CN1849879A - Selective breeding method preserved sichuan pickle cytoplasm male sterile line - Google Patents

Abstract

The present invention relates to a method for selecting and breeding tuber muslard cytoplasmic male sterile line. It is characterized by that it uses natural mustard rape cytoplasmic male sterile line 6-102A as female parent, uses high-quality tuber mustard 6-170 as maintainer line, utilizes continuous backcross and molecular making auxiliary selection to obtain genetic stable tuber mustard cytoplasmic male sterile line CMS 6-170, breeding said sterile line so as to obtain the tuber mustard cytoplasmic male sterile line for production application.

Description

A kind of selection of preserved sichuan pickle cytoplasm male sterile line

Technical field

The invention belongs to the brassicaceous vegetable breeding technical field, be specifically related to a kind of seed selection of new preserved sichuan pickle cytoplasm male sterile line, the method for breeding.

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 cytoplasmic male sterile line with practical value in the world that the Fu Tingdong of Hua Zhong Agriculture University professor found in 1972.Data shows during the 1985-1994, have the cross rape more than 70% to breed by pol CMS 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 Guilan etc. (1992) utilize cabbage type rape Pori horse sterile source many for transformation for material and Chinese cabbage, obtained allo-plasm Chinese cabbage male sterile line CMS3411-7, its sterile plant rate can reach 100%, sterile degree is more than 95%, but its deadly defect is to 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 under the hostile environment condition, 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 because there are problems such as low temperature yellow in seedling stage, nectary be undeveloped in initial Ogu CMS, so far do not obtain utilization and extention (Zhang Deshuan etc., the characteristic analysis of Chinese cabbage CMS96 cytoplasmic male sterile line, North China agronomy newspaper aborning, 2005,20 (1): 59-62).

Leaf mustard is a Cruciferae, and rape belongs to this plant of sward one or two years.Leaf mustard is long at China's cultivation history, is distributed in each province on the south the Chinese the Changjiang river more, and its type and kind are a lot, mainly contains mustard seed dish, leaf mustard, stem usefulness leaf mustard, a kind of sedge usefulness leaf mustard, bud usefulness leaf mustard, root-mustard etc.Usually said leaf mustard refers generally to leaf mustard.Leaf mustard has leafy mustard, little leaf mustard, potherb mustard, envelopped senvy etc., and the Chinese Guangdong area is the most common with leafy mustard, little leaf mustard.Stem mainly refers to hot pickled mustard tube with leaf mustard.

Up to the present, still there is not the report that utilizes juncea sterile cytoplasm seed selection preserved sichuan pickle cytoplasm male sterile line.

Summary of the invention

The objective of the invention is to overcome the defective that existing preserved sichuan pickle cytoplasm male sterile line seed selection exists, propose a kind of selection of preserved sichuan pickle cytoplasm male sterile line of new stable fertility, for the hot pickled mustard tube heterosis utilization provides new germ plasm resource.Utilize advantages such as the sterility of preserved sichuan pickle cytoplasm male sterile line of seed selection of the present invention is stable, thorough and not affected by environment, cultivate novel preserved sichuan pickle cytoplasm male sterile material and vegetables crossbreed.

The present invention is achieved by the following technical programs:

A kind of selection of preserved sichuan pickle cytoplasm male sterile line, it is maternal to select natural juncea cytoplasmic male sterile line 6-102A to do, 6-170 makes male parent with the maintenance line hot pickled mustard tube, hybridization obtains F1, by molecular marker assisted selection and continuous backcross, obtain the preserved sichuan pickle cytoplasm male sterile line and the maintenance line of inheritance stability, breed this preserved sichuan pickle cytoplasm male sterile line, obtain described preserved sichuan pickle cytoplasm male sterile line CMS6-170.

Concrete 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 6-170 makes male parent with the maintenance line hot pickled mustard tube, and hybridization obtains hybrid F1;

(2) hybrid F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with maintenance line hot pickled mustard tube 6-170, and hybridization obtains BC1F1 cytoplasmic male sterile line;

(3) BC1F1 is carried out fertility and identify, make female parent with the BC1F1 cytoplasmic male sterile line that step (2) obtains, 6-170 makes male parent with the maintenance line hot pickled mustard tube, 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 maintenance line hot pickled mustard tube 6-170, and hybridization obtains BC3F1 cytoplasmic male sterile line;

(5) BC3F1 cytoplasmic male sterile line is carried out fertility and identify, make female parent, do 5 generations of recurrent parent continuous backcross, obtain BC8 preserved sichuan pickle cytoplasm male sterile line CMS6-170 with maintenance line 6-170 with the BC3F1 cytoplasmic male sterile line that step (4) obtains.

The male sterile series of envelopped senvy cytoplasm that above-mentioned breeding obtains is bred under autumn sowing or summer sowing isolation condition, promptly obtain the alleged preserved sichuan pickle cytoplasm male sterile line CMS6-170 of the present invention.

Good effect of the present invention is:

1, the sterility of preserved sichuan pickle 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 Wuhan, 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, preserved sichuan pickle cytoplasm male sterile line of the present invention is without any phenomenons such as dead flower bud and yellows in seedling stage.

