CN1849880A - Selective breeding for potherb mustard cytoplasm male sterile line - Google Patents

Abstract

The present invention relates to a potherb mustard cytoplasmic male sterile line selecting and breeding method. Said invention is characterized by that it uses natural mustard rape cytoplasmic male sterile line 6-102A as female parent, uses potherb mustard 6-130 as maintainer line, utilizes continuous backcross and molecular marking auxiliary selection to obtain genetic stable potherb mustard cytoplasmic male sterile line and maintainer line, breeding said sterile line so as to obtain the potherb mustard cyloplasmic male sterile line CMS 6-130 for production application.

Description

A kind of selection of potherb mustard cytoplasm male sterile line

Technical field

The invention belongs to potherb mustard vegetable breeding technical field, be specifically related to a kind of seed selection, propagation method of new potherb mustard cytoplasm male sterile line.

Background technology

Cytoplasmic male sterility (being called for short CMS, down together) is the most important approach of rape heterosis.The cytoplasmic male sterility type of having reported both at home and abroad is a lot of at present, can be divided into allogeneic cytoplasm male sterile and homologous cell matter male sterile two big classes according to its source difference: in allos CMS, have most utilize to be worth also begun the commercialization breeding ogu CMS, kosena CMS and tour CMS arranged; In homology CMS, what have commercial exploitation value mainly is pol cytoplasmic male sterility (pol cms) system (Pol CMS).Pol CMS is first rape Pori horse male sterile line with practical value in the world that the Fu Tingdong of Hua Zhong Agriculture University professor found in 1972,1985-1994 breeds by pol CMS in the hybridization of the rape more than 70% in the world, and it still is that current domestic cabbage type rape hybrid produces the main sterile cytoplasm source of using.But the main deficiency of Pol CMS is a male sterile line because of the nuclear different existence of background ecological sensitivity in various degree, occurs the fertile flower powder usually under low temperature or hot conditions, influences seed production purity.Nap CMS more is subject to environment and Temperature Influence, and the value on hybrid produces is restricted.

Pol CMS sterile source not only is widely used in the seed selection of male sterile line of rape, but also is widely applied in the seed selection of other brassicaceous vegetable crop male sterile line.Ke Gui waited in blue 1992 utilize cabbage type rape Pori horse sterile source for material with more than the Chinese cabbage for transformation, obtained allo-plasm Chinese cabbage male sterile line CMS3411-7, its sterile plant rate can reach 100%, sterile degree is more than 95%, but deadly defect be under the hostile environment condition, can occur trace-pollen and slight flower bud abortion phenomenon (Ke Guilan etc. the seed selection of allo-plasm of Chinese cabbage male sterile line CMS3411-7 and application [J]. gardening journal, 1992.19(4)：333-340)。

The research and utilization progress of relevant radish cytoplasmic male sterility, since Ogura1968 found Ogu CMS, Chinese scholars had been carried out a large amount of backcross transformations to this sterile material.At present transformation in crop in cruciferae such as rape, wild cabbage, Chinese cabbage, broccoli.But, do not obtain utilization and extention so far aborning because there are problems such as low temperature yellow in seedling stage, nectary be undeveloped in initial Ogu CMS.(Zhang Deshuan, Zhang Fenglan, Xu JiaBing. the characteristic analysis of Chinese cabbage CMS96 cytoplasmic male sterile line. North China agronomy newspaper, 2005,20 (1): 59-62).

Potherb mustard is a Cruciferae, and rape belongs to, and is biennial vegetable, is divided into flower leaf type and plate blade profile two big classes, has another name called potherb mustard, potherb mustard.

Up to now, do not see the report of relevant potherb mustard cytoplasm male sterile line selection as yet.

Summary of the invention

Purpose of the present invention is exactly the weak point that overcomes existing vegetables cytoplasmic male sterile line, proposes a kind of selection of potherb mustard cytoplasm male sterile line of new stable fertility, for the utilization of potherb mustard vegetable heterosis provides new germ plasm resource.Utilize advantages such as the sterility of potherb mustard cytoplasm male sterile line of seed selection of the present invention is stable, thorough and not affected by environment, cultivate novel potherb mustard cytoplasm male sterile material and vegetables crossbreed.

