CN103468805B - Marker and method for identifying cytoplasmic sterility homozygous restorer line of Gossypium harknessii - Google Patents
Marker and method for identifying cytoplasmic sterility homozygous restorer line of Gossypium harknessii Download PDFInfo
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- CN103468805B CN103468805B CN201310409805.7A CN201310409805A CN103468805B CN 103468805 B CN103468805 B CN 103468805B CN 201310409805 A CN201310409805 A CN 201310409805A CN 103468805 B CN103468805 B CN 103468805B
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- gossypium harknessii
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- 241001479463 Gossypium harknessii Species 0.000 title claims abstract description 34
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- 238000000034 method Methods 0.000 title claims abstract description 33
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- 230000036512 infertility Effects 0.000 title claims abstract description 27
- 239000003550 marker Substances 0.000 title abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 241000196324 Embryophyta Species 0.000 claims description 26
- 238000012797 qualification Methods 0.000 claims description 16
- 238000011084 recovery Methods 0.000 claims description 15
- 230000003321 amplification Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 230000035558 fertility Effects 0.000 claims description 8
- 238000011835 investigation Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 abstract description 24
- 241000219146 Gossypium Species 0.000 abstract description 24
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 230000001976 improved effect Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000003147 molecular marker Substances 0.000 abstract description 2
- 208000000509 infertility Diseases 0.000 description 19
- 208000021267 infertility disease Diseases 0.000 description 19
- 238000012360 testing method Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
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- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
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- 229920000936 Agarose Polymers 0.000 description 1
- 101100433727 Caenorhabditis elegans got-1.2 gene Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 235000009429 Gossypium barbadense Nutrition 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
- 206010021929 Infertility male Diseases 0.000 description 1
- 208000007466 Male Infertility Diseases 0.000 description 1
- 101000708283 Oryza sativa subsp. indica Protein Rf1, mitochondrial Proteins 0.000 description 1
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
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- 235000018322 upland cotton Nutrition 0.000 description 1
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Abstract
The invention relates to the technical field of molecular breeding of cotton, in particular relates to a marker and a method for identifying a cytoplasmic sterility homozygous restorer line of Gossypium harknessii. Compared with the prior art, the marker and the method have the beneficial effects of high speed and high accuracy as well as practicability and simplicity, wherein when a primer provided by the invention is used for identifying the molecular marker, the homozygosity of the restorer line (or strain) can be quickly identified, and the workload can be reduced by more than 70% compared with that of traditional identification methods; the primer has the advantages of simplicity, stability, reliability, high repeatability and the like and can be used for quickly identifying the homozygosity of restore genes rapidly, the genetic condition of the restore genes can be truly identified in the level of DNA, and thus the selection accuracy is improved to be above 97% at least; all procedures of the identification method provided by the invention are easy to operate, and thus the method has good scientific research and commercial application prospects.
Description
Technical field
The present invention relates to Molecular breeding in upland cotton: technical field, specifically a kind of for the identification of gossypium harknessii brand cytoplasmic sterility isozygoty mark and the method for restorer.
Background technology
Hybrid cotton is the important channel that Cotton in China obtains high yield.Artificial hybridization cotton is in cotton region, the Yangtze valley and the widespread use of cotton region, the Huanghe valley.Along with the increase of recruitment cost, artificial emasculation hybrid seeding cost is high in recent years, and hybrid cotton is promoted and is affected.Utilize cotton cytoplasmic male sterilty three series mating seed production way to there is the advantages such as saving of labor, seed purity is high, cost is lower.In the situation that Cotton in China cultivated area glides, utilizing cotton cytoplasmic male sterilty three series mating seed production way is the inexorable trend of cotton crossbreed production development, is to ensure total basicly stable grand strategy means of producing.
