CN103468805A - 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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- CN103468805A CN103468805A CN2013104098057A CN201310409805A CN103468805A CN 103468805 A CN103468805 A CN 103468805A CN 2013104098057 A CN2013104098057 A CN 2013104098057A CN 201310409805 A CN201310409805 A CN 201310409805A CN 103468805 A CN103468805 A CN 103468805A
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- gossypium harknessii
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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 the 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.The 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 the 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 guarantee total basicly stable grand strategy means of producing.
At present, take gossypium harknessii brand cytoplasmic male sterility material as basic three series mating material in China's application of succeeding.But, with the artificial seed, to compare, still seldom, the Elite restorer line material that particularly has strong restorer remains the key constraints of three-line cotton hybrid seed selection to available parent's resource.Therefore, in the three-line cotton hybrid Breeding Process, Elite restorer line seed selection and improvement with strong restorer are the Focal point and difficult points of research always.And adopt the 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 evaluation 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 the restorer seed selection and improve the key link that becomes the three-line cotton hybrid development.
Summary of the invention
The present invention is in order to solve gossypium harknessii brand cytoplasmic sterility restorer field test cross and the problems such as the work of evaluation required time is long, consumption is 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, guarantee 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 present invention also provides the application of SSR mark NAU5121 in evaluation gossypium harknessii brand cytoplasmic sterility isozygotys restorer.
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 that simultaneously has 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.Take traditional method (field test cross and evaluation) as checking, whether proof adopts SSR mark NAU5121 of the present invention is the isozygoty evaluation 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 the codominant marker, can the Rapid identification fertile plant be the restorer of isozygotying, and secondly with traditional method, compares, and the evaluation workload of gossypium harknessii brand cytoplasmic sterility restorer reduces 70%.
Further, the larger offspring for breeding population, a kind of method that the present invention also provides Rapid identification gossypium harknessii brand cytoplasmic sterility to isozygoty restorer, provide a kind of for the identification of whether carrying the labeled primer PF1 that recovers gene in the method, 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 gossypium harknessii brand of the Rapid identification for the larger offspring of breeding population cytoplasmic sterility, 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 that simultaneously has 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 the described fertile plant of primer amplification step a shown in SEQ ID NO.1 and 2; The fertile plant that simultaneously has 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 the dominant marker, but precise Identification goes out to recover gene, simultaneously equal common amplified band at the 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 the codominant marker, but whether Rapid identification carries the fertile plant of recovery gene for isozygotying; Take traditional method (field test cross and evaluation) as checking, prove and 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%.
During concrete enforcement, the fertile plant of the gossypium harknessii brand sample to be measured described in each authentication method for obtaining through pollen fertility investigation.Sample after the pollen fertility investigation, shortened evaluation 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 identified recovering gene rapidly not in time, certainly will be delayed the cultivation process of cotton three series cross-fertilize seed.And the present invention only need to extract the DNA that restorer is separated progeny material, carry out molecular markers for identification with primer of the present invention, just can identify very soon the homozygosity of restorer (strain), reduce workload more than 70% than traditional authentication method; 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 the target gene close linkage or be divided into from, but whether Rapid identification is recovered gene and is isozygotied, really from DNA level, identify the hereditary situation of recovering gene, improved the accuracy of selecting, 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.
The accompanying drawing explanation
Fig. 1 is that specific mark PF1 Primer selection can be educated the analytical results schematic diagram with sterile individual plant, and in figure, S means sterile type, and F means to educate type, the X failed sample that means 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 means the sterile line parent, and P2 means the restorer parent, and B means the restorer of isozygotying, and H means the heterozygosis restorer.
Embodiment
The preparation of material and method:
(1) (19R * Shanxi cotton 50) BC
3f
2: with the recovery of gossypium harknessii brand cytoplasmic sterility, be maternal, Shanxi cotton 50 is male parent (recurrent parent), hybridizes for 1 generation with restorer, after 3 generations that backcrossed, BC
3f
1fertile plant selfing in segregating population adds generation acquisition BC
3f
2segregating population.
