CN102827837A - Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker and use thereof - Google Patents
Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker and use thereof Download PDFInfo
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Abstract
The invention discloses a Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker and a use thereof, and relates to the field of biotechnology-assistant breeding. The Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker has a sequence of SSR06791-F/SSR06791-R: TTTGTAGTTTGAGAACTCAAGTTGG/TGGTGTTTGGTTGTCCTGAG. Amplified characteristic bands of the Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker are shown in the formula of Seq ID No: 1 (219bp) and in the formula of Seq ID No: 2 (234bp). Through the Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker, tender-fruit white-peel material screening can be carried out in any stages of Cucumis sativus L. candidate materials. The Cucumis sativus L. tender-fruit white-peel gene w linked SSR marker has high efficiency and a low limit, improves efficiency of white-peel Cucumis sativus L. breeding, and shortens a breeding period.
Description
Technical field
The present invention relates to chain SSR mark and the application in seed of Fructus Cucumidis sativi money is selected thereof of the tender fruit white of biotechnology assistant breeding, particularly cucumber peel gene w.
Background technology
Cucumber (Cucumis sativus L.) is one of the world ten big vegetables, because of its local flavor delicate fragrance, mouthfeel are crisp and refreshing, receives liking of various countries human consumer deeply.Along with the raising of the level of consumption, people grow with each passing day to the attention degree of cucumber quality.Quality breeding has also become one of emphasis of breed cucumber.The cucumber fruits quality comprises exterior quality and interior quality.Usually the quality trait of saying refers to that mainly commodity is an exterior quality, comprises melon look, melon length, fruit thorn, fruit knurl, fruit color uniformity, glossiness etc.Tender fruit fruit colour is as important commodity property, and is very big to the merchandise sales influence.The genetic development and the molecule marker of the tender fruit fruit colour of research cucumber have important practical significance.
The gene of the tender fruit fruit colour of relevant controlling cucumber has been reported w (white pericarp), DG (deep green pericarp), dg (green pericarp), yellow-green colour pericarp (yg), D (dark-coloured pericarp) (Pierce and Wehner, 1990; Wehner and Staub, 1997; Gu Xingfang etc., 2005).Forefathers have carried out multinomial research for the genetic development of tender fruit fruit colour.Cochran (1938) the tender fruit white skin of report (w) is recessive in green.Youngner (1952) thinks that tender fruit yellow-green colour pericarp proterties controlled by yg, and yg is bottle-green gene to the control pericarp and shows as recessiveness, the control fruit colour is jade-green gene shows as epistasis.SUN Xiaodan etc. (2011) research shows that the gene (w) of controlling the white pericarp of tender fruit is recessiveness to the gene (yg) of controlling tender fruit pericarp green, and w and the mutual work of other modifying factor existence, is recessive epistasis as type mutually.SUN Xiaodans etc. (2011) have also been studied the genetic affinity between w and other proterties simultaneously, find yg, w, and the genetic affinity between Tu (fruit knurl) and the Se (shape of fruit end, name temporarily) is independent inheritance.
Research for cucumber fruit colour molecular level is few; Li Yali (2008) has made up F2 colony with WD3 * B-2-2; Adopt the BSA method to combine the SRAP molecular marking technique; Find relevant molecule marker ME9EM1-309 and the ME8EM14-425 of the green proterties of cucumber pericarp, genetic distance is respectively 6cM and 8.3cM.The investigator is also arranged with the tender fruit white of AFLP molecule marking research cucumber fruit colour genetic development, found 2 the AFLP marks chain with w, E43M61 and E34M59, genetic distance are respectively 5.2cM and 5.6cM (SUN Xiaodan, 2011).
This test is that the parent makes up F2 and backcross population with tender fruit green pericarp self-mating system 04796 (P1) and tender fruit white pericarp self-mating system Bai Yesan (P2); Through the phenotypic evaluation to 6 generation population, w has carried out the genetic development analysis to the tender fruit white skin of cucumber gene.With F2 colony is mapping population, utilizes BSA method and SSR technology, realizes the chromosomal localization of w gene, and obtains the close linkage mark.Fine Mapping and molecular cloning that this test is not merely the tender fruit white skin of cucumber gene w lay the foundation, simultaneously also for utilizing molecular marking supplementary breeding white pericarp new breeds of cucumbers that theoretical foundation is provided.