At present, taking gossypium harknessii brand cytoplasmic male sterility material as basic three series mating material is in China's application of succeeding.But compared with artificial seed, available parent's resource is still little, the Elite restorer line material particularly with strong restorer remains the key constraints of three-line cotton hybrid seed selection.Therefore,, in three-line cotton hybrid Breeding Process, Elite restorer line seed selection and the improvement with strong restorer are the Focal point and difficult points of research always.And adopt traditional breeding method seed selection and improve new restorer line material require year limit for length; and all need to carry out a large amount of field test crosses and qualification work after annual transformation; to guarantee that recover gene does not lose in transformation and improved, process, thereby need to expend a large amount of manpower and financial resources.Therefore, how by Molecular Marker Assisted Selection Technology, not only reliable and stable but also carry out quickly and easily restorer seed selection and improve the key link that becomes three-line cotton hybrid development.
Summary of the invention
The present invention for solve gossypium harknessii brand cytoplasmic sterility restorer field test cross and qualification work required time long, consume the problems such as large, poor accuracy, provide a kind of for the identification of gossypium harknessii brand cytoplasmic sterility isozygoty primer and the method for restorer, thereby can accelerate restorer seed selection and improvement process, ensure the homozygosity of restorer simultaneously.
The present invention is achieved by the following technical solutions: for the identification of the isozygoty mark (SSR mark NAU5121) of restorer of gossypium harknessii brand cytoplasmic sterility, it is the primer of nucleotide sequence as shown in SEQ ID NO.1 and 2.
SSR mark NAU5121 primer:
Forward primer 5
,-AGGGCAAATTTCATCTCTCT-3
,(SEQ ID NO.1)
Reverse primer 5
,-AAAGCTTGACCGAAATCAAC-3
,(SEQ ID NO.2)
SSR mark NAU5121 primer of the present invention is from CMD website www.cottonmarkers.org.
The application that the present invention also provides SSR mark NAU5121 to isozygoty in restorer at qualification gossypium harknessii brand cytoplasmic sterility.
The present invention adopts the primer shown in SSR mark NAU5121(SEQ ID NO.1 and 2) authentication method be: with the genomic dna of the primer amplification gossypium harknessii brand sample to be measured shown in SEQ ID NO.1 and 2; The sample simultaneously with two amplified bands at 170bp place and 190bp place is to have the heterozygosis strain that recovers gene, the sample that has 170bp place amplified band and do not have 190 bp place amplified bands is the recovery strain of isozygotying of gossypium harknessii brand cytoplasmic sterility, and numerous acquisition restorer of isozygotying is expanded in this recovery strain.Taking traditional method (field test cross and qualification) as checking, whether prove to adopt SSR mark NAU5121 of the present invention is the isozygoty qualification of restorer of gossypium harknessii brand cytoplasmic sterility to sample to be tested, its accuracy rate is more than 97%, and SSR mark NAU5121 and gossypium harknessii brand cytoplasmic sterility recover gene
rf 1 whether genetic distance is 0.4cM, is codominant marker, can Rapid identification fertile plant be the restorer of isozygotying, and secondly, compared with traditional method, the qualification workload of gossypium harknessii brand cytoplasmic sterility restorer reduces 70%.
Further, for the larger offspring of breeding population, a kind of method that the present invention also provides Rapid identification gossypium harknessii brand cytoplasmic sterility to isozygoty restorer, in the method, provide a kind of for the identification of whether carrying the labeled primer PF1 that recovers gene, it is the primer of nucleotide sequence as shown in SEQ ID NO.3 and 4.
Labeled primer PF1:
Forward sequence: 5
,-CAAGATATGGGTAAGCTTCC-3
,(SEQ ID NO.3)
Reverse sequence: 5
,-TAACAACATTAGGCTCGATTT-3
,(SEQ ID NO.4)
The isozygoty method of restorer of the above-mentioned Rapid identification gossypium harknessii brand cytoplasmic sterility for the larger offspring of breeding population, its concrete steps are: the genomic dna of the primer amplification gossypium harknessii brand sample to be measured shown in SEQ ID NO.3 and 4 for a.; The sample simultaneously with two amplified bands in 1200 bp places and 1045bp place is fertile plant, and the sample that has 1045bp place amplified band and do not have 1200 bp place amplified bands is sterile strain; B. use the genomic dna of fertile plant described in the primer amplification step a shown in SEQ ID NO.1 and 2; The fertile plant simultaneously with two amplified bands at 170bp place and 190bp place is to have the heterozygosis strain that recovers gene, the fertile plant that has 170bp place amplified band and do not have 190 bp place amplified bands is the recovery strain of isozygotying of gossypium harknessii brand cytoplasmic sterility, and numerous acquisition restorer of isozygotying is expanded in this recovery strain.