The present invention is achieved by the following technical programs:

A kind of selection of potherb mustard vegetables cytoplasmic male sterile line, it is maternal to select natural juncea cytoplasmic male sterile line 6-102A to do, 6-130 makes male parent with the maintenance line potherb mustard, hybridization obtains F1, by molecular marker assisted selection and continuous backcross, obtain the potherb mustard cytoplasm male sterile line and the maintenance line of inheritance stability, breed this potherb mustard cytoplasm male sterile line, obtain potherb mustard cytoplasm male sterile line CMS6-130.

2, according to right 1 described method, its seed selection step is as follows:

(1) utilizes the RFLP molecule labelling method that natural mustard type rape cytoplasmic male sterile line 6-102A is carried out genetic typing, obtain rapeseed cytoplasmic male sterile material 6-102A;

(2) 6-102A that obtains with step (1) makes female parent, and 6-130 makes male parent with the maintenance line potherb mustard, and hybridization obtains F1;

(3) F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with maintenance line potherb mustard 6-130, and hybridization obtains BC1F1 cytoplasmic male sterile line;

(4) BC1F1 is carried out fertility and identify, the BC1F1 CMS that obtains with step (2) makes female parent, and 6-130 makes male parent with the maintenance line potherb mustard, obtains BC2F1 cytoplasmic male sterile line;

(5) BC2F1 cytoplasmic male sterile line is carried out fertility and identify, the BC2F1 cytoplasmic male sterile line that obtains with step (3) is maternal, makes male parent with maintenance line potherb mustard 6-130, and hybridization obtains BC3F1 cytoplasmic male sterile line;

(6) BC3F1 cytoplasmic male sterile line is carried out fertility and identify, make female parent, do 5 generations of recurrent parent continuous backcross, obtain BC8 potherb mustard cytoplasm male sterile line CMS6-130 with maintenance line 6-130 with the BC3F1 cytoplasmic male sterile line that step (4) obtains.

3, method according to claim 1 and 2 is characterized in that, breeding potherb mustard cytoplasm male sterile line CMS6-130 under autumn sowing or summer sowing isolation condition.The potherb mustard cytoplasm male sterile line that above-mentioned breeding obtains is bred under autumn sowing or summer sowing isolation condition, promptly obtain the alleged potherb mustard cytoplasm male sterile line of the present invention.

Good effect of the present invention is:

1, the sterility of potherb mustard cytoplasm male sterile line of the present invention is very stable and thorough, and this male sterile line is not affected by environment, in Lanzhou, Chinese Gansu and the plantation of continuous 5 years 10 generations of Wuchang, Hubei and carry out fertility and identify and show that its fertility all shows 0 grade.Sterile plant rate and sterile degree are 100%.

2, potherb mustard cytoplasm male sterile line of the present invention is without any phenomenons such as dead flower bud and yellows in seedling stage.

3, potherb mustard cytoplasm male sterile line of the present invention can be used for the brassicaceous vegetable breeding.

Description of drawings

Fig. 1: be the technology of the present invention flow chart

Fig. 2: be juncea cytoplasmic male sterile line 6-102A, in the anther development cytological observation figure RFLP molecular hyridization figure spectrogram of 6-102B: A is the paraffin section figure of the flower pesticide of mustard type rape cytoplasmic male sterile line 6-102A and 6-102B; B and C are RFLP molecular labeling polymorphism analysis (wherein the probe that uses of B are atp6, and the probe that C uses is atp9), and the 2nd of B and C the swimming lane is mustard type rape cytoplasmic male sterile line 6-102A of the present invention among Fig. 2.

Fig. 3: the florescence of the potherb mustard cytoplasm male sterile line CMS6-130 of seed selection of the present invention and the phenotypic map in seedling stage

Wherein: A, B, C, D are the florescence of potherb mustard cytoplasm male sterile line CMS6-130; E, F are respectively the contrast in seedling stage of potherb mustard cytoplasm male sterile line CMS6-130 and maintenance line 6-130

Fig. 4: the blade of potherb mustard cytoplasm male sterile line CMS6-130 and maintenance line 6-130 and potherb mustard cytoplasm male sterile line CMS6-130 cane phenotypic map

A is the blade contrast of potherb mustard cytoplasm male sterile line CMS6-130 and maintenance line 6-130 among the figure; B and C are the potherb mustard cytoplasm male sterile line CMS6-130 cane phenotypes of seed selection of the present invention

Embodiment

The seed selection of embodiment 1 potherb mustard cytoplasm male sterile line

One, mustard type rape cytoplasmic male sterile line is carried out genetic typing, its step is as follows:

1, the mensuration of extensive guarantor's relation:

(1) by the mensuration of extensive guarantor relation, juncea cytoplasmic male sterile line has been carried out correct genetic typing, the material of determining to be numbered 6-102A is new cytoplasmic male sterility kytoplasm type.