Summary of the invention
The present invention is based on the blank in above-mentioned field; The chain SSR mark with tender fruit white peel gene w is provided; And provide it in the application of selecting on the adularescent pericarp cucumber germ plasm resource; For Fine Mapping and the molecular cloning of the tender fruit of cucumber white peel gene w lays the foundation; Can any stage of cucumber candidate material to its screening of carrying out white pericarp, have efficient, restriction less, advantage accurately, for utilizing molecular marking supplementary breeding cucumber white pericarp kind practical, approach efficiently is provided.
The closely linked SSR mark of the tender fruit white pericarp w of cucumber, sequence is following:
The characteristic band of its amplification of SSR06791-F/SSR06791-R:TTTGTAGTTTGAGAACTCAAGTTGG/TGGTGTTT GGTTGTCCTGAG is shown in Seq ID No.1 shown in (219bp) and the Seq ID No.2 (243bp);
With the chain fragment of the tender fruit green pericarp of fruit W shown in SEQ ID No.1: (219bp)
TTTGTAGTTTGAGAACTCAAGTTGGTTAGTTATTCTTCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTACCTTTACCTTTATACTTCATCTTTGGTTTAAAAAGTATCCATTGCAATATTATTCTTACCGTTCATAAGTATTTGTAAACTACCATTTGGAGTAGAACTCAGGACAACCAAACACCA
With the chain fragment of the tender fruit of fruit white pericarp w shown in SEQ ID No.2: (243bp)
TTTGTAGTTTGAGAACTCAAGTTGGTTAGTTATTCTTCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTGGCTTTACCTTTACCTTTATACTTCATCTTTGGTTTAAAAAGTATCCATTGCAATATTATTCTTACCGTTCATAAGTATTTGTAAACTACCATTTGGAGTAGAACTCAGGACAACCAAACACCA
Above-mentioned SSR is marked at the application of screening in the cucumber germ plasm resource with tender fruit white peel gene w, and its step is following:
(1) adopt above-mentioned SSR to be labeled as primer the genomic dna of cucumber variety to be selected carried out pcr amplification respectively,
(2) amplification is carried out detected through gel electrophoresis;
(3) amplify the segmental material shown in SEQ ID No.2 from screening the detected result.
Said PCR reaction system is: 7.5ng DNA; Each 50ng of forward and reverse primer; 5 μ L Go
Green MasterMix adds distilled water to 10ul.
Said pcr amplification program is: 94 ℃ of preparatory sex change 4 minutes; 94 ℃ of sex change 15 seconds, 55 ℃ of annealing 15 seconds, 72 ℃ were extended 35 circulations 30 seconds; 72 ℃ are incubated 5 minutes, 16 ℃ of preservations.
Said detected through gel electrophoresis: refer to adopt 6% non-denaturing polyacrylamide gel, in the permanent power electrophoretic separation of 160V, last silver dyes colour developing.
This test is that the parent makes up F with green self-mating system 04796 (P1) of the tender fruit pericarp of cucumber and tender fruit white skin self-mating system Bai Yesan (P2)
2And backcross population, through phenotypic evaluation, tender fruit white skin gene w has been carried out the genetic development analysis to 6 generation population.Again with F
2Colony is a mapping population, utilizes the BSA method to screen the mark SSR06791 chain with w, with the genetic distance of w be 8.6cM.
Through other the 30 parts of material identifications with different genetic backgrounds, the rate of accuracy reached 80% of mark.
Fine Mapping and molecular cloning that this test is not merely the tender fruit white skin of cucumber gene w lay the foundation, simultaneously also for utilizing molecular marking supplementary breeding white pericarp new breeds of cucumbers that theoretical foundation is provided.
Description of drawings
Fig. 1. be SSR mark SSR06791 to cucumber parents material 04796, Bai Yesan, F1 be individual plant and F from generation to generation
2Colony is the detected result of individual plant at random;
Wherein: P
1: 04796 (pericarp is green); P
2: Bai Yesan (white skin).
Embodiment
Materials and methods
The test materials that this institute uses is ' 04796 ' (P
1), ' Bai Yesan ' (P
2).
' 04796 ' be the height that forms by the excellent No. 1 inbreeding of more generation directive breeding in elite hybrid Tianjin for self-mating system, the pericarp deep green, the thorn knurl is close, thorn white, the fruit knurl is little.Existing known kind; Also on the books in article " seed selections that heat-resisting new breeds of cucumbers middle peasant the is No. 106 " literary composition that Gu Xingfang etc. delivered in " China's Vegetable " 2008 the 6th phases; There is preservation in this laboratory, guarantees in 20 years applyings date, to be used for confirmatory experiment to public's granting.
