CN104082054A - Method for identifying genotypes of sterile plants of oilseed rape by high temperature stress - Google Patents
Method for identifying genotypes of sterile plants of oilseed rape by high temperature stress Download PDFInfo
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- CN104082054A CN104082054A CN201410231271.8A CN201410231271A CN104082054A CN 104082054 A CN104082054 A CN 104082054A CN 201410231271 A CN201410231271 A CN 201410231271A CN 104082054 A CN104082054 A CN 104082054A
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Abstract
The invention discloses a method for identifying genotypes of sterile plants of an oilseed rape by high temperature stress. By virtue of certain high temperature stress treatment, the genotypes of the sterile plants to be tested are distinguished according to the sterility stability of two sterile plants, and the advantages of a test crossing method and a molecular marking method are combined. The method is easy to operate, the contemporary detection is realized, requirements on laboratory conditions and technologies are low, and a result is reliable.
Description
Technical field
The present invention relates to the sterile pnca gene V-neck V of rape territory, is exactly a kind of method of utilizing the sterile pnca gene type of high temperature stress qualification rape.
Background technology
Recessive epistatic interaction core is sterile is a kind of important channel that rape heterosis utilizes, this utilized approach to obtain national technological invention patent (ZL97125803.1) in 2003, this patented method successfully realizes the sterile dual-purpose system of isozygotying of rapeseed plant recessive core, interim maintainer and the production of hybrid seeds of restorer three series mating completely, when the production of hybrid seeds, first produce complete sterile line by the sterile strain of isozygotying in dual-purpose system and interim maintainer completely, then with complete sterile line and restorer production crossbreed.Therefore in line with genic sterile recessive epistatic interaction in system, have two kinds of sterile strains of genotype, in the dual-purpose system of isozygotying, sterile pnca gene type isozygotys, and is homozygous sterile strain; Complete sterile line genotype is heterozygosis, is the sterile strain of heterozygous.
The sterile strain of isozygotying in theory in dual-purpose system is homozygous sterile strain, and it can produce sterile plant rate with complete temporary maintainer line test cross is 100% complete sterile line.Because rape is Cross Pollinated, in reproductive process, very easily mix, in the dual-purpose system of therefore isozygotying, very easily sneak into the sterile strain of heterozygous.These two kinds of sterile strains of genotype are difficult to distinguish in appearance, cannot in the time producing complete sterile line, sterile heterozygous strain be extracted, and cause the complete sterile line sterile plant rate of producing to decline, and have a strong impact on seed production efficiency and purity.Therefore differentiate that these two kinds sterile strain types are extremely important on breed and production, identify that at present the method for these two kinds of sterile strains of genotype has test cross method and molecular labeling method.
Test cross method with interim maintainer and sterile strain test cross to be identified completely, is recently differentiated sterile pnca gene type according to test cross offspring's Fertility segregation conventionally, if test cross offspring is all sterile strain, sterile strain to be identified is homozygous sterile strain; If test cross offspring fertile plant separates with sterile strain than being 1:1, sterile strain to be identified is the sterile strain of heterozygous.This authentication method advantage is simple to operate, and shortcoming is to need test cross, and (year) just can know the genotype of sterile strain to need the second season, and spended time is long.
Molecular labeling method is will first find and the closely linked molecular labeling in upper suppressor site, then with this molecular labeling, sterile strain is detected, and has or not to judge genotype according to key band.The method advantage is that detection speed is fast, can realize the present age and detect.Shortcoming is higher to laboratory and operating personnel's requirement, only applies in minority unit at present.
