CN1283139C - Hybridizing method for obtaining allotranssituation system for anti downy mildew of cucumbus - Google Patents
Hybridizing method for obtaining allotranssituation system for anti downy mildew of cucumbus Download PDFInfo
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- CN1283139C CN1283139C CN 200410041933 CN200410041933A CN1283139C CN 1283139 C CN1283139 C CN 1283139C CN 200410041933 CN200410041933 CN 200410041933 CN 200410041933 A CN200410041933 A CN 200410041933A CN 1283139 C CN1283139 C CN 1283139C
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Abstract
The present invention relates to a method for breeding an alien translocation line CT-01 of cucumber with downy mildew resistance, which is especially used for the disease resistant breeding of cucumber. A new variety of the didiploid of muskmelons and a parent of Beijing Jietou for culturing cucumber are in backcross, single plants with downy mildew resistance and the chromosome number of 2n=14 are directly sieved from a backcross generation by a mounting technology of root-end meristematic cell chromosomes and a field inoculation test of downy mildew resistance. Selfing descendants of the single plants are tracked and identified to obtain an alien translocation line CT-01 of cucumber with high downy mildew resistance.
Description
One, technical field
The present invention relates to the selection of cucumber downy mildew resistance alien translocation line CT-01, belong to the biotechnology breeding field.
Two, background technology
For over ten years, the cucumber Hybrid parent resource-constrained of domestic cultivation, even the phenomenon that a parent reuses in a plurality of kinds appears.Thereby in present breed cucumber, the shortage of special fine germplasm resources is a problem demanding prompt solution, and provides a valid approach by the innovation that distant hybridization can be the distinguished germ plasm resource such as disease-resistant, pest-resistant, degeneration-resistant of cucumber.But owing to the existence of heredity or physiological disorder, be difficult to hybridize successfully between the edge kind far away.
(patent No. ZL 00 1 12271.1 obtaining patent " the Cucumis wild species are used for the method for breed cucumber " in seminar, publication number: CN 1279009A, open day: January 10 calendar year 2001), we save by rataria, first the wild species C.hystrix of high downy mildew resistance and cultivated cucumber hybridization are obtained the double haploid hybrid, cultivate first Cucumis interspecific cross amphidiploid novel species C.hytivus in the world by chromosome doubling again.Amphidiploid novel species C.hytivus carries a complete set of chromosome set of wild species C.hystrix and cultivated cucumber, two cover chromosome groups often exchange in the reduction division process, produce the n=19 and n=7 gamete (the Chen J of transposition, Staub J, Qian CH, et al, Reproduction and cytogeneticcharacterization of interspecific hybrids derived from Cucumis hystrixchakr. * Cucumis sativus L.Theor Appl Genet.2003,106:688～695).Utilize amphidiploid novel species (effectively gamete n=19) to backcross and cultivate the allotriploid cucumber with cultivated cucumber, and patent applied for " seed selection of allotriploid cucumber and be used for the method for breed cucumber " (application number: 02148497.X, publication number: CN1413447, open day: 2003.04.30).Utilize simultaneously that amphidiploid novel species (effectively gamete n=7) and cultivated cucumber are backcrossed, selfing, obtain the colony of many chromosome number of somatic 2n=14.Carry out the analysis of peroxidase isozyme by self progeny that these are backcrossed, find that its genetic variation is than the abundant (Chen Jingfeng of common cultivation cucumber, appoint just, Yu Jizhu etc. the peroxidase enzymes analysis that inbreeding population is backcrossed in Cucumis distant hybrid. Wuhan botany research, 2002,20 (5): 336-337), show its part exogenous genetic material of may having transduceed, perhaps produced alien translocation line.
The translocation line that carries the useful gene of affinity species can be used as the parent of breed improvement, and some translocation lines with good genetic background itself are exactly improved seeds.At present, the method that produces alien translocation line of inducing commonly used mainly contains: (1) spontaneous generation transposition; (2) utilize the homeologous pairing to induce translocation line; (3) utilize ionizing radiation to bring out chromosome translocation; (4) utilize Plant Tissue Breeding to bring out transposition.Generally, the frequency of spontaneous generation translocation line is lower, but genetic equilibrium is better, and then three's occurrence frequency is but higher relatively.
