CN103355160A - Gradually oozing between the Cucumis kind is the method that colony made up and be used for breed cucumber - Google Patents
Gradually oozing between the Cucumis kind is the method that colony made up and be used for breed cucumber Download PDFInfo
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Abstract
Gradually oozing between Cucumis kind of the present invention is the method that colony made up and be used for breed cucumber, belongs to the biotechnology breeding field.By wild pickled cucumber and seed of Fructus Cucumidis sativi intermolecular hybrid-backcross-selfing, in conjunction with the SSR molecular marker assisted selection, made up successfully that gradually to ooze between the first kind of the Cucumis that is comprised of 86 parts of strains be colony.This gradually oozes is that colony has comprised the introgressed segment that covers the wild pickled cucumber of the full genome 54% of cucumber, and identify by Field inoculation, screen disease resistance stronger three parts of resources " IL2 ", " IL24 ", " IL49 ", it can be obtained again a series of cucumber new lines with stronger resistance against diseases respectively with after the good cultivated cucumber hybridization.Gradually oozing between cucumber/pickled cucumber kind is the hereditary basis that colony has not only widened cucumber, and for the excellent genes that develops wild pickled cucumber provides new germ plasm resource, also established material foundation for the important gene of locating from now on cucumber or genetic locus and functional study.
Description
Technical field
The present invention relates to gradually ooze between the Cucumis kind is the construction method of colony, belongs to the biotechnology breeding field.
Background technology
Cucumber is a kind of important worldwide vegetable crop of Curcurbitaceae Cucumis, and nutritious, the wild germplasm resource is more, and damage by disease and insect is a lot of simultaneously.And hereditary basis is narrow in the seed of Fructus Cucumidis sativi, and polymorphism only is 3%-8%, and the long-term use of Core Germplasms resource causes genetic background more and more single, and offspring's variation type is fewer and feweri, and the ability of resisting various damage by disease and insect and natural adverse circumstance is also more and more lower.Therefore in the face of the day by day exhaustion of planting interior excellent resources and the bottleneck that conventional breeding is difficult to break through, excavate excellent wild germplasm, outside seed of Fructus Cucumidis sativi, seek the various excellent proterties of its urgent need, become an important channel of acceleration breed cucumber process.
Gradually ooze between kind is that colony backcrosses by many generations after the interspecific cross to form with selfed breeding.It is that colony at first will select the good kind of Comprehensive Traits as receptor parent that structure gradually oozes, have the wild resource of the excellent proterties of other or sibling species as donor parents, can have complementary advantages like this, gradually oozing is the deficiency that has also remedied receptor parent when obtaining the receptor parent merit.In addition, gradually oozing is that it has overcome the defective of traditional QTL target group as a kind of new breeding population resource, can carry out meticulous location to the quantitative trait locus of crop, and gene location and breed breeding are perfectly united.
Shifting the useful proterties of external source by interspecific cross is used widely in paddy rice, tomato and Soybean and Other Crops.Chen Jingfeng broke through the various obstacles of distant hybridization in 2000, utilizing cultivated cucumber ' Beijing butt ' is receptor parent, (the Cucumis hystrix Chakr. of the wild species pickled cucumber with multiple disease-resistant adversity gene from Xishuangbanna, 2n=24) synthesized at present unique in the world species hybrid allotetraploid (Chen Jingfeng for donor parents, village scud, Qian Chuntao. Cucumis one new species (amphidiploid) synthetic and qualitative [J]. Wuhan botany research, 2001,19 (5): 357-362).Day by day poor and damage by disease and insect spreads unchecked day by day in the face of the resource in the present breed cucumber, utilize wild pickled cucumber and cultivated cucumber to carry out the germ plasm resource that interspecific cross can be enriched cultivated cucumber, gradually oozing between structure is planted on this basis is that colony not only can transfer to the merit of external source wild species in the cultivated species, the excellent gene of Cucumis wild species can also be deeply excavated, the disease-resistant adversity gene of the wild pickled cucumber in meticulous location might be realized.
