CN108719047A - Molecular breeding method for improving heat resistance of rice young ear in meiosis stage by using single-segment replacement line - Google Patents
Molecular breeding method for improving heat resistance of rice young ear in meiosis stage by using single-segment replacement line Download PDFInfo
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 41
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- 238000009395 breeding Methods 0.000 title claims abstract description 29
- 230000021121 meiosis Effects 0.000 title abstract description 5
- 240000007594 Oryza sativa Species 0.000 title description 41
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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Abstract
The invention belongs to the field of molecular breeding, and relates to a molecular breeding method for cultivating a heat-resistant restoring line of a rice young ear in a meiosis stage. The method is to use the medium indica two-line bone with the preservation number of CGMCC NO.12532The dry recovery line is female parent, wild rice chromosome segment introgression line YJ03-03-12 with preservation number CGMCC NO.12533 is male parent, 1 generation is hybridized, 3 generation is backcrossed, and 3 generation is selfed to obtain BC3F4Each generation is single plant harvest; and then a new strain with obviously improved heat resistance is obtained by combining molecular marker-assisted selection and artificial climate chamber simulation high temperature stress identification. By using the method, an improved restorer line 'Yuanye No. 4' with remarkable heat resistance in the meiotic stage of the young ears of the rice is cultivated. By selecting target characters with the aid of molecular markers, a restorer line with remarkably improved heat resistance in the meiotic stage of the young ears can be rapidly and efficiently cultured within 3 to 4 years.
Description
Technical field
The invention belongs to field of molecular breeding, are related to a kind of utilization single slice substitution line improvement rice young panicle meiophase
The molecular breeding method of heat resistance, and in particular to a kind of to improve rice using Yuanjiang River common wild-rice chromosome single slice substitution line
The molecular breeding method of young fringe meiophase heat resistance.
Background technology
Climatic regionalization is the Major Natural for influencing one of the Main Factors of global yield and quality of rice, and Chinese rice makees
Disaster occurs mainly on the south the Yangtze river basin (Beijing Xiong Zhenmin, Cai Hongfa chief editor rice in China:Chinese agriculture science and technology goes out
Version society, 1992).Shanghai Inst. of Plant Physiology, Chinese Academy of Sciences, Rice & Chines Sorghum Inst., Sichuan Academy of Agricultural Sciences, Hunan Province
The units such as rice research institute of Academy of Agricultural Sciences, 20 century 70s are just proved high temperature to rice anthesis and young fringe subtrahend point
There is injury in the phase of splitting, and defining the reason of obtain the critical-temperature of injury, the sensitive periods of injury and high temperature injury etc.
Conclusion.Rice is in bud, heading period, and extremely sensitive to temperature, optimum temperature is 25~30 DEG C, and 30 DEG C or more just
It will produce adverse effect.Such as 35 DEG C or more of continuous high temperature weather of young fringe meiophase, Rice Floral Organ by hypoplasia,
Pollen declines depauperation, vigor.Heading flowering period such as 35 DEG C or more continuous high temperature weather, just will produce hot evil,
High temperature prevents pollen maturation, anther dehiscence, pollen in column cap germination and the elongation of pollen tube, leads to nonfertilization, this period
It it is sensitive periods of the rice to high temperature, the same day of especially blooming meets high temperature stress, easily induces little Hua infertility, fertilization is caused to hinder
Hinder, seriously affects setting percentage and yield.
Using traditional breeding way and molecular mark technology cultivate rice heat resistant variety be it is most effective at present and
The method of feasible control high temperature stress harm.Yuanjiang County of Yunnan wild rice is long term growth in Yuanjiang County of Yunnan Dry-hot Valley Area
Common wild-rice has the characteristic for adapting to the extreme natural environments such as high temperature and drought.Therefore Yuanjiang County of Yunnan wild rice is important
The important sources of heat-resisting and drought-enduring resource.Breeders want always by way of interspecific hybridization resistance in Yuanjiang County of Yunnan wild rice
In the channel genes to cultivated rice of heat, but limited due to being not easy stabilization etc. Linkage drag and filial generation heat-resisting
The cultivation of kind.What molecular marking technique selected in the application in improving rice Heat-tolerance Breeding greatly reduces breeding process
Blindness fast implements external source elite germplasm and is permeated to cultivar, widened the hereditary basis of kind.Domestic and international researcher
A large amount of QTL Position Research is carried out to heat resistance, some has been used to marker assisted selection breeding practice.