(2) mensuration by extensive guarantor relation (seeing Fu Tingdong, " breeding of cross-bred rape and utilization ", Hubei science tech publishing house, the method that version in 2000 is introduced), choose respectively 39 Wild cabbage types and mustard type rape kind or strain respectively with pol CMS; Ogu CMS, tour CMS, napCMS, 6-102A carries out test cross, and its extensive guarantor concerns that measurement result is as shown in table 1.

Extensive guarantor between several rape cytoplasmic male sterile lines of table 1 concerns mensuration

Kind (being) code name	6-102A	tour CMS	pol CMS	Ogu CMS
					5900	S	S	F	S
5148	S	S	F	S
					5200	S	S	F	S
Extensive 10	S	S	F	S
					Floral leaf is extensive	S	S	F	S
tour B	S	S	S	S
					Tour is extensive	S	F	S	S
02-102	S	S	S	S
					02-106	S	S	S	S
Radish is extensive	S	S	S	F
					3706	S	S	F	S
3721	S	S	F	S
					Canopy 71-1	S	S	F	S

Illustrate: 1, CMS is the english abbreviation of cytoplasmic male sterile line, and the Chinese and English alphabetical implication of table 1: S represents cytoplasmic male sterility, and F represents that cytoplasm is male and educates);

2, said determination material 5900 to canopy 71-1 respectively from Hua Zhong Agriculture University rapeseed breeding research department.

(3) F1 that obtains is carried out the fertility investigation, the result shows that 6-102A concerns different with the extensive guarantor of other a few class male sterile lines.

2, the microscopic examination of anther development:

The bud of getting 6-102A is made the paraffin wax section (with reference to lingering remnants of past customs group etc., the cytology research of the several kind anther developments of cabbage type rape, China's oil plant, 1988, (4): 23-25), displaing microstructure observing shows: period is broken up period for the stamen original hase in the abortion of the anther development of mustard type rape cytoplasmic male sterile line 6-102A of the present invention, its main feature is that the stamen original hase departs from normal differentiation track, form the petal original hase, do not form flower pesticide, the position of giving birth to stamen and grow small-flowered, abortion is different with the rapeseed cytoplasmic male sterile of existing report with mode period.

3, RFLP molecular marker analysis

(1) extracts genomic DNA respectively totally six kinds of rape materials from natural mustard type rape cytoplasmic male sterile line 6-102A (mustard type rape), maintenance line 6-102B (mustard type rape), pol CMS (cabbage type rape), ogu CMS (cabbage type rape), tour CMS (cabbage type rape), nap CMS (cabbage type rape), with reference to CTAB method (murray and thompson, 1980) extract genome DNA

Concrete steps are as follows:

A, add 2%CTAB and extract buffer solution (Cetyltriethylammonium bromide) to 25ml, 65 ℃ of heating 1hr, jog is cooled to room temperature..

B, add isopyknic chloroform/isoamyl alcohol (24: 1), mix gently, mixing, to lower floor be bottle green, left standstill 10 minutes;

C, the mixed liquor of B step is centrifugal, 3000rpm, 13～20 minutes;

D, get supernatant, pour out supernatant as far as possible;

The 10%CTAB extract of E, adding 1/10 volume, 65 ℃ were heated 10 minutes;

F, adding 25ml chloroform/isoamyl alcohol (24: 1), purifying, 3000rpm, 12 minutes;

G, add the isopropanol precipitating DNA of 1 times of volume, under-20 ℃ of conditions, place 20min;

H, ethanol washing DNA 2～3 times (3200rpm, centrifugal 8 minutes) with 76%;

I, add TE 200 μ l dissolving, 4 ℃ are spent the night, treat the DNA dissolving after, handle the dna solution (37 ℃ heating 1 hour) of gained with the RNase enzyme, detect DNA concentration and quality.

(2) preparation seven kinds of mitochondria probes (seeing table 2 for details):

Several mitochondria probes that are used for the Southern hybridization analysis of table 2

Gene	Insert fragment	Cloning site	The source	Selected marker
					atp6	2.7Kb	HindIII	Corn	Amp r
atp9	2.2Kb	XbaI	Corn	Amp r
					atpA	4.2Kb	HindIII	Corn	Amp r
coxI	4.5Kb	BamHI	Wheat	Amp r
					coxII	1.7Kb	KpnI/BamHI	Wheat	Amp r
Orf222	0.66Kb	EcoRI	Rape	Amp r

(3) with restriction enzyme EcoRI EcoRV, HindIII, BamHI, PstI the genomic DNA of above-mentioned six kinds of crop materials being carried out enzyme cuts

(4) (operating procedure is with reference to sambrook, and J waits work to carry out Southern hybridization; " molecular cloning experiment guide ", Beijing, Science Press, 2002).