The paper that this seminar delivers in earlier stage " structure of chromosome introgressive line and genetic evaluation between the cultivated cucumber kind " (Shi Jianlei, Agricultural University Of Nanjing's master thesis, 2009) and " cucumber-structure of pickled cucumber chromosome segment introgressive line colony and the location of fruit correlated quantitative traits gene " (Meng Jiali, Scientia Agricultura Sinica, 2012,45 (8): 1558-1567), we have also made up gradually to ooze between cucumber/pickled cucumber kind, and it is estimated, excellent proterties for the wild pickled cucumber of transduceing, the cultivation new breeds of cucumbers is significant, has greatly advanced the process of cucumber genetic breeding.But be limited to the inbreeding population that backcrosses of getting up early in these researchs to gradually oozing the screening that is, the individual plant introgressed segment is more, and the mark of use is also less, and colony's coverage rate is on the low side, does not also carry out the screening of disease-resistant strain.Therefore, in order to excavate more fully and utilize the excellent gene of wild species, we have carried out widely screening to high for the inbreeding population that backcrosses by the binding molecule mark, and having made up new gradually oozing is colony, improve coverage rate, and laid material foundation for the breeding of new variety in later stage.
Summary of the invention
The object of the invention provides between a kind of cucumber/pickled cucumber kind gradually oozes the breeding method that is, comprises that mainly gradually oozing is the contents such as the structure of colony and evaluation.
The concrete steps of technical scheme are as follows:
1. gradually oozing between the Cucumis kind is the structure of colony:
Take the wild species pickled cucumber as maternal, cultivated cucumber " Beijing butt " obtains hybrid F1 for paternal hybrid, hybrid F1 by embryo rescue in conjunction with somaclonal variation chromosome doubling technology, allotetraploid novel species between the kind that acquisition can be educated, called after Cucumis hytivus, take allotetraploid novel species Cucumis hytivus as maternal, backcross and selfed breeding by system with cultivar " Beijing butt ", a series of offsprings that carry the wild species chromosome segment have been obtained, comprise BC6F1, BC7F1, BC8F1, and BC3F4, BC3F5, BC3F6 etc.
2. gradually ooze is the Screening and Identification of colony:
Utilization is distributed in the SSR mark on the Cucumber Genetic Map, the a series of progeny materials that obtain are carried out first foreground selection, namely obtain really to contain the strain of wild species chromosome segment, then carrying out background selects, the recycling molecular labeling is eliminated Linkage drag, and gradually oozing between the kind that has finally obtained to be made of 86 parts of strains is colony, length average out to 4 centimorgans of introgressed segment, 7 linkage groups of cucumber include 337 wild species fragments altogether, and the coverage rate that ties up in the genome of gradually oozing of screening is 54%.
3. goodly gradually ooze the application that is:
By field paired observation and inoculated identification, gradually ooze between above-mentioned kind is to have filtered out high downy mildew resistance strain ' IL2 ' and anti didymella and fusarium wilt strain ' IL24 ' and three parts of disease-resistant materials of anti-Meloidogyne incognita strain ' IL49 ' the colony, after being purified, a series of cucumber new lines with stronger resistance against diseases have been obtained again respectively with after the good cultivated cucumber hybridization.
Beneficial effect
1. utilization of the present invention unique seed of Fructus Cucumidis sativi species hybrid in the world, having made up by the selfing that backcrosses that a Cucumis gradually oozes is colony, not only can widen the hereditary basis of cultivated cucumber, is more conducive to from now on to the excellent heterogenic exploitation of wild pickled cucumber and efficient the utilization.
2. the present invention utilizes the published covering full genome of cucumber and whole SSR molecular labelings of being distributed on seven chromosomes are that colony identifies and estimates to gradually oozing of obtaining, determined every portion gradually ooze be in position and the size of wild pickled cucumber fragment, and the clear and definite coverage rate of fragment on Cucumber germplasm of gradually oozing, for later on location and clone and functional analysis provides more efficiently approach from specific gene or the QTL of wild pickled cucumber.
3. the present invention is by being that colony carries out that Field inoculation is identified and sick level statistics to gradually oozing of obtaining, three parts of disease-resistant resources have been obtained, they have shown stable resistance against diseases to pathogen, and itself and cultivated species continued hybridization, and then disease-resistant gene transferred in the good cultivated species widely, therefore these resources can be used as Bridge parent from now on, accelerate the molecular breeding process of cucumber, will exert far reaching influence to the cucumber variety improvement.