Single slice substitution line (single segment substitution lines, SSSL) is to utilize hybridization, backcrossing
With the nurse crop whole gene group of molecular marker assisted selection (marker-assisted selection, MAS) structure
A series of near isogenic lines.An only homozygous chromosome segment from donor parents in its genome, and genome
Rest part it is identical as recurrent parent.It is to carry out genome research, the ideal of especially QTL positioning and Molecular design breeding
Material.Peleman etc.（Peleman J .D. and Vander Voort J.R., Breeding by design [J]
Trends Plant Sci, 2003,8 (7): 330-334）Propose the new concept of Molecular design breeding, including positioning correlation
The QTL of character, the allelism variation for evaluating these sites and development design and context, are expected to break through the side such as accurate high-efficient breeding of rice
Obstacle current Guangdong Province's plant molecular breedings key lab in face has cultivated more than 1 600 parts of rice single slice substitution line (Xi
Z.Y., He F.H., Zeng R.Z., et al. Development of a wide population of
chromosome single segment substitution lines( SSSLs) in the genetic
background of an elite cultivar in rice ( Oryza sativaL) [J] .Genome, 2006,
49:476-484), the QTLs and its allele of many Main Agronomic Characters are identified with these materials（Yang Zifeng, Zhu Hai
Great waves, Liu Ziqiang wait rice ear sprouting period QTL upper Journal of Sex Research [J] Agricultural University Of South China journals of the based on single slice substitution line,
2014,35 (6):24-28.）, and carried out Molecular design breeding extensively（High yields of the beam sea good fortune based on single slice substitution line
The Guangzhou high-grade rice design and context [D]:Agricultural University Of South China, 2011.）.
Invention content
The purpose of the present invention is the above-mentioned deficiencies for the prior art, provide a kind of utilization Yuanjiang River common wild-rice single slice
Substitution line improves the molecular breeding method of rice young panicle meiophase heat resistance.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of molecular breeding for improveing rice young panicle meiophase heat resistance using Yuanjiang River common wild-rice single slice substitution line
Method comprises the following steps：
（1）Take the long-grained nonglutinous rice elite restorer lines 9311 that preserving number is CGMCC NO.12532 as maternal, preserving number CGMCC
The single slice substitution line YJ03-03-12 of NO.12533 be male parent, hybridize 1 generation, be returned 3 generations, then be selfed 3 generations obtain stablize it is pure
Close strain BC3F4, per generation is all single fringe harvest；
（2）Molecular labeling auxiliary selects for the first time：Plant BC1F1, DNA is extracted using SDS methods, using the Yuanjiang River wild rice
The corresponding molecular labeling primers of single slice substitution line YJ03-03-12 carry out PCR amplification to RM3204 and RM7403, and selection is simultaneously
The molecular labeling band for deriving from Yuanjiang River common wild-rice containing above-mentioned 2, molecular weight is the single plant of 195bp and 234bp respectively
It is returned with recurrent parent 9311, seed is harvested by single plant；Wherein molecular labeling primer it is positive/negative to RM3204 to sequence be SEQ ID
NO.1/SEQ ID NO.2, RM7403 it is positive/negative to sequence be SEQ ID NO.3/SEQ ID NO.4；
（3）Second of the selection of molecular labeling auxiliary：Plant BC2F1, after every plant is extracted DNA, the molecular labeling is recycled to draw
Object is to determining BC2F1The genotype of single plant, while there are 2 molecular labeling bands for deriving from above-mentioned wild rice, molecular weight difference
It is single plant continuation and the backcrossing of recurrent parent 9311 of 195bp and 234bp；
（4）Molecular labeling auxiliary third time selects：Plant BC3F1, after every plant is extracted DNA, continue with the molecular labeling
Primer pair determines BC3F1The genotype of single plant, while there is 2 molecular labeling bands for deriving from above-mentioned wild rice, molecular weight point
It is not the individual plant selfing of 195bp and 234bp, seed is harvested by single plant；
（5）Molecular labeling assists the 4th selection：Plant BC3F2, after every plant is extracted DNA, continue with the molecular labeling
Primer pair determines BC3F2The genotype of single plant, while there is 2 molecular labeling bands for deriving from above-mentioned wild rice, molecular weight point
It is not the homozygous genotype individual plant selfing acquisition BC of 195bp and 234bp3F3, seed is harvested by single plant.