(19R * GK44) F
2: the recovery with the gossypium harknessii brand cytoplasmic sterility is maternal, and GK44 is male parent, after hybridizing for 2 generations with restorer, obtains (19R * GK44) F
2.
(2) pollen fertility investigation: the 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), the hand of take is twisted with the fingers broken flower pesticide and whether is occurred that pollen granule can educate and sterile standard as distinguishing.
(3) molecular mark detection method: DNA extraction with reference to the 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 the centrifuge tube that tender leaf is put into 1.5ml, and adds the freshly prepared Extraction buffer 600 μ l of precooling to be extracted.PCR reaction system 15 μ L, comprise the total DNA of 20ng, 0.2mmol/L dNTP, 0.5U
taqarchaeal dna polymerase, 1 *
taqarchaeal dna polymerase buffer, forward and reverse primer each 0.33 μ mol/L, MgCl
22mmol/L.The PCR reaction conditions is 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 70s, 30 circulations; 72 ℃ 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, Shanxi Province plantation (19R * Shanxi cotton 50) BC
3f
2segregating population, plant 8 row, every row 23-25 strain.Because female parent derives from the restorer of cytoplasmic sterility, the offspring, Fertility segregation appears, this segregating population has 196 strains, at first twist with the fingers broken flower pesticide, identify the 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 the field test cross in Hainan in winter in 2010 and identify the above-mentioned 170bp of having place amplified band and do not have the individual plant of 190 bp place amplified bands, the 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, Shanxi Province plantation (19R * GK44) F
2segregating population, plant 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, 126 strains that have of 1045bp banding pattern are only arranged, 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 evaluation, 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 that only have 170bp place amplified band
1carry out the fertility restorer evaluation, 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 (5)
1. for the identification of the isozygoty mark of restorer of gossypium harknessii brand cytoplasmic sterility, it is the primer of nucleotide sequence as shown in SEQ ID NO.1 and 2.
2. claimed in claim 1ly be marked at the application of identifying that the gossypium harknessii brand cytoplasmic sterility isozygotys in restorer.
3. 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 step gossypium harknessii brand sample to be measured shown in SEQ ID NO.1 and 2; The sample that simultaneously has 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.
4. 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 that simultaneously has 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 the described fertile plant of primer amplification step a shown in SEQ ID NO.1 and 2; The fertile plant that simultaneously has 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.
5. according to claim 3 or 4 is described, 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 investigation obtains through pollen fertility fertile plant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105316345A (en) * | 2015-10-15 | 2016-02-10 | 四川省农业科学院作物研究所 | Chuanyou 36 oilseed rape fertility restorergene, purity and homozygosity detecting method |
| CN105755140A (en) * | 2016-04-15 | 2016-07-13 | 中国农业科学院棉花研究所 | InDel marker for cotton cytoplasm male sterility restoring lines and method for identifying molecules by aid of InDel marker |
| CN108728571A (en) * | 2018-06-08 | 2018-11-02 | 中国农业科学院棉花研究所 | The InDel molecular labeling and application chain with gossypium harknessii brand fertility restorer gene |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316345A (en) * | 2015-10-15 | 2016-02-10 | 四川省农业科学院作物研究所 | Chuanyou 36 oilseed rape fertility restorergene, purity and homozygosity detecting method |
| 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 |
| CN105755140A (en) * | 2016-04-15 | 2016-07-13 | 中国农业科学院棉花研究所 | InDel marker for cotton cytoplasm male sterility restoring lines and method for identifying molecules by aid of InDel marker |
| 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 |
| CN108728571A (en) * | 2018-06-08 | 2018-11-02 | 中国农业科学院棉花研究所 | The InDel molecular labeling and application chain with gossypium harknessii brand fertility restorer gene |
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