Description of drawings
Fig. 1: gradually ooze the structure flow process that is between the Cucumis kind
Fig. 2: the FISH of allotetraploid identifies (danger signal is the cucumber centromeric sequence, and signal has 14)
Fig. 3: the polymorphic primer between wild species and cultivated cucumber ' Beijing butt '
Fig. 4: SSR labeled primer 16305 gradually oozes the amplified band of fastening parent and 86 parts
Fig. 5: 86 parts gradually ooze is the distribution of introgressed segment on Cucumber germplasm of strain
Fig. 6: the plant forms of disease-resistant material ' IL2 ', ' IL24 ', ' IL49 '
Embodiment
Cucumber is the important vegetable crop of China, expanding day along with the facility cultivation area, the difficulty of its disease control is also increasing, and the good disease-resistant gene of shortage in the cucumber cultivation kind, the present invention is on the basis of interspecific cross, the binding molecule marker assisted selection, identify and filtered out and contain wild pickled cucumber fragment and 86 parts of less strains of individual plant introgressed segment number and formed that gradually to ooze be colony, and utilize GGT software that the position of introgressed segment on cucumber chromosome of this colony positioned, therefrom screen three parts of strains that resistance against diseases is stronger by Field inoculation, and continue itself and common cucumber are hybridized, it is some to have obtained good cucumber new lines.
Gradually ooze the structure flow process that is between kind and see figure one, detailed process is as follows:
1. the acquisition of backcross population between seed of Fructus Cucumidis sativi:
Wild species pickled cucumber (Cucumis hystrix, 2n=24) be short-day plant, and the initial stage growing way is slower, therefore than the sowing in 2 months in advance of common cucumber, and its photoperiod of carrying out more than the 40d processed (illumination 16 hours every days, 8 hours dark), make itself and cultivated cucumber flower synchronization, take the wild species pickled cucumber as maternal, cultivated cucumber " Beijing butt " is paternal hybrid, Hybrid F1 passes through embryo rescue (MS medium) in conjunction with somaclonal variation chromosome doubling technology, allotetraploid novel species (cytological Identification is seen figure two) between obtaining to plant, called after Cucumis hytivus is afterwards take the allotetraploid novel species as maternal, backcross more than 3 generations with " Beijing butt ", in 3～5 generations of selfing, obtain progeny population strain, i.e. BC6F1 again, BC7F1, BC8F1, and BC3F4, BC3F5, BC3F6 etc., show that through the somatic chromosome qualification result its chromosome number is identical with the common cultivation cucumber, 2n=14.
2. gradually oozing between planting is the screening of strain:
After the plant seedling, get 2～3 of each strain young leaves, liquid nitrogen flash freezer is preserved, and then utilizes the CTAB method to extract genomic DNA, and (final concentration is 100 μ gmL to use RNaseA again
-1) removed RNA in 20 minutes at 37 ℃ of incubations, carry out at last the electrophoresis of DNA with 1.0% Ago-Gel, and with the ethidium bromide development process concentration of DNA is carried out quantitatively.DNA behind the purifying is kept in-20 ℃ of refrigerators.
At first carry out foreground selection, namely with the complete genomic SSR molecular labeling of published covering cucumber to pickled cucumber with ' Beijing butt ' two parent carries out polymorphism screening, obtains altogether polymorphic mark 359 to (seeing figure three); Then carrying out background selects, utilize these 359 pairs of SSR marks that backcrossing of obtaining in the process 1-each generation population of selfing is analyzed, wherein there are 112 pairs to be marked at the specific band (seeing figure four, take mark 16305 as example) that has detected wild pickled cucumber in the progeny population.It is the least possible that selection contains the donor chromosome segment, the higher material of while coverage rate, and elimination background burden has been set up afterwards gradually to ooze between the Cucumis kind that is made of 86 parts of strains and has been.Concrete technology is as follows:
1) PCR system: total reaction volume is 20 μ L, wherein comprises ddH
2011.3 μ L, 10 * Buffer2.0 μ L, Mg
2+1.5mmol.L
-1, dNTP 150 μ molL
-1, each 0.5 μ molL of forward primer and reverse primer
-1, template DNA 40ng, Taq archaeal dna polymerase 1.0U.The program of amplification is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 80s, 35 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations.The instrument that the PCR reaction is adopted is PTC-100TM PCR instrument (MJR esearch Inc., USA).
2) detection of PCR product: the method that adopts polyacrylamide gel electrophoresis and silver to dye.Pcr amplification product separates at 7% polyacrylamide gel, voltage 200V, electrophoresis 1～2h.A. fixing: as gel to be taken off from glass plate, place pallet, add the 200mL fixer that contains 5% acetic acid, 10% absolute ethyl alcohol, jiggle fixedly 15min at shaking table.B. dyeing: reclaim fixer, then add 200mL in the gel and contain 0.2%AgNO
3Dyeing liquor, jiggle on the shaking table dyeing 15min (lucifuge).C. rinsing: reclaim dyeing liquor, use first rinsed with deionized water 30～60s, again with containing 0.2%Na
2S
2O
3200mL rinsed with deionized water 60s.D. colour developing: abandon rinsing liquid, with containing the 200mL developing liquid developing of 1.5%NaOH, 1%HCHO, clear to band till.Stop for twice with the running water flushing at last.The preservation liquid d (NaHCO that can add 200mL
315gL
-1) preserve.