The molecular breeding method further includes step（6）：By step（5）The BC containing homozygous genotype screened3F3Strain
System kind is moved into phjytotron in basin alms bowl, at young fringe meiosis initial stage, and carrying out young fringe using manual simulation's high temperature stress subtracts
The heat resistance screening of number division stage obtains the Improved lines that young fringe meiophase heat resistance significantly increases.
Advantageous effect：A kind of utilization Yuanjiang River common wild-rice single slice substitution line provided by the present invention improves long-grained nonglutinous rice backbone
The molecular breeding method of the young fringe meiophase heat resistance of restorer 9311, has compared with traditional backcrossing pyramiding breeding technology
Following advantage：There are crossing work amounts for traditional backcrossing pyramiding breeding technology greatly, it is scarce to select poor reliability, breeding cycle to grow
It falls into.By molecular marker assisted selection objective trait, it is aobvious that young fringe meiophase patience can be rapidly and efficiently cultivated in 3 ~ 4 years
Write conventional kind of the early rice of enhancing.The present invention is restored with Yuanjiang River common wild-rice single slice substitution line YJ03-03-12 and long-grained nonglutinous rice backbone
Be 9311 be material, the new lines that young fringe meiophase heat resistance significantly improves are obtained by molecular marker assisted selection
" member wild No. 4 ", " member is No. 4 wild " setting percentage in the case where young fringe meiophase simulates high temperature stress improves 15% than control 9311 ~
25%。
Description of the drawings
Fig. 1 molecular marker assisted selection children's fringe meiophase heat resistances are significantly better than the new lines of recurrent parent.
Young fringe meiophase heat-resisting sex expression under target interval different genotype high temperature stress in 9311 backgrounds of Fig. 2
（2014-2015）.
Biomaterial preservation proves
9311 be long-grained nonglutinous rice（Oryza sativasubsp. indica.）, it is preserved in China Microbiological bacterium on May 20th, 2016
Kind preservation administration committee common micro-organisms center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are micro-
Biological study institute, preserving number are CGMCC NO.12532；
YJ03-03-12 is long-grained nonglutinous rice（Oryza sativasubsp. indica.）, it is micro- to be preserved in China on May 20th, 2016
Biological inoculum preservation administration committee common micro-organisms center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology of institute, preserving number are CGMCC NO.12533.
Specific implementation mode
1 Selection parent of embodiment
Rice national engineering laboratory of Jiangxi Academy of Agricultural Sciences（Nanchang）Super rice research and development center is wheel with another name for Sichuan Province extensive 527
Parent is returned, with the Yuanjiang River wild rice lotus pool 3 for donor parents（Nonrecurrent parent）, being returned, 5 generations selfing, 3 generations combination covering is complete
The SSR molecular marker assisted Selection of genome has been cultivated the Yuanjiang River common wild-rice single slice that a set of another name for Sichuan Province extensive 527 is background and has been replaced
It it is 112, the coverage rate of genome is up to 73.5%.It 2010, in young fringe meiosis initial stage, chooses and grows consistent plant
It is packed into basin alms bowl, moves into phjytotron.Per 3 plants of potting, every part of material sets 3 repetitions, conventional water and fertilizer management.Setting is relatively wet
Degree is 85 %, illumination 12h/d .0 DEG C of constant temperature 38 (daytime)/28 DEG C (night).5 d are handled, after processing, will be tested
Material moves on under room temperature, until ripe.Setting percentage is investigated, each processing is all provided with control, and (control is field normal growing conditions
Under each test material), metrics evaluation children's fringe meiophase heat resistance is fallen to setting percentage.Filter out young fringe meiosis
The introgressive line 5 that phase heat resistance significantly improves, including introgressive line YJ03-03-12, in simulation high temperature stress, knot
Real rate improves 18.4% than control.9311 be cultivation long-grained nonglutinous rice（Oryza sativasubsp. indica.）, May 20 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica, preserving number are CGMCC NO.12532.Introgressive line YJ03-03-12 is cultivation
Long-grained nonglutinous rice（Oryza sativasubsp. indica.）, it is preserved in Chinese microorganism strain preservation management committee on May 20th, 2016
Member's meeting common micro-organisms center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
Preserving number is CGMCC NO.12533.