' Bai Yesan ' is the pure lines that filter out among the domestic good local variety Bai Yesan, white skin, and the thorn knurl is rare, thorn white, the fruit knurl is bigger.Existing known kind; Also on the books in " agriculture of gansu science and technology " article that the 12nd phase delivered in 2008 " 9 white Beijing opera melon kinds are in the comparative test result of introducing a fine variety of Long Dong " literary composition at Zhang Zhanjun; There is preservation in this laboratory, guarantees in 20 years applyings date, to be used for confirmatory experiment to public's granting.
The SSR primer is from international cucumber gene group plan (CUGI), and totally 2112 to (Ren et al., 2009).Detailed results can be referring to Ren etc. at the PloS One online article of delivering of 2009 the 4th phases " An Integrated Genetic and Cytogenetic map of the cucumber Genome ".The GoTaq Green Master Mix of Shang hai Promega company is used in the PCR experiment; Gel electrophoresis is used and to be full of 40% non-denaturing polyacrylamide of Xinyang light company, it is diluted to 6% back uses.
The screening of the chain SSR mark of embodiment 1. cucumber fruit white skin gene w
Step 1. cucumber fruit colour macroscopical identification
Test in 2008-2009 and carry out, with 04796 (P in Vegetable & Flower Inst., Chinese Academy of Agriculture Science experiment base
1) and Bai Yesan (P
2) originally carry out reciprocal cross, selfing, backcross for father and mother, obtain F
1, F
2And BC
1P
1, BC
1P
2Colony.
Spring in 2009 was planted P in Vegetable & Flower Inst., Chinese Academy of Agriculture Science's experiment booth
1, P
2And F
1, F
1' each 10 strain, F
2189 strains, BC
1P
178 strains, BC
1P
280 strains.Spacing in the rows 25cm, line-spacing 55cm is by conventional field management.
In the phase of yielding positive results, investigate (the fruit colour of the back 7 ~ 10d) of blooming of commodity melon on each individual plant of six generation population.
In six generation population of plantation, the The Characters of investigation fruit colour is calculated and is separated ratio, uses Microsoft Excel2003 software to carry out data statistic analysis, uses SAS8.0 that the result is carried out Chi-square test.
The result shows: reciprocal cross offspring F no matter
1Pericarp all shows as green; At F
2In the colony; It is that the backcross population of backcross parent all shows as the pericarp green with 04796 that the segregation ratio of green pericarp and white pericarp plant meets 3:1 through Chi-square test; And backcross in the backcross population that obtains with another parent Bai Yesan, the green ratio with white skin of pericarp is 1:1.Hence one can see that, and tender fruit white skin proterties is controlled by 1 pair of nuclear gene, and be w with this unnamed gene, and pericarp green (W) is a dominance to white skin (w).
Step 2.DNA extracts and ssr analysis
Get the tender leaf of cucumber plant, extract the genomic dna of parent and each individual plant of colony with improved CTAB method (cetyl trimethylammonium bromide) method.
The PCR reaction system is: total reaction system 10 μ L; 3 μ L DNA (2.5ng μ L-1); Each 1 μ L of forward and reverse primer (50ng μ L-1), 5 μ L Go
Green Master Mix (Promega Company products).Primer uses SSR primer (Ren et al., 2009 of the full gene sequencing exploitation of cucumber; Cavagnaro et al., 2010).The pcr amplification program is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 15s, 55 ℃ of annealing 15s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of insulation 5min, 16 ℃ of forever.Amplified production separates with 6.0% non-denaturing polyacrylamide gel, and electrophoretic buffer is 0.5 * TBE, the permanent power electrophoretic separation of 150V 1.5h, and silver dyes colour developing behind the electrophoresis, the statistics banding pattern.
Step 3.SSR label screening, data statistics and linkage map make up
Mainly comprised for 4 steps: (1) screening is at the polymorphic SSR mark of this performance of father and mother; (2) in F2 colony, choose each 7 of the DNA of pericarp green, white skin individual plant, it is green to make up pericarp according to the BSA method, and polymorphism primer is screened with the pond in 2 near isogene ponds of white; (3) utilize the polymorphism primer that obtains to analyze each individual plant genotype of F2 colony; (4) utilize JoinMap4.0 software to carry out the drafting of linkage map.