Cucumber downy mildew is by Cuba false Pseudoperonospora cubensis Pseudoperonospora cubensis (Berk.﹠amp; Curt.) Rostov causes, is one of cucumber three big diseases, under suitable condition, PD is very fast, several days time just can spread to full field, caused yellow leaf, and is withered, production loss is big, the lighter's underproduction 10%-20%, heavy person reaches more than 30%, produces to cucumber and brings grave danger (Huang Zhongsheng, Zhang Zhili etc. identification of cucumber damage by disease and insect and control. Beijing: the .2002 of Chinese agriculture publishing house, 31-41).Because the cucumber hereditary basis is narrow, both at home and abroad to the resistant material of cucumber downy mildew only concentrate on a few anti-source (Wang Longzhi. the vegetable disease-resistant thremmatology. Beijing: the .1998 of Chinese agriculture publishing house, 142-144), and different anti-source material to the resistance mechanism difference of cucumber downy mildew.Have at present two kinds of results of study, promptly polygenic resistance and single recessive gene resistance (usury, Pang Jinan. the main disease genetics of resistance of cucumber progress.China's Vegetable .2004 (1): 63-66).Therefore, utilize the downy mildew resistance gene of wild species, cultivate the cucumber alien translocation line of downy mildew resistance, not only can introduce new anti-source, widen the existing hereditary basis of cucumber by distant hybridization; And can on producing, cultivate anti-cucumber downy mildew new varieties, enriched the content of cucumber downy mildew resistance mechanism in theory.
Three, summary of the invention
Technical problem the objective of the invention is at the present situation of directly utilizing at present Cucumis external source disease-resistant gene difficulty, the anti-source of the narrow and anti-cucumber downy mildew of cucumber hereditary basis deficiency, the selection of one cover cucumber downy mildew resistance alien translocation line is provided, obtain the disease-resistant alien translocation line of Cucumis, cucumber breeding for disease resistance new material is provided, cultivates disease-resistant new breeds of cucumbers.
The selection of technical scheme cucumber downy mildew resistance provided by the present invention alien translocation line CT-01, its embodiment is as follows:
1) utilizes patent No. ZL 00 1 12271.1, publication number: CN 1279009A, open day: the cucumber amphidiploid novel species that obtain January 10 calendar year 2001 was a male parent, with common cucumber " Beijing butt " is female parent, first backcross generation utilizes tip of a root meristematic cell chromosome sectioning technology and the inoculation test of downy mildew resistance field directly to screen, and obtains individual plant, this individual plant chromosome number 2n=14, high downy mildew resistance, the i.e. sick level 1.8 ± 0.5 of downy mildew;
2) to above-mentioned individual plant selfing offspring's screening, selecting individual plant is high downy mildew resistance, and promptly sick level is 2.8 ± 0.2, its leaf area 182.1 ± 26.1cm
2, the long 5.9 ± 3.1cm of joint, the long 15.6 ± 1.3cm of fruit, fruit footpath 3.3 ± 0.4cm, the fruit look is that dark green background color adds the light green color striped, it is brown that 7.5 ± 4.2 of number of branch, fruit are stung color; Its pollen mother cells process I in mid-term multivalent frequency 56 ± 8% and later stage I bivalent segregation lag rate 72 ± 5%;
3) can repeatedly amplify the specific DNA segment of original parent wild species therein with RAPD primer E-19:ACGGCGTATG and AI-20:CCTGTTCCCT to selected individual plant, clip size is 1200bp, be the cucumber alien translocation line of acquisition, name and be CT-01.
The selection of beneficial effect cucumber downy mildew resistance of the present invention alien translocation line CT-01 compared with prior art, has following advantage and good effect:
The present invention obtains by the selfing again of backcrossing of Cucumis interspecific cross amphidiploid novel species and common cucumber, be spontaneous fully, compare with the alien translocation line that brings out generation, have higher genetic equilibrium, stability, can be used for the practice of cucumber breeding for disease resistance as soon as possible.
Utilize technology such as field downy mildew resistance inoculation evaluation, economical character observation, cytology and RAPD molecular labeling to carry out the comprehensive evaluation of cucumber alien translocation line, conventional method and modern molecular biology means are organically combined, authenticate technology is comparatively comprehensive, confidence level is higher, the evaluation of Cucumis alien translocation line is had important reference be worth.