Summary of the invention
The object of the invention is to solve the technical problem of two kinds of sterile pnca gene type qualifications in line with genic sterile recessive epistatic interaction in system,
Above-mentioned purpose realizes by following scheme:
A method of utilizing the sterile pnca gene type of high temperature stress qualification rape, is characterized in that:
Comprise the following steps:
(1), field planting: the normal plantation in season of rape to be measured;
(2), transplant: move into rape with the basin alms bowl of bottom outlet squaring period, every basin one strain, rape is planted into land for growing field crops together with basin alms bowl;
(3), high temperature stress: peduncle-growing period for rapeseed is in the time of the clear and legible rape fertile plant of naked eyes and sterile strain, to place in phytotron or illumination box with the sterile strain of basin alms bowl, be controlled under the illumination condition of 34-36 DEG C and process 14-16 hour, remove immediately basin alms bowl, sterile strain is transplanted to land for growing field crops;
(4), fertility investigation: the flowers are in blossom decontrols and begin from first of sterile strain, investigates day by day the open single flower fertility of four the primary branch main sequences in every main inflorescence of sterile strain and top, extracts immediately after having investigated, and continues one week of investigation; By 7 grades of Fertility rating statistic laws, single flower fertility is divided into 0-6 level according to the number of its Fertile stamen, and wherein 0 grade is 6 stamen complete sterilities, and 1-6 level has respectively 1-6 Fertile stamen successively;
Fertility index M FI calculates: occur in one week of joint account those days of 1 grade and above flower all by the average stamen number of average fertility index M FI=of investigation flower=(1 × 1 grade spend+2 × 2 grades of numbers spend number+... spend number for+6 × 6 grades)/investigation flower number; If all investigated flowers are 0 grade in the week, MFI is 0.
(5), genotype is judged: work as MFI=0, sterile strain to be measured is homozygous sterile strain; Work as MFI>1, sterile strain to be measured is the sterile strain of heterozygous.
A kind of described method of utilizing the sterile pnca gene type of high temperature stress qualification rape, is characterized in that: the illumination temperature of described step (3) is 35 DEG C, the processing time is 15 hours.
Operation principle of the present invention is: research finds that two types of sterile strain sterility there are differences the susceptibility of temperature in rapeseed plant recessive epistatic interaction caryon sterile line system, at a lower temperature, two types of sterile strains all show thoroughly sterile, be difficult to distinguish, and under certain high temperature stress, the sterility of homozygous sterile strain is more stable than the sterility of the sterile strain of heterozygous, according to this phenomenon (principle), just can identify the genotype of these two types of sterile strains.
Beneficial effect of the present invention is: by certain high temperature stress processing, differentiate the genotype of sterile strain to be measured according to the difference of processing the sterile strain sterile stability of latter two, combine the advantage of test cross method and molecular labeling method, simple to operate, realizing the present age detects, low to laboratory condition and technical requirement, reliable results.
Embodiment
A method of utilizing the sterile pnca gene type of high temperature stress qualification rape, comprises the following steps:
(1), field planting: the normal plantation in season of rape to be measured;
(2), transplant: move into plastic basin alms bowl with the diameter 10cm of bottom outlet by rape squaring period, every basin one strain, rape is planted into land for growing field crops together with basin alms bowl;
(3), high temperature stress: peduncle-growing period for rapeseed is in the time of the clear and legible rape fertile plant of naked eyes and sterile strain, sterile strain with basin alms bowl is placed on to phytotron or illumination box, under 35 DEG C of illumination conditions, process 15 hours, remove immediately basin alms bowl, sterile strain is transplanted to land for growing field crops;
(4), fertility investigation: the flowers are in blossom decontrols and begin from first of sterile strain, investigates day by day the open single flower fertility of four the primary branch main sequences in every main inflorescence of sterile strain and top, extracts immediately after having investigated, and continues one week of investigation; By 7 grades of Fertility rating statistic laws, single flower fertility is divided into 0-6 level according to the number of its Fertile stamen, and wherein 0 grade is 6 stamen complete sterilities, and 1-6 level has respectively 1-6 Fertile stamen successively;
Fertility index M FI calculates: occur in one week of joint account those days of 1 grade and above flower all by the average stamen number of average fertility index M FI=of investigation flower=(1 × 1 grade spend+2 × 2 grades of numbers spend number+... spend number for+6 × 6 grades)/investigation flower number; If all investigated flowers are 0 grade in the week, MFI is 0.