3, preserved sichuan pickle cytoplasm male sterile line of the present invention provides new breeding approaches and methods for hot pickled mustard tube hybrid vigour.

Description of drawings

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

Fig. 2: be juncea cytoplasmic male sterile line 6-102A, the anther development cytological observation figure of 6-102B and RFLP molecular hyridization collection of illustrative plates:

Among the figure: 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 preserved sichuan pickle cytoplasm male sterile line CMS6-170 floral organ phenotype and the flowering stage that are seed selection of the present invention scheme:

A is male sterile line CMS6-170 floral organ figure among the figure; B and C represent the florescence figure of preserved sichuan pickle cytoplasm male sterile line CMS6-170 and maintenance line 6-170 respectively;

Fig. 4: the blade of preserved sichuan pickle cytoplasm male sterile line CMS6-170 and maintenance line 6-170 and the contrast of field phenotype:

Among the figure: A is the blade contrast of preserved sichuan pickle cytoplasm male sterile line CMS6-170 (a figure left side) and maintenance line 6-170 (figure is right); B and C are the field contrasts of preserved sichuan pickle cytoplasm male sterile line CMS6-170 and maintenance line 6-170 plant.

Embodiment

The seed selection of embodiment 1 preserved sichuan pickle cytoplasm male sterile line

One, the used mustard type rape cytoplasmic male sterile line of the present invention 6-102A 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, nap CMS, 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 extract (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 (prescription as follows), 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-3 ²P-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, airing on filter paper, till the no visible moisture film in film surface (avoid too dried, in case probe is difficult to wash-out, influence reuse) use the preservative film coating, compressing tablet is 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, Orf263-atp6 testing result show: 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 preserved sichuan pickle cytoplasm male sterile line is as follows:

(1) with described natural juncea male sterile plant 6-102A (referring to the Chinese invention patent application number: 200510086942.7, this seed on November 12nd, 2005 in China's typical culture collection center preservation, preserving number is: CCTCC NO:P200504)) the work female parent, make male parent with maintenance line hot pickled mustard tube conventional variety 6-170 (seed of this breeding material is public offering before the applying date), hybridization obtains hybrid F1;

(2) hybrid F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with hot pickled mustard tube conventional variety 6-170 (maintenance line), and hybridization obtains BC1F1 cytoplasmic male sterile line CMS.

(3) BC1F1 is carried out fertility and identify, the BC1F1 CMS that obtains with step (2) makes female parent, makes male parent with hot pickled mustard tube conventional variety 6-170 (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 hot pickled mustard tube conventional variety 6-170 (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 6-170, obtains BC8 preserved sichuan pickle cytoplasm male sterile line CMS6-170.

The preserved sichuan pickle cytoplasm male sterile line propagation steps is:

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

Good effect of the present invention is:

The phenotype of table 3 preserved sichuan pickle cytoplasm male sterile line CMS6-170 and Pol CMS, 6-102A relatively

The cytoplasmic male sterility type	Pol CMS	6-102A	Preserved sichuan pickle cytoplasm male sterile line CMS6-170
				Investigation strain number (strain)	2500	1900	1850
Sterile degree (%)	Trace-pollen is arranged	100％	100％
				Sterile strain (strain)	2500	1900	1850
Flower bud loses phenomenon	Slight flower bud loses	Do not have	Do not have

Claims (3)

1, a kind of selection of preserved sichuan pickle cytoplasm male sterile line, it is characterized in that, it is maternal to select natural juncea cytoplasmic male sterile line 6-102A to do, 6-170 makes male parent with the maintenance line hot pickled mustard tube, hybridization obtains F1, by molecular marker assisted selection and continuous backcross, obtains the preserved sichuan pickle cytoplasm male sterile line and the maintenance line of inheritance stability, breed this preserved sichuan pickle cytoplasm male sterile line, obtain described preserved sichuan pickle cytoplasm male sterile line CMS6-170.

2, according to right 1 described method, its step comprises:

(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 6-170 makes male parent with the maintenance line hot pickled mustard tube, and hybridization obtains hybrid F1;

(2) hybrid F1 is carried out fertility and identify, the F1 plant that obtains with step (1) is maternal, makes male parent with maintenance line hot pickled mustard tube 6-170, and hybridization obtains BC1F1 cytoplasmic male sterile line;

(3) BC1F1 is carried out fertility and identify, make female parent with the BC1F1 cytoplasmic male sterile line that step (2) obtains, 6-170 makes male parent with the maintenance line hot pickled mustard tube, 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 maintenance line hot pickled mustard tube 6-170, and hybridization obtains BC3F1 cytoplasmic male sterile line;

(5) BC3F1 cytoplasmic male sterile line is carried out fertility and identify, make female parent, do 5 generations of recurrent parent continuous backcross, obtain BC8 preserved sichuan pickle cytoplasm male sterile line CMS6-170 with maintenance line 6-170 with the BC3F1 cytoplasmic male sterile line that step (4) obtains.

3, according to claim 1-or 2 described methods, it is characterized in that the described preserved sichuan pickle cytoplasm male sterile line CMS6-170 of breeding under autumn sowing or summer sowing isolation condition.

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