3. gradually ooze the evaluation that is between planting:
The computational methods of introgressed segment length: do not consider the interval double crossing over event that occurs of two adjacent markers, when the genotype of adjacent marker on the chromosome is identical, just think that the chromosome segment between these two marks is comprised of identical marker genetype; When the genotype of adjacent marker not simultaneously, just think that these two marker genetypes form respectively half of this interval.
In pcr amplification product, banding pattern performance and receptor parent ' Beijing butt ' consistent being designated as " a ", wild with donor parents ' pickled cucumber ' consistent being designated as " b ", the disappearance band is designated as " u ", and new banding pattern is designated as " n ", and the heterozygosis banding pattern is designated as " h ".Be marked at position on the Cucumber germplasm according to SSR, respectively gradually oozing with the EXCEL statistics is the size of middle introgressed segment and the number of introgressed segment, the genotype diagram (seeing figure five) that recycling GGT genotype mapping software is drawn whole individual plants, analyze each gradually ooze be the size of introgressed segment in the strain and in Cucumber germplasm shared ratio.
86 parts gradually ooze is the introgressed segment that has carried altogether 337 wild pickled cucumbers, and average every part gradually to ooze be that 4 introgressed segments are arranged, and 78 different introgressed segments are wherein arranged, and total length is 312.6cM, fragment average length 4cM, and the genome coverage rate is 54%.In addition, the chromosome segment number that imports on 7 cucumber chromosome does not wait, and wherein the introgressed segment number on No. 1 chromosome is maximum, has 105; Introgressed segment minimum number on No. 7 chromosome has 7; 48 introgressed segments on average every chromosome.
4. gradually ooze the screening of disease-resistant material in being:
(cucumber-pickled cucumber downy mildew resistance gradually oozes the expression analysis of the sifting machine apoptosis related gene that is molecular labeling with reference to Zhao Zhenguo, Agricultural University Of Nanjing's master thesis, 2011) downy mildew resistance authentication method, (it is the resource evaluation of anti didymella that cucumber-pickled cucumber gradually oozes to Lou Lina, genetic analysis and main effect QTL location, Agricultural University Of Nanjing's doctorate paper, 2011) climing rot Resistance Identification method, (cytogenetics of cucumber alien translocation line forms mechanism and to the genetics of resistance characteristics of fusarium wilt to Qian Chuntao, Agricultural University Of Nanjing's doctorate paper, 2006) fusarium wilt disease resistance authentication method, Zhang Yanxia (patent of invention, the anti-Meloidogyne incognita cucumber is gradually oozed the selection of based material, publication number CN102257953A, 2011) resistant to southern root knot nematode authentication method is that colony carries out the Field inoculation evaluation to gradually oozing of acquisition.Therefrom selected the most obvious three parts of resources of resistance (seeing figure six), the result is: IL2 shows as high resistance (disease-resistant grade 1, blade almost not or a small amount of little scab arranged) to downy mildew; IL24 shows as anti-(disease index is between 15～45) to climing rot, and IL24 shows as anti-(visible scab appears in seedling hypocotyl, cotyledon margin etc. in disease-resistant grade 2, or the local yellow of cotyledon) to fusarium wilt; IL49 shows as anti-(disease index is 2,3～10 root knots and pieces of an egg) to Meloidogyne incognita.
Gradually oozing between the Cucumis kind that application the present invention obtains is that colony is a permanent colony with wide application prospect.At first, genotype is isozygotied unanimously in its each strain, only be that a genotype has difference, be not embodied on the full genomic level and this species diversity only is confined between being in the minority chromosome segment, so it can carry out the location of cucumber QTL and the analysis of hereditary effect more quickly than traditional segregation population; Secondly, it also is more efficient utilizing this gradually to ooze to be colony that the disease-resistant adversity gene from wild species is positioned and clones, and is that colony can be further converted to the overlapping system that near-isogenic line maybe can be carried out the chromosomal substitution mapping because gradually ooze; Again, utilizing this gradually to ooze is the QTL that colony can also detect very minor effect, and reaches the mutual work between QTL and environment between convenient research QTL.