Above-mentioned labeled primer PF1 and recovery gene
rf 1 be divided into from, it is dominant marker, can go out to recover gene by precise Identification, simultaneously equal common amplified band at 1045bp place in fertile plant and sterile strain, also can determine whether to increase successfully; SSR mark NAU5121 primer and recovery gene
rf 1 genetic distance is 0.4cM, is codominant marker, can Rapid identification whether carry the fertile plant of recovering gene for isozygotying; Taking traditional method (field test cross and qualification) as checking, prove to adopt two labeled primers of the present invention jointly to use, get final product the Rapid identification restorer that goes out to isozygoty, accuracy rate is more than 99%.
When concrete enforcement, the gossypium harknessii brand sample to be measured described in each authentication method is the fertile plant that investigation obtains through pollen fertility.Sample after pollen fertility investigation, has shortened qualification workload and qualification time.
The present invention compared with prior art has following beneficial effect: 1) speed is fast: in cotton three series restorer Breeding Process, normal field test cross and qualification test need to detect a large amount of phenotypic characters, be accompanied by and drop into a large amount of time, man power and material, if identify recovering gene rapidly not in time, certainly will delay the cultivation process of cotton three series cross-fertilize seed.And the present invention only need to extract restorer and separates the DNA of progeny material, carry out molecular markers for identification with primer of the present invention, just can identify very soon the homozygosity of restorer (strain), than traditional authentication method minimizing workload more than 70%; 2) accuracy rate is high: the expression that the western cytoplasmic sterility of Hackney recovers gene is subject to such environmental effects, and pollen fertility can not be accurately, the hereditary situation of reacting recovery gene truly; Primer of the present invention has the advantages such as simple, reliable and stable, reproducible, be easy to by PCR rapid amplifying and agarose or polyacrylamide gel electrophoresis detection, and Molecular Identification is not subject to the impact of environmental factors, and with target gene close linkage or be divided into from, can whether isozygoty by Rapid identification recovery gene, the hereditary situation of really recovering gene from DNA level qualification, has improved the accuracy of selecting, and accuracy rate is at least more than 97%; 3) practical simple: of the present invention for the identification of the isozygoty whole program easy handling of restorer method of gossypium harknessii brand cytoplasmic sterility, there is good scientific research and commercial application prospect.
Brief description of the drawings
Fig. 1 is that specific mark PF1 Primer selection can be educated and the analytical results schematic diagram of sterile individual plant, and in figure, S represents sterile type, and F represents to educate type, the X failed sample that represents to increase.
Fig. 2 is the analytical results schematic diagram that SSR mark NAU5121 primer is identified the restorer of isozygotying, and in figure, P1 represents sterile line parent, and P2 represents restorer parent, and B represents the restorer of isozygotying, and H represents heterozygosis restorer.
Embodiment
The preparation of material and method:
(1) (19R × Shanxi cotton 50) BC
3f
2: be maternal with the recovery of gossypium harknessii brand cytoplasmic sterility, Shanxi cotton 50 is male parent (recurrent parent), hybridizes for 1 generation, backcrosses after 3 generations, BC with restorer
3f
1fertile plant selfing in segregating population adds generation acquisition BC
3f
2segregating population.
(19R × GK44) F
2: be maternal with the recovery of gossypium harknessii brand cytoplasmic sterility, GK44 is male parent, hybridizes after 2 generations with restorer, obtains (19R × GK44) F
2.