Codominant marker's statistical method: be designated as a with maternal (04796) consistent banding pattern, be designated as b with the consistent banding pattern of male parent (Bai Yesan), the banding pattern of heterozygosis is designated as h.Dominant marker's statistical method: if female parent is the dominant marker, then the individual plant consistent with maternal banding pattern is designated as d in the segregating population, consistent with the male parent banding pattern b that is designated as; If male parent is the dominant marker, then the individual plant consistent with maternal banding pattern is designated as a in the segregating population, consistent with male parent banding pattern c, the u that is designated as that do not amplify or ambiguous of being designated as.
The result shows:
With 2112 pairs of SSR primers to P
1(04796) and P
2(Bai Yesan) screen, wherein the polymorphic primer of performance has 211 pairs, polymorphic rate 10% between two parents.
Build the pond in conjunction with the BSA method, further screening has obtained 16 SSR polymorphism marks.
The polymorphism mark that utilization filters out is analyzed the F2 colony of 04796 * Bai Yesan combination, utilizes Joinmap4.0 to make up linkage map in conjunction with investigation proterties data.14 marks and w are positioned in (LOD=10) on the same linkage group as a result, obtain the mark SSR06791 chain with w, and genetic distance is 8.6cM.With this test linkage map that obtains and cucumber genetic map (Ren et al., 2009 of having delivered; Miao et al, 2011) relatively, find to have 12 total marks on this linkage group and the 3rd karyomit(e), in view of the above with the w assignment of genes gene mapping on the 3rd karyomit(e).
The recovery purifying and the order-checking of the difference band of step 4. mark SSR06791PCR amplification
(1) the segmental recovery of purpose
Adopt boiling method.Concrete operations are: 2 of the difference bands of mark SSR06791PCR amplification dug down in the Eppendorf pipe of the 1.5mL that packs into from the glue earlier, and adding 100 μ L ultrapure waters in pipe, amount of water is looked the adhesive tape shade and is decided; Normal temperature soaks down 24h, change over to boil 30min in 95 ℃ of water-baths (or PCR appearance) after, the centrifugal 3min of 5000rpm.Product is that desirable supernatant 3 μ L do template and carry out pcr amplification, and it is subsequent use that resultant product is put-20 ℃ of preservations.
(2) the segmental purifying of purpose
With PCR product direct purification method.The absolute ethyl alcohol that in the PCR product, adds 2 times of volumes ,-20 ℃ of placements of spending the night, 1, the centrifugal 5min of 2000rpm just can obtain purified product.
(3) purpose fragment and carrier is connected
Reaction system is 10 μ L:PMD18-T Vector1.0 μ L; Ligation buffer I 5.0 μ L; Purpose fragment 4.0 μ L.
Application of sample on Bechtop, the mixing reactant, of short duration centrifugal, 16 ℃ connect about 1h, and spending the night does not influence joint efficiency yet.
(4) conversion of connection product
1) take out competent cell, SolutionA, SolutionB places on ice and melts;
2) competence (50 μ L)+5 μ L SolutionA+4 μ L SolutionB+46 μ L precooling deionized water;
3) with the aseptic rifle head of refrigerative above-mentioned suspension-s branch is installed to the 1.5mL centrifuge tube, every pipe adds 105 μ L, adds the target DNA of 5 μ L again, gently revolves mixing;
4) 42 ℃ of water-bath heat shock 90s note not rocking centrifuge tube;
5) fast pipe is transferred in the ice bath, made cell cooling 3 ~ 5min;
6) add 500 μ L LB liquid nutrient mediums.At 37 ℃, cultivate 1h on the 150rpm shaking table in advance;
7) bacterium liquid is applied to contains 100 μ gmL
-1Amp, 25 μ gmL
-1IPTG and 40 μ gmL
-1On the LB solid medium of X-GAL,, place room temperature to be absorbed until liquid with an aseptic elbow glass rod bacterium liquid evenly is coated with out gently;
8) be inverted flat board, cultivate 12 ~ 16h for 37 ℃.
(5) the blue hickie screening of recombinant plasmid
After 37 ℃ of cultivations, a small amount of blue colonies and more white colony appear on the LB flat board of coating X-Gal/IPTG, and wherein white colony is recombinant clone.The single bacterium colony of picking white spreads upon in the LB liquid nutrient medium of drawing good grid, and 37 ℃, the 150rpm incubated overnight.
(6) detection of bacterium colony PCR
Draw 1 μ L bacterium liquid and carry out pcr amplification as template.Get 4 μ L PCR products, detect through 1.5% agarose gel electrophoresis, compare with PCR Marker standard molecular weight to detect and insert segmental size, consistent clone is positive colony with insertion purpose clip size.