The alien translocation line that the present invention obtains has been widened the hereditary basis of cucumber, has enriched the cucumber anti-source material of downy mildew resistance, identifies that by inoculation the resistance of this translocation line is extremely strong, reaches high anti-level (sick level 2.8 ± 0.2).Its economical character is different from common cucumber, as its leaf area 182.1 ± 26.1cm
2, the long 5.9 ± 3.1cm of joint, the long 15.6 ± 1.3cm of fruit, fruit footpath 3.3 ± 0.4cm, the fruit look is that dark green background color adds the light green color striped, it all is its particular shape feature that 7.5 ± 4.2 of number of branch, fruit are stung color brown etc., has enriched the morphotype of common cucumber.
Utilize the present invention and comprehensive proterties good but need the cucumber combo of downy mildew resistance, be expected to cultivate the new breeds of cucumbers of downy mildew resistance.
Four, description of drawings
The seed selection of Fig. 1, cucumber downy mildew resistance alien translocation line CT-01 and be used for the family tree of breed cucumber
The less blade of Fig. 2, CT-01 and female flower (left side), blade that common cucumber " Beijing butt " is bigger and female flower (right side)
The fruit of the fruit of Fig. 3, CT-01 and common cultivation cucumber " Beijing butt "
Fig. 4, CT-01 chromosome number of somatic (A), I multivalent meiosis metaphase (B) and later stage I bivalent segregation lag (C)
Fig. 5, be primer has amplified wild species C.hystrix in CT-01 (swimming lane 2) characteristic strip with E-19
Fig. 6, be primer has amplified wild species C.hystrix in CT-01 (swimming lane 1) characteristic strip with AI-20
Five, embodiment
The selection of embodiment 1 cucumber downy mildew resistance alien translocation line CT-01 comprises:
1) (patent No. ZL 00 1 12271.1 to utilize the amphidiploid novel species of synthetic, publication number: CN 1279009A, open day: be male parent January 10 calendar year 2001), with common cucumber " Beijing butt " (public kind, country of the Chinese Academy of Agricultural Sciences preserves in vegetables germ plasm resource storehouse in mid-term) be maternal, backcross, in backcross population, utilize tip of a root meristematic cell chromosome sectioning technology and the inoculation test of downy mildew resistance field directly to screen and obtain chromosome number 2n=14 (Fig. 4 .A), downy mildew resistance, sick level is the individual plant HH of 1.8 ± 0.5 (tables 1)
1-8
2) to individual plant HH
1-8Carry out selfing and reserve seed for planting, and proceed the inoculation of disease resistance field in sowing in 1 year and identify, found that individual plant HH
1-8-57High downy mildew resistance, sick level 2.8 ± 0.2 (tables 1).It is carried out the economical character observed result find that this strain is at its leaf area 182.1 ± 26.1cm
2, the long 5.9 ± 3.1cm of joint, the long 15.6 ± 1.3cm of fruit, fruit footpath 3.3 ± 0.4cm, fruit look be that dark green background color adds aspect such as light green color striped between parent cucumber and wild species, sting aspects such as color is brown and be partial to wild species (table 2, Fig. 2, Fig. 3) more 7.5 ± 4.2 of number of branch, fruit.Observe its pollen mother cell (PMC) reduction division process, find its PMC I in mid-term multivalent frequency 56 ± 8% and later stage I bivalent segregation lag rate 72 ± 5% all higher (Fig. 4 .B, C).This phenomenon is seldom observed in common cucumber reduction division process, and preliminary supposition is caused by chromosome translocation, and this translocation line called after CT-01.Further utilize the RAPD molecular marker analysis, the result has found two primer E-19:ACGGCGTATG and AI-20:CCTGTTCCCT in 40 random primers, the size that can repeatedly amplify original parent wild species Cucumis hystrix in CT-01 is 1200bp specific DNA segment (Fig. 5, Fig. 6), has identified that from molecular level it is the cucumber alien translocation line.
The correlation technique that adopts in the above-mentioned cucumber downy mildew resistance alien translocation line seed selection process and and authentication method, comprising:
1) the downy mildew resistance inoculation identification method is:
1.1) the sick level of the downy mildew criteria for classifying: 0 grade, no scab; 1 grade, lesion area accounts for the 0-1% of whole leaf area; 2 grades, lesion area accounts for the 2-3% of whole leaf area; 3 grades, lesion area accounts for the 4-6% of whole leaf area; 4 grades, lesion area accounts for the 7-12% of whole leaf area; 5 grades, lesion area accounts for the 13-25% of whole blade area; 6 grades, lesion area accounts for the 26-50% of whole leaf area; 7 grades, lesion area accounts for the 51-75% of whole leaf area; 8 grades, lesion area accounts for the 76-100% of whole leaf area; 9 grades, plant death.