(2) pollen fertility investigation: fertility investigation method is with reference to Justus(Justus N, Leinweber CL. A heritable partially male sterile character in cotton. J Hered (1960) 51 (4): method 191-192), twist with the fingers broken flower pesticide taking hand and whether occur that pollen granule can educate and sterile standard as distinguishing.
(3) molecular mark detection method: DNA extraction with reference to CTAB method (Paterson AH, Brubaker CL, Wendel JF. A rapid method for extraction of cotton (
gossypiumspp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep, 1993,11,2:122-127), be improved to without liquid nitrogen electric drill polishing, every strain is got 1 and is not launched tender leaf and put into the centrifuge tube of 1.5ml, and adds the freshly prepared Extraction buffer 600 μ l of precooling to extract.PCR reaction system 15 μ L, comprise the total DNA of 20ng, 0.2mmol/L dNTP, 0.5U
taqarchaeal dna polymerase, 1 ×
taqthe each 0.33 μ mol/L of archaeal dna polymerase buffer, forward and reverse primer, MgCl
22mmol/L.PCR reaction conditions is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 70s, 30 circulations; 72 DEG C fill extension 10min.PCR product 1% agarose gel electrophoresis of specific mark PF1, volts lost 3V/cm, electrophoresis 1h, ethidium bromide staining; PCR product 6% denaturing polyacrylamide gel electrophoresis of SSR mark NAU5121, the permanent power electrophoresis of 70W 40min, silver dyes development.
embodiment 1
Summer in 2010 is at Cotton Research Institute farm, academy of agricultural sciences of Shanxi Province plantation (19R × Shanxi cotton 50) BC
3f
2segregating population, plantation 8 row, every row 23-25 strain.Because female parent derives from the restorer of cytoplasmic sterility, there is Fertility segregation offspring, this segregating population has 196 strains, first twist with the fingers broken flower pesticide, qualification individual plant fertility, wherein fertile plant 125 strains, in fertile plant, that chooses that SSR mark NAU5121 primer amplification goes out has 170bp place amplified band and does not have individual plant totally 48 strains of 190 bp place amplified bands.
Carry out field test cross and the qualification above-mentioned 170bp of having place amplified band in Hainan in winter in 2010 and do not have the individual plant of 190 bp place amplified bands, result demonstration wherein has 47 strains to turn out to be the recovery strain of isozygotying, and accuracy rate is 97.9%.
embodiment 2
Summer in 2010 is at Cotton Research Institute farm, academy of agricultural sciences of Shanxi Province plantation (19R × GK44) F
2segregating population, plantation 20 row, every row 23-25 strain.This segregating population has 483 strains, before blooming, this segregating population individual plant is extracted to DNA.In this segregating population, choose individual plant totally 349 strains with two amplified bands in 1200 bp places and 1045bp place that mark PF1 primer amplification goes out, only there are 126 strains that have of 1045bp banding pattern, without 8 strains that have of amplification banding pattern, pcr amplification the results are shown in Figure 1; Do second with 349 strains of SSR mark NAU5121 primer pair and take turns qualification, have 170bp place amplified band and do not have individual plant totally 117 strains of 190 bp place amplified bands, pcr amplification the results are shown in Figure 2, by 117 individual plants and the combination of sterile line test cross.
Winter in 2010, Hainan was to above-mentioned 117 the individual plant field test cross F only with 170bp place amplified band
1carry out fertility restorer qualification, result shows that wherein 116 strains turn out to be the restorer of isozygotying (strain), and accuracy rate is 99.1%.