A. prehybridization

Nylon membrane is put into hybridization bag or hybrid pipe, add hybridization solution (20 * SSC, concrete compound method is as described below), catch up with bubble, seal, put into 65 ℃ hybridization case,＞3hrs, hybridization solution submergence film.Usually, 12hrs.

B. label probe

Reaction system: 1-2blots (19.5 * 9.5cm ²)

DNA 100ng

DNTP 2.0μl

Random primer 5.0μl

Klenow(1u/μl) 1μl

α-dCTP* 3.0μl

Add water to 17.0 μ l

Concrete steps are: with the dna probe sex change (100 ℃, 10min)-the sex change probe place ice bath-by reaction system add reaction mixture in denatured DNA-again on the isotope operating desk, add ³²The dNTP of P mark (α- ³²P dCTP*, α- ³²P-dCTP) (specific radioactivity lives＞3000Ci/mmol)-30 ℃ incubation more than 2 hours.

C. hybridization

The probe that mark is good is added 100 ℃ of sex change of 300 μ l hybridization solutions (perhaps 0.4N NaOH sex change), adds in the hybridization bag (box) (avoid directly and be added on the film), and hybridization previous crops labeling effciency is measured.Can down do hybridization more than＞25%.The hybridization of spending the night.Hybridization efficiency is hybridized speed and hybrid stability influences.

D. wash film

According to washing film from the low rigorous rigorous degree of height of spending.

1 * SSC/0.1%SDS wash film twice (cold 5min, 65 ℃ of heat, 15min) → 0.5 * SSC/0.1%SDS heat washes 65 ℃, 15min.

E. coating

Film is pulled out from film washing liquid (as mentioned above), and airing on filter paper (is avoided too dried till the no visible moisture film in film surface, in case probe is difficult to wash-out, influence reuses) use preservative film coating, compressing tablet, put-20 ℃ or-70 ℃, basis signal power expose (3-7 days)

F. wash the X-mating plate

Under the red light of darkroom, take out the X-mating plate, insert in the developer solution to hybrid belt and display (developing time basis signal power and time for exposure length can by several seconds to 2 minute), change rinsing in the clear water over to, put into the fixing solution photographic fixing then to limpid (about 10 minutes).After running water is rinsed well, dry, read sheet.

Hybridization buffer Southern Blot Hybridization Buffer (Saghai ' s Lab)

Final concentration. the stoste volume.

5×SSC 20× 250ml

50mM PB(pH 6.8) 0.5M 100ml

5×Denhardt’s 50× 100ml

2.5mM EDTA(pH 8.0) 0.5M 5ml

100μg/ml ssDNA 5mg/ml 20ml

0.4％SDS 20％ 20ml

Dextran sulfate 50g

ddH ₂O to 1000ml

20×SSC

NaCl 175.3g 701.2g

Na ₃Citrate 88.2g 352.8g

dH ₂O to 1000ml 4000ml

(5) RFLP interpretation of result

The result shows: probe orf222, atp6, atp9,12 kinds of polymorphism marks of Orf263-atp6 testing result performance.In 28 kinds of mitochondria probe/enzyme combinations, 12 kinds of probes/enzyme combine detection is arranged to polymorphism, the juncea cytoplasmic male sterile line 6-102A that proof the present invention obtains and Pol CMS, Nap CMS, ogu CMS, tour CMS are diverse in the mitochondrial DNA level, are different cytoplasm types.Results of hybridization as shown in Figure 2, the cytoplasmic mtDNA of male sterile line 6-102A and other five class hybridization collection of illustrative plates is different fully, shows tangible polymorphism.Simultaneously, there is tangible polymorphism in the mtDNA banding pattern that 12 kinds of combinations of four chondriogens also detect 6-102A and 6-102B, and the number that shows as hybrid belt is different with the position, the difference of having only orf222/EcoRI to exist banding pattern to have or not.