Divide resistance level according to sick level: high anti-, sick level 1.3-3.0; In anti-, sick level 3.3-5.0; Middle sense, sick level 5.3-7.0; High sense, sick level 7.3-9.0.(Todd C.Wehner.Downy mildewresistance of the cucumber germplasm collection in North Carolina fieldtests.Crop Sci.1997，37：1331-1340)
1.2) field downy mildew resistance inoculation identifies: parent, novel species, backcross material vernalization and sowings such as self progeny and susceptible variety Beijing spinule, and in 2 weeks of back of emerging by distance between rows and hills (the unified land for growing field crops that is colonizated in of 70cm * 30cm).In order to strengthen the infection dynamics and to remove marginal effect, every triplex row plantation delegation Beijing spinule.After 4 weeks of field planting, be 2.5 * 10 with spore concentration
4Individual/ml inoculates at field spray.Carry out the sick level statistics in field after inoculating for 2 weeks.
2) its economical character observation procedure is:
Long and the leaf area of the joint of observation parent, the amphidiploid hybrid and the self progeny's that backcrosses the 1st, 5,10,15 and 20 joints, number of branch, fruit length, the fruit look, fruit is stung proterties such as color, and the gravimetric detemination leaf area is adopted in every processing 5 strains.
3) tip of a root meristematic cell chromosome flaking method:
Getting the cucumber lateral root that is in the growth animated period places the 0.002mol.L that now joins is housed
-1The 10ml centrifuge tube of oxine pre-treatment medicaments in, handle 1-4h 19 ℃ of left and right sides lucifuges.With improvement Carnoy ' the s II fixer of now joining (absolute alcohol: chloroform: glacial acetic acid=5: 2: 3) more than the immobilization material 12h of low temperature dark place, change over to then in 70% alcohol, preserve standby in the refrigerator.In ultra thermostat (50l type), under 60 ℃ of strictness, use 1mol.L
-1HCl dissociates.Drip and dye 5min with the precious dye liquor of card, use 1% acetic acid color separation again, little roasting, strike sheet, compressing tablet.OLYMPUS (BX-51) sediments microscope inspection, photograph.30 meristematic cells that division is mutually good are observed in each film-making.
4) pollen mother cells conformational analysis:
Directly is the immature bud of 1-4mm at the climing growth of plant master 10 joints with interior cut-off, with improvement Carnoy ' s II fixer (absolute alcohol: chloroform: glacial acetic acid=5: 2: 3) fix 24 hours, change then that to be positioned over 4 ℃ of refrigerators preservations in 70% alcohol standby over to.When carrying out chromosome sectioning,, use rinsed with deionized water 5 minutes, get one of them flower pesticide and carry out preliminary microscopy at first to fixing bud rip cutting.The PMC that is in reduction division period is dyeed with carbolfuchsin, and the dyeing back covers with clean cover glass, does not press or minute-pressure, adopts the little roasting and 45% acetic acid color separation film-making of flame.With OLYMPUS (BX-51) microscopic examination and photograph.
5) RAPD molecular marker identification method:
Select for use Shanghai to give birth to 40 random primers of worker company (AI group and E group).Reaction system is 20 μ l, comprises Tris-HCl (pH=9.0) 10mM, KCl 50mM, and MgCl22.5mM, 4 kinds of nucleotide respectively are 150 μ M (Dalian is precious to produce), primer 0.4 μ M, template DNA 40ng, the Tag archaeal dna polymerase of 1.0u (Shanghai is given birth to the worker and produced).In PTC-100 PCR instrument (MJ, the U.S.), carry out.The response procedures condition is as follows: 94 ℃ of pre-sex change 4min of elder generation; 94 ℃ of sex change 15s, 35 ℃ of renaturation 15s, 72 ℃ are extended 75s, circulated for 3 weeks; 94 ℃ of sex change 15s, 40 ℃ of renaturation 15s, 72 ℃ are extended 75s, circulated for 40 weeks; 72 ℃ are extended 7min, preserve down for last 4 ℃.Product is electrophoretic separation in containing 1.2% Ago-Gel of EB, detects under the uviol lamp and takes pictures.
Cucumis downy mildew resistance alien translocation line CT-01 of the present invention carries out the application of breed cucumber:
With integral economic trait female series material good, the anti-cucumber downy mildew of needs is female parent, carries out combo with cucumber downy mildew resistance alien translocation line CT-01 of the present invention.By viewing test, variety comparative test and regional test, can filter out the new breeds of cucumbers of disease-resistant, high yield and high-quality.