Claims (3)
1. gradually oozing between the Cucumis kind is the method that colony made up and be used for breed cucumber, mainly comprises:
Gradually ooze between kind is that the structure of colony at first will obtain a plurality of progeny populations by the hybridization of allotetraploid and cultivated cucumber and the again selfing that backcrosses; Then utilize the full genome SSR of cucumber mark to carry out foreground selection and background selection, it is burdensome to eliminate background, and it is the least possible to select to contain the donor chromosome segment, simultaneously the higher material construction colony of coverage rate; The main statistics band consistent with the donor parents wild species identified colony and estimated; And this colony carried out inoculated identification, and filter out the stronger strain of resistance against diseases as female parent, continue and good cultivated species hybridization, then shift resistant gene.
2. gradually oozing between kind according to claim 1 is the construction method of colony, it is characterized in that:
1) take the allotetraploid cucumber as maternal, cultivated cucumber is that male parent is hybridized, and backcrosses as recurrent parent with cultivated cucumber again; And the number of times that backcrosses is in 3～8 generations, and the selfing number of times is more than 1～6 generation;
2) to utilize mark to screen to the self progeny colony that backcrosses, at first sieve parent's polymorphism, sieve again colony, improve efficiency of selection;
3) evaluation of introgressed segment mainly is will select to amplify that gradually to ooze be mark with the consistent band of the wild pickled cucumber of donor parents; In addition, also will add up the heterozygosis band, new band lacks generation;
4) to gradually ooze be the Disease Resistance Identification of colony after, be to do maternal and a plurality of good cultivated speciess hybridization with disease-resistant gradually oozing, come the seed selection new varieties.
According to claim 1 and 2 described methods make up that gradually to ooze between the Cucumis kind of the covering Cucumber germplasm 54% that 86 parts of strains forming form be colony and the application in breed cucumber thereof.
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Cited By (6)
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| CN104737901A (en) * | 2015-04-15 | 2015-07-01 | 南京农业大学 | Method for screening mycosphaerella melonis resistance IL14 of cucumber-pickled cucumber introgression line |
| CN106171963A (en) * | 2016-08-25 | 2016-12-07 | 南京农业大学 | The breeding method of Fructus Cucumidis sativi anti didymella Introgressed line HH1 812 and resistant gene site molecular marker |
| CN106258945A (en) * | 2016-10-24 | 2017-01-04 | 新疆农垦科学院 | A kind of method gradually oozing method transformation high-quality color sea island cotton |
| CN108866233A (en) * | 2018-08-23 | 2018-11-23 | 中国农业科学院郑州果树研究所 | For identifying marker site, primer pair, kit and application of the peach to disease-resistant/susceptible character of Meloidogyne incognita |
| CN110150050A (en) * | 2018-03-15 | 2019-08-23 | 中国农业科学院蔬菜花卉研究所 | A kind of anti-root-knot nematode pumpkin rootstock and its selection and application |
| CN111713400A (en) * | 2020-07-03 | 2020-09-29 | 南京农业大学 | Creation and identification method of cucumber-pickled cucumber two-body additional line |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104737901A (en) * | 2015-04-15 | 2015-07-01 | 南京农业大学 | Method for screening mycosphaerella melonis resistance IL14 of cucumber-pickled cucumber introgression line |
| CN106171963A (en) * | 2016-08-25 | 2016-12-07 | 南京农业大学 | The breeding method of Fructus Cucumidis sativi anti didymella Introgressed line HH1 812 and resistant gene site molecular marker |
| CN106258945A (en) * | 2016-10-24 | 2017-01-04 | 新疆农垦科学院 | A kind of method gradually oozing method transformation high-quality color sea island cotton |
| CN110150050A (en) * | 2018-03-15 | 2019-08-23 | 中国农业科学院蔬菜花卉研究所 | A kind of anti-root-knot nematode pumpkin rootstock and its selection and application |
| CN108866233A (en) * | 2018-08-23 | 2018-11-23 | 中国农业科学院郑州果树研究所 | For identifying marker site, primer pair, kit and application of the peach to disease-resistant/susceptible character of Meloidogyne incognita |
| CN108866233B (en) * | 2018-08-23 | 2021-06-25 | 中国农业科学院郑州果树研究所 | Marker locus, primer pair, kit and application for identifying disease resistance/infection traits of peach trees to meloidogyne incognita |
| CN111713400A (en) * | 2020-07-03 | 2020-09-29 | 南京农业大学 | Creation and identification method of cucumber-pickled cucumber two-body additional line |
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