Embodiment 2
（1）Summer in 2011 plants each a line of YJ03-03-12 and 9311 respectively in Nanchang experiment station of Jiangxi Academy of Agricultural Sciences,
9311（CGMCC NO.12532）For female parent, YJ03-03-12（CGMCC NO.12533）It combines and generates for male parent preparing hybrid
F1, all mix and receive after ripe.Winter in 2011 plants 3 row F in Sanya, Hainan1, backcrossing acquisition BC1F1, all mix and receive after ripe.
Field management carries out according to a conventional method.
（2）Molecular marker assisted selection：Summer in 2012 plants 50 rows in Nanchang experiment station of Jiangxi Academy of Agricultural Sciences
BC2F1, often 8 plants of row, every plant of acquisition blade are put into the eppendorf pipes of 2 ml, fill the small steel ball of people, ground using high-flux tissue
Grind the SDS extracting solutions 600 that instrument high speed concussion grinding is added 1.25% laterμL extracts DNA, using YJ03-03- using SDS methods
12（CGMCC NO. 12532）With 9311（CGMCC NO. 12533）Molecular labeling on introgressed segment（Table 1）Carry out PCR expansions
Increase, amplification system 10μL, DNA profiling 1μL, 94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 0.5min, 57 DEG C of renaturation 0.5min, 72 DEG C
Extend 1min, after 32 recycle, 72 DEG C re-extend 10 min, and amplified production is through native polyacrylamide gel electrophoresis：Gel
A concentration of 8%, electrophoretic buffer is 0.5 times of TBE, 175V constant pressures electrophoresis 1.5 hours.Select 10 lists containing 2 molecular labelings
Strain continues backcrossing and obtains BC3F1Seed.
（3）Winter in 2012 is in Sanya, Hainan by BC3F13 rows are planted, often 8 plants of row, every plant of acquisition blade, after extracting DNA,
The molecular labeling provided using table 1 determines BC3F1The genotype of single plant selects 2 the single plant all contained is marked to continue selfing and obtains
BC3F2（There are 2 molecular labeling bands that above-mentioned wild rice is derived from table 1 simultaneously）.
The SSR molecular marker of 1 YJ03-03-12 introgressed segments of table
（4）Winter in 2013 is in Sanya, Hainan by BC3F210 rows are planted, often 8 plants of row, every plant of acquisition blade, after extracting DNA, profit
The molecular labeling provided with table 1 determines BC3F2The genotype of single plant selects 2 homozygous individual plant selfings of label to obtain BC3F3（Together
When in table 1 derive from above-mentioned wild rice 2 molecular labeling bands）.
（5）Phjytotron simulates high temperature stress identification：2014-2015, by homozygous BC3F4And BC3F5Strain is in children
Fringe meiophase is chosen and grows consistent plant loading basin alms bowl, moves into phjytotron.Per 3 plants of potting, every part of material
If 3 repetitions, conventional water and fertilizer management.Relative humidity is set as 80 %, illumination 11h/d .0 DEG C of constant temperature 38 (daytime)/28
DEG C (night).5 d are handled, after processing, test material is moved on under room temperature, until ripe.Investigate setting percentage, and other
Main Agronomic Characters, each processing is all provided with control (control is each test material under the normal growing conditions of field), with setting percentage
Fall to metrics evaluation children's fringe meiophase heat resistance.
By variance analysis, manual simulation is identified in being selected from the pure lines more than two eposides identified in the controlled environment chamber
Setting percentage heat resistance declines inapparent family, as improved line " 9311 change No. 1 " in the case of high temperature stress.