(7) order-checking and the analysis of clone back carrier
Get 3 positive colony bacterium liquid and in glycerine (adding 1000 μ L bacterium liquid in the 330 μ L glycerine), respectively preserve two parts, a-20 ℃ of preservations, portion is sent to order-checking.Sequencing result is shown in SEQ ID No.1 and SEQ ID No.2.
The checking of embodiment 2.w gene flank mark
Utilize the cucumber material of 30 parts of different fruit colour types that the flank mark SSR06791 with w gene linkage that embodiment 1 obtains is verified; To confirm that mark is used for the accuracy of molecular marker assisted selection: these 30 parts of materials have 28 parts of green pericarp materials, 2 parts of white pericarp materials.The field phenotype of warp and selected materials is compared, and the phenotypic data that is marked at the banding pattern reflection that has 24 parts of materials in 30 parts of materials is consistent with the field investigation result, is 80% through calculating accuracy.Wherein 2 parts of white pericarp materials all amplify white pericarp characteristic band.
Made up the SSR linkage map of the tender fruit white of cucumber peel gene in this research; SSR mark (SSR06791) that is obtained and the genetic distance 8.6cM of w, this and the chain SSR of cucumber white peel gene are labeled as molecular marker assisted selection breeding screening cucumber white pericarp new variety and have laid a good foundation.Identify quick and precisely that for setting up a cover method of the tender fruit white of cucumber pericarp provides theoretical foundation.
Claims (5)
1. the chain SSR mark of the tender fruit of cucumber white peel gene w, the characteristic band of its amplification of the following SSR06791-F/SSR06791-R:TTTGTAGTTTGAGAACTCAAGTTGG/TGGTGTTT GGTTGTCCTGAG of sequence is respectively shown in Seq ID No.1, Seq ID No.2.
2. the described SSR of claim 1 is marked at the application of screening in the cucumber germ plasm resource with tender fruit white peel gene w, and its step is following:
(1) adopt above-mentioned SSR to be labeled as primer the genomic dna of cucumber variety to be selected carried out pcr amplification respectively,
(2) amplification is carried out detected through gel electrophoresis;
(3) amplify the segmental material shown in SEQ ID No.2 from screening the detected result.
3. application according to claim 2; Said PCR reaction system is: 7.5ng DNA; Each 50ng of forward and reverse primer; 5 μ L Go
Green Master Mix adds distilled water to 10ul.
4. application according to claim 2, said pcr amplification program is: 94 ℃ of preparatory sex change 4 minutes; 94 ℃ of sex change 15 seconds, 55 ℃ of annealing 15 seconds, 72 ℃ were extended 35 circulations 30 seconds; 72 ℃ are incubated 5 minutes, 16 ℃ of preservations.
5. according to the arbitrary described application of claim 2～4: said detected through gel electrophoresis refers to adopt 6% non-denaturing polyacrylamide gel, and in the permanent power electrophoretic separation of 160V, last silver dyes colour developing.
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| CN103146659A (en) * | 2013-02-06 | 2013-06-12 | 中国农业科学院蔬菜花卉研究所 | Cucumber fruit beta carotene content related SNP marker and its application |
| CN103740711A (en) * | 2014-01-29 | 2014-04-23 | 中国农业科学院蔬菜花卉研究所 | Indel marker linked with yellow flesh gene yf of cucmis sativus L. and application of Indel marker |
| CN103937788A (en) * | 2014-04-24 | 2014-07-23 | 中国农业科学院蔬菜花卉研究所 | SSR (simple sequence repeat) marker for cucumber leaf color mutant genes v-1 and application thereof |
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2012
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| CN103146659A (en) * | 2013-02-06 | 2013-06-12 | 中国农业科学院蔬菜花卉研究所 | Cucumber fruit beta carotene content related SNP marker and its application |
| CN103740711A (en) * | 2014-01-29 | 2014-04-23 | 中国农业科学院蔬菜花卉研究所 | Indel marker linked with yellow flesh gene yf of cucmis sativus L. and application of Indel marker |
| CN103937788A (en) * | 2014-04-24 | 2014-07-23 | 中国农业科学院蔬菜花卉研究所 | SSR (simple sequence repeat) marker for cucumber leaf color mutant genes v-1 and application thereof |
| CN103937788B (en) * | 2014-04-24 | 2016-04-13 | 中国农业科学院蔬菜花卉研究所 | The SSR marker of Folium Cucumidis sativi look mutator gene v-1 and application thereof |
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