The inoculation qualification result of table 1 application downy mildew resistance in 2 years
| Material | Sick level | Resistance level | ||
| Autumn in 2003 | Spring in 2004 | On average | ||
| Beijing butt | 5.5±0.2 | 6.1±0.7 | 5.8±0.5 | Middle sense |
| Wild species | 1.5±0.2 | 1.9±0.6 | 1.7±0.4 | High anti- |
| The amphidiploid novel species | 3.8±0.4 | 4.8±0.2 | 4.3±0.3 | In anti- |
| HH 1-8 | 1.8±0.5 | - | 1.8±0.5 | High anti- |
| HH 1-8-57(CT-01) | - | 2.8±0.2 | 2.8±0.2 | High anti- |
| Beijing spinule | - | 8.2±0.7 | 8.2±0.7 | High sense |
Table 2 application relates to the economical character of main material
| Morphological feature | Wild species | " Beijing butt " | The amphidiploid novel species | CT-01 |
| Joint long (cm) | 4.8±2.7 | 10.6±2.4 | 5.7±1.4 | 5.9±3.1 |
| Number of branch | 12.0±2.0 | 2.6±1.4 | 5.4±1.2 | 7.5±4.2 |
| Leaf area (cm 2) | 102.1±13.9 | 321.5±34.5 | 276.4±36.2 | 182.1±26.1 |
| Fruit long (cm) | 5.5±1.2 | 25±2.3 | 8.1±1.8 | 15.6±2.4 |
| Fruit footpath (cm) | 2.2±0.3 | 4.5±0.4 | 4.0±0.3 | 3.3±0.2 |
| The fruit look | Green | Blackish green | Pale green | Dark green background color adds the light green color striped |
| Fruit is stung color | Black | White | Black | Brown |
Claims (1)
1, the selection of cucumber downy mildew resistance alien translocation line CT-01 is characterized by:
1) utilizes patent No. ZL 00112271.1, publication number: CN 1279009A, open day: the Cucumis interspecific cross amphidiploid novel species C.hytivus that obtain January 10 calendar year 2001 was a male parent, with common cucumber " Beijing butt " is female parent, first backcross generation, utilize tip of a root meristematic cell chromosome flaking method and the inoculation test of downy mildew resistance field directly to screen, obtain individual plant, this individual plant chromosome number 2n=14, the sick level 1.8 ± 0.5 of downy mildew;
2) to above-mentioned individual plant selfing offspring's screening, selecting individual plant is that the sick level of downy mildew is 2.8 ± 0.2, its leaf area 182.1 ± 26.1cm
2, the long 5.9 ± 3.1cm of joint, the long 15.6 ± 1.3cm of fruit, fruit footpath 3.3 ± 0.4cm, the fruit look is that dark green background color adds the light green color striped, it is brown that 7.5 ± 4.2 of number of branch, fruit are stung color; Its pollen mother cells process I in mid-term multivalent frequency 56 ± 8% and later stage I bivalent segregation lag rate 72 ± 5%;
3) selected individual plant repeatedly amplifies the dna fragmentation of wild species C.hystix with RAPD primer E-19:ACGGCGTATG and primer AI-20:CCTGTTCCCT, and clip size is 1200bp, and this individual plant is the cucumber alien translocation line of acquisition, names to be CT-01.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104004749B (en) * | 2013-02-27 | 2016-05-04 | 中国农业科学院蔬菜花卉研究所 | Chain SSR labeled primer and the application thereof of cucumber black fruit Ci Jiyin B |
| CN103115811A (en) * | 2013-03-14 | 2013-05-22 | 南京农业大学 | Method for producing cucumber pachytene chromosomes |
| CN105821135B (en) * | 2016-04-29 | 2019-04-26 | 新疆农业科学院哈密瓜研究中心 | Molecular labeling and application with muskmelon downy mildew resistance gene close linkage |
| CN107711494A (en) * | 2017-11-24 | 2018-02-23 | 扬州大学 | A kind of breeding method of cucumber chromosomal Single Segment Substitution Lines in Rice |
| CN109601367B (en) * | 2018-12-06 | 2019-10-08 | 南京农业大学 | The breeding method of downy mildew resistance cucumber interspecific hybrid new varieties and application |
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