It is elite restorer lines for middle Xian two since the heat-resisting strain background parent that the present invention selects is 9311, therefore logical
" member is No. 4 wild " the restorer distinguishing feature for crossing the molecular breeding method selection and breeding is coordinate force height, has young fringe meiophase aobvious
Write heat-resisting feature.
Note:The primer of RM numbers, which comes from, utilizes Temnykh etc.（2000）With McCouch etc.（2002）The SSR primers of announcement
The SSR marker of sequent synthesis（Temnykh S, Park W D, Ayres N, et al. Mapping and genome
organization of microsatellite sequences in rice (Oryza sativa L.).
Theoretical and Applied Genetics, 2000, 100(5): 697-712；McCouch S R,
Teytelman L, Xu Y, et al. Development and mapping of 2240 new SSR markers for
rice (Oryza sativaL.). DNA research, 2002, 9(6): 199-207.）.
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24
Claims (2)
1. a kind of molecular breeding method for improveing rice young panicle meiophase heat resistance using single slice substitution line, including：
1) the middle Xian elite restorer lines 9311 for being CGMCC NO.12532 with preserving number are maternal, preserving number CGMCC
The Yuanjiang River common wild-rice chromosome segment single slice substitution line YJ03-03-12 of NO.12533 is male parent, hybridizes 1 generation, backcrossing 3
Generation, then be selfed the acquisition of 3 generations and stablize homozygous lines BC3F4, per generation is all single fringe harvest；
2) BC is planted1F1, DNA is extracted using SDS methods, using the Yuanjiang River wild rice chromosome single slice substitution line YJ03-
The corresponding molecular labeling primers of 03-12 carry out PCR amplification to RM3204 and RM7403 (table 1), and selection comes containing above-mentioned 2 simultaneously
Derived from the molecular labeling band of Yuanjiang River common wild-rice, molecular weight is the single plant and recurrent parent 9311 of 195bp and 234bp respectively
Backcrossing, seed are harvested by single plant；Wherein molecular labeling primer it is positive/negative to RM3204 to sequence be SEQ ID NO.1/SEQ ID
NO.2, RM7403 it is positive/negative to sequence be SEQ ID NO.3/SEQ ID NO.4；
1 primer characteristic strip size of table and sequence
3) second of selection of molecular labeling auxiliary：Plant BC2F1, after every plant is extracted DNA, recycle the molecular labeling primer
To determining BC1F1The genotype of single plant, while there are 2 molecular labeling bands for deriving from above-mentioned wild rice, molecular weight is respectively
The single plant of 195bp and 234bp continues to be returned with recurrent parent 9311；
4) molecular labeling auxiliary third time selects：Plant BC3F1, after every plant is extracted DNA, continue with the molecular labeling and draw
Object is to determining BC3F1The genotype of single plant, while there are 2 molecular labeling bands for deriving from above-mentioned Yuanjiang River common wild-rice, point
Son amount is the individual plant selfing of 195bp and 234bp respectively, and seed is harvested by single fringe；
5) molecular labeling assists the 4th selection：Plant BC3F2, after every plant is extracted DNA, continue with the molecular labeling and draw
Object is to determining BC3F2The genotype of single plant, while there are 2 molecular labeling bands for deriving from above-mentioned Yuanjiang River common wild-rice, point
Son amount is that the individual plant selfing of 195bp and 234bp obtains BC respectively3F3, seed is harvested by single fringe.
2. a kind of point for improveing rice young panicle meiophase heat resistance using single slice substitution line according to claim 1
Sub- breeding method, it is characterised in that：The strain shows as setting percentage under manual simulation's high temperature stress in 2014 and is
11.8%, increase by 5.3%, single plant yield 4.72g than recurrent parent setting percentage, increases 2.14g than recurrent parent；In young fringe subtrahend
The bear fruit grains of the choosing system (" member is No. 4 wild ") and single plant yield average value are dramatically increased compared with 9311 poles under division stage high temperature stress, strain
The effective fringe of height, spike length, single plant, Defined daily doses, every total grain panicle numerical value are compared with 9311 without significant difference.
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| 洪剑鸣等: "《中国水稻病害及其防治》", 30 November 2006, 上海科学技术出版社 * |
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