(5), genotype is judged: work as MFI=0, sterile strain to be measured is homozygous sterile strain; Work as MFI>1, sterile strain to be measured is the sterile strain of heterozygous.
The rape of needs qualification is planted large Tanaka normal season.Moved into squaring period in the plastic basin alms bowl with the diameter 10cm of bottom outlet at rape, every basin one strain, then rape is planted into large field together with basin alms bowl.By observing, the sterile strain of androecium abortion is chosen to 40 strains at rape peduncle-growing period for rapeseed, front 20 strains are placed under 35 DEG C of conditions and process 15 hours (phytotron, illumination box), and rear 20 strains are processed 15 hours as a control group at normal temperatures, remove afterwards basin alms bowl, transplant into land for growing field crops.From first of sterile strain, the flowers are in blossom decontrols and begin, and investigates day by day the open single flower fertility of four the primary branch main sequences in every main inflorescence of sterile strain and top, after having investigated, extracts immediately, continues one week of investigation.The those days that record 1 grade of interior appearance of a week and above flower are all by the average fertility indexes (MFI) of investigation flower; If all investigated flowers are 0 grade in the week, MFI is 0.The results are shown in Table 1.
Claims (2)
1. a method of utilizing the sterile pnca gene type of high temperature stress qualification rape, is characterized in that:
Comprise the following steps:
(1), field planting: the normal plantation in season of rape to be measured;
(2), transplant: move into rape with the basin alms bowl of bottom outlet squaring period, every basin one strain, rape is planted into land for growing field crops together with basin alms bowl;
(3), high temperature stress: peduncle-growing period for rapeseed is in the time of the clear and legible rape fertile plant of naked eyes and sterile strain, to place in phytotron or illumination box with the sterile strain of basin alms bowl, be controlled under the illumination condition of 34-36 DEG C and process 14-16 hour, remove immediately basin alms bowl, sterile strain is transplanted to land for growing field crops;
(4), fertility investigation: the flowers are in blossom decontrols and begin from first of sterile strain, investigates day by day the open single flower fertility of four the primary branch main sequences in every main inflorescence of sterile strain and top, extracts immediately after having investigated, and continues one week of investigation; By 7 grades of Fertility rating statistic laws, single flower fertility is divided into 0-6 level according to the number of its Fertile stamen, and wherein 0 grade is 6 stamen complete sterilities, and 1-6 level has respectively 1-6 Fertile stamen successively;
Fertility index M FI calculates: occur in one week of joint account those days of 1 grade and above flower all by the average stamen number of average fertility index M FI=of investigation flower=(1 × 1 grade spend+2 × 2 grades of numbers spend number+... spend number for+6 × 6 grades)/investigation flower number; If all investigated flowers are 0 grade in the week, MFI is 0;
(5), genotype is judged: work as MFI=0, sterile strain to be measured is homozygous sterile strain; Work as MFI>1, sterile strain to be measured is the sterile strain of heterozygous.
2. a kind of method of utilizing the sterile pnca gene type of high temperature stress qualification rape according to claim 1, is characterized in that: the illumination temperature of described step (3) is 35 DEG C, and the processing time is 15 hours.
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| EP0139674B1 (en) * | 1983-03-16 | 1987-11-04 | Institut National De La Recherche Agronomique | Method for the somatic hybridization of colza |
| US4658084A (en) * | 1985-11-14 | 1987-04-14 | University Of Guelph | Hybridization using cytoplasmic male sterility and herbicide tolerance from nuclear genes |
| US4658085A (en) * | 1985-11-14 | 1987-04-14 | University Of Guelph | Hybridization using cytoplasmic male sterility, cytoplasmic herbicide tolerance, and herbicide tolerance from nuclear genes |
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