 2020-03-27 | 安徽农业大学 | Linkage KASP molecular marker for rice high temperature resistance character and application thereof |
| 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 |
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