Two, the seed selection step of potherb mustard cytoplasm male sterile line is as follows:

(1) (this breeding material is seen the Chinese invention patent application number: 200510086942.7 to make female parent with mustard type rape cytoplasmic male sterile line 6-102A, this seed on November 12nd, 2005 in China's typical culture collection center preservation, preserving number is: CCTCC NO:P200504), make male parent with maintenance line potherb mustard 6-130 (this seed is public offering before the applying date), hybridization obtains F1;

(2) hybrid F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with potherb mustard 6-130 (maintenance line), and hybridization obtains BC1F1CMS;

(3) BC1F1 is carried out fertility and identify, the BC1F1 CMS that obtains with step (2) makes female parent, and makes male parent with potherb mustard conventional variety 6-130 (maintenance line), obtains BC2F1 cytoplasmic male sterile line;

(4) BC2F1 cytoplasmic male sterile line is carried out fertility and identify, the BC2F1 cytoplasmic male sterile line that obtains with step (3) is maternal, makes male parent with potherb mustard conventional variety 6-130 (maintenance line), and hybridization obtains BC3F1 cytoplasmic male sterile line;

(5) BC3F1 cytoplasmic male sterile line being carried out fertility identifies; The BC3F1 cytoplasmic male sterile line that obtains with step (4) is done maternal, does six generations of recurrent parent continuous backcross with maintenance line 6-130, obtains the BC8 potherb mustard cytoplasm male sterile line.

The potherb mustard cytoplasm male sterile line propagation steps is:

Use under autumn sowing potherb mustard cytoplasm male sterile line (for example Mei Nian September) or Chinese Gansu Lanzhou summer sowing potherb mustard cytoplasm male sterile line (for example Mei Nian May) the isolation condition in Wuhan, Chinese Hubei, by male parent: maternal row is bred potherb mustard cytoplasm male sterile line than the planting proportion that is 2: 4, and other cultivation managements are with the breeding of common male sterile line.

Good effect of the present invention is:

The phenotype of table 3 potherb mustard cytoplasm male sterile line CMS6-130 and Pol6-CMS, 6-102A relatively

The cytoplasmic male sterility type	Pol CMS	6-102A	Potherb mustard cytoplasm male sterile line CMS6-130
				Investigation strain number (strain)	2500	1900	3200
Sterile degree (%)	Trace-pollen is arranged	100％	100％
				Sterile strain (strain)	2500	1900	3200
Flower bud loses phenomenon	Slight flower bud loses	Nothing	Nothing

Claims (3)

1, a kind of selection of potherb mustard vegetables cytoplasmic male sterile line, it is characterized in that, it is maternal to select natural juncea cytoplasmic male sterile line 6-102A to do, 6-130 makes male parent with the maintenance line potherb mustard, hybridization obtains F1, by molecular marker assisted selection and continuous backcross, obtains the potherb mustard cytoplasm male sterile line and the maintenance line of inheritance stability, breed this potherb mustard cytoplasm male sterile line, obtain potherb mustard cytoplasm male sterile line CMS6-130.

2, according to right 1 described method, its seed selection step is as follows:

(1) utilizes the RFLP molecule labelling method that natural mustard type rape cytoplasmic male sterile line 6-102A is carried out genetic typing, obtain rapeseed cytoplasmic male sterile material 6-102A;

(2) 6-102A that obtains with step (1) makes female parent, and 6-130 makes male parent with the maintenance line potherb mustard, and hybridization obtains F1;

(3) F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with maintenance line potherb mustard 6-130, and hybridization obtains BC1F1 cytoplasmic male sterile line;

(4) BC1F1 is carried out fertility and identify, the BC1F1 CMS that obtains with step (2) makes female parent, and 6-130 makes male parent with the maintenance line potherb mustard, obtains BC2F1 cytoplasmic male sterile line;

(5) BC2F1 cytoplasmic male sterile line is carried out fertility and identify, the BC2F1 cytoplasmic male sterile line that obtains with step (3) is maternal, makes male parent with maintenance line potherb mustard 6-130, and hybridization obtains BC3F1 cytoplasmic male sterile line;

(6) BC3F1 cytoplasmic male sterile line is carried out fertility and identify, make female parent, do 5 generations of recurrent parent continuous backcross, obtain BC8 potherb mustard cytoplasm male sterile line CMS6-130 with maintenance line 6-130 with the BC3F1 cytoplasmic male sterile line that step (4) obtains.

3, method according to claim 1 and 2 is characterized in that, breeding potherb mustard cytoplasm male sterile line CMS6-130 under autumn sowing or summer sowing isolation condition.

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