﹤ 110 ﹥ Cotton Research Institute, Shanxi Academy of Agricultural Sciences
﹤ 120 ﹥ are for the identification of gossypium harknessii brand cytoplasmic sterility isozygoty mark and the method for restorer
﹤160﹥4
﹤210﹥1
﹤211﹥20
﹤212﹥DNA
﹤ 213 ﹥ artificial sequences
﹤400﹥AGGGCAAATTTCATCTCTCT 20
﹤210﹥2
﹤211﹥20
﹤212﹥DNA
﹤ 213 ﹥ artificial sequences
﹤400﹥AAAGCTTGACCGAAATCAAC 20
﹤210﹥3
﹤211﹥20
﹤212﹥DNA
﹤ 213 ﹥ artificial sequences
﹤400﹥CAAGATATGGGTAAGCTTCC 20
﹤210﹥4
﹤211﹥20
﹤212﹥DNA
﹤ 213 ﹥ artificial sequences
﹤400﹥TAACAACATTAGGCTCGATTT 20
Claims (4)
1. the application that the primer of nucleotide sequence as shown in SEQ ID NO.1 and 2 isozygotys in restorer at qualification gossypium harknessii brand cytoplasmic sterility.
2. for the identification of the isozygoty method of restorer of gossypium harknessii brand cytoplasmic sterility, it is characterized in that, step is: with the genomic dna of the primer amplification gossypium harknessii brand sample to be measured shown in SEQ ID NO.1 and 2; The sample simultaneously with two amplified bands at 170bp place and 190bp place is to have the heterozygosis strain that recovers gene, the sample that has 170bp place amplified band and do not have 190 bp place amplified bands is the recovery strain of isozygotying of gossypium harknessii brand cytoplasmic sterility, and numerous acquisition restorer of isozygotying is expanded in this recovery strain.
3. for the identification of the isozygoty method of restorer of gossypium harknessii brand cytoplasmic sterility, it is characterized in that, step is:
A. use the genomic dna of the gossypium harknessii brand sample to be measured of the primer amplification shown in SEQ ID NO.3 and 4; The gossypium harknessii brand sample simultaneously with two amplified bands in 1200 bp places and 1045bp place is fertile plant, and the gossypium harknessii brand sample that has 1045bp place amplified band and do not have 1200 bp place amplified bands is sterile strain;
B. use the genomic dna of fertile plant described in the primer amplification step a shown in SEQ ID NO.1 and 2; The fertile plant simultaneously with two amplified bands at 170bp place and 190bp place is to have the heterozygosis strain that recovers gene, the fertile plant that has 170bp place amplified band and do not have 190 bp place amplified bands is the recovery strain of isozygotying of gossypium harknessii brand cytoplasmic sterility, and numerous acquisition restorer of isozygotying is expanded in this recovery strain.
According to described in claim 2 or 3 for the identification of the isozygoty method of restorer of gossypium harknessii brand cytoplasmic sterility, it is characterized in that, described gossypium harknessii brand sample to be measured is the fertile plant that investigation obtains through pollen fertility.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310409805.7A CN103468805B (en) | 2013-09-11 | 2013-09-11 | Marker and method for identifying cytoplasmic sterility homozygous restorer line of Gossypium harknessii |
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|---|---|---|---|
| CN201310409805.7A CN103468805B (en) | 2013-09-11 | 2013-09-11 | Marker and method for identifying cytoplasmic sterility homozygous restorer line of Gossypium harknessii |
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| CN103468805B true CN103468805B (en) | 2014-10-22 |
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| CN105316345B (en) * | 2015-10-15 | 2018-08-28 | 四川省农业科学院作物研究所 | River 36 rape restoring genes of oil and purity and homozygosity detection method |
| CN105755140B (en) * | 2016-04-15 | 2019-02-01 | 中国农业科学院棉花研究所 | The method that cotton cells matter male sterile restoring line InDel is marked and its identified |
| CN108728571B (en) * | 2018-06-08 | 2021-07-16 | 中国农业科学院棉花研究所 | InDel molecular marker linked with male sterility restoring gene of cotton harknessi and application |
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| CN102156824B (en) * | 2010-12-23 | 2013-06-12 | 山西省农业科学院棉花研究所 | Bioinformatics analyzing method for redundancy of SSR (Simple Sequence Repeat) molecular marker |
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