CN102690823B - SSR (Simple Sequence Repeat) marker tightly interlocked with cucumber fruit bitter gene Bt and application thereof - Google Patents
SSR (Simple Sequence Repeat) marker tightly interlocked with cucumber fruit bitter gene Bt and application thereof Download PDFInfo
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Abstract
The invention provides an SSR (Simple Sequence Repeat) marker tightly interlocked with a cucumber fruit bitter gene Bt and an application thereof, and relates to a biological breeding auxiliary technology. The sequence of the marker is as follows: SSR21558-F/SSR221558-R: GTGGGGGATGTGATT CAGAC/CATCATCCATTCCCCTCAAC, wherein the amplified characteristic stripe is (175bp) represented by Seq ID No.1 and (192bp) represented by Seq ID No.2; and SSR20054-F/SSR20054-R: GTTTGTGAGGGAAACGCAAT/TCAAAAAGCTTCCTTCCTTCA, wherein the amplified characteristic stripe is (127bp) represented by Seq ID No.3 and (108bp) represented by Seq ID No.4. Two markers are more tightly interlocked with the gene Bt so as to be more helpful to the establishment of a cucumber fruit bitter molecular marker breeding auxiliary system; with the adoption of the SSR marker obtained by the system, the screening of fruit bitterness or no fruit bitterness is carried out on a cucumber candidate material at any phase, so as to have the advantages of high efficiency, less limitation and accuracy, and improve the selection efficiency and accuracy for selecting bitter-free materials for cucumber breeding.
Description
Technical field
The present invention relates to Biology Breeding ancillary technique, particularly a kind of cucumber fruits picryl is because of the closely linked SSR mark of Bt and application thereof.
Background technology
During cucumber (Cucumis sativus L.) produces, particularly, in facility cultivation process, sometimes have bitter taste fruit and produce, cause loss in revenue (Rehm et al., 1957; Gu Xingfang etc., 2000; Kano et al., 2002).The bitter taste of mellon plant is that the material that is called cucurbitacin (Cucurbitacins) by a class causes (Rice et al., 1981; Balkema et al., 2003).Cucurbitacin belongs to cucurbitane type tetracyclic triterpenoids compound, to most of organisms poisonous (Smyth et al., 2002).Fruit bitter taste is one of problem affecting cucumber production.Holland cucumber breeding man is as far back as the 1959 seed selection work that started without bitter taste cucumber, and is bred as corresponding kind, and the vegetative organ of these kinds and fruit are all without bitter taste (AndeweG and Bruyn, 1959).Research causes molecule marker and the heredity location of the gene of cucumber fruits bitter taste generation, significant without bitter taste cucumber for utilizing molecular marker assisted selection (Marker Assisted Selection, MAS) technology to cultivate.
Two genes of the control cucumber fruits bitter taste of having reported are Bt and Bt-2(Barham, 1953; Walters, 2001).The cucumber material PI183967(genotype that preserve in this laboratory is BiBiBtBt) contain Bt gene.Forefathers focus mostly in aspects such as genetic development, gene linkage, environmental influence, breed breedings about the research of cucumber fruits bitter taste.There are some researches show, Bt and Bt-2 gene all meet single-gene dominant inheritance, meet feature (Barham, 1953 of qualitative character heredity; InGGamer and Deponti, 1981; Walters et al., 2001).There are not chain (Cowen and Helsel, 1983 in Bt gene and cucumber female gene F; Gu Xingfang etc., 2005).Bt-2 gene and fruit look consistent gene u, dark-coloured peel gene D, spinule gene ss chain (Wehner and Liu, 1998; Walters et al., 2001).Cultivation condition affects generation (the Kano et al. of cucumber bitter taste, 2003), nitrogen in soil too much or not enough, fertilizer lacks, temperature is too low or the condition such as too high, sun exposure is not enough, malnutritive, upper earth drought and water shortage, disease and pest and the weak many diseases of plant under, all can bring out formation and the accumulation of cucurbitacin, thereby form bitter taste fruit.
The research that determines gene Bt molecular level for cucumber fruits bitter taste is mainly carried out and is carried out by this laboratory.We from molecular level to fruit picryl because being studied and having obtained two dominant AFLP marks with Bt gene linkage: E23M66-101 and E25M65-213.The genetic distance of these two marks and Bt gene is respectively 5cM and 4cM, and is positioned at the both sides (Gu Xingfang etc., 2006) of Bt gene.The fruit of take for 2011 has bitter taste 46GBt(BtBt without the isozygoty close thorn in self-mating system Xintai City (btbt) and fruit of the cucumber of bitter taste) F that contains 184 individual plants that builds as parent
2colony, for examination material, carries out SSR mark genetic linkage analysis to it, has built the SSR linkage group of Bt gene.The both sides nearest with Bt gene are labeled as SSR10795 and SSR07081, and genetic distance is respectively 0.8cM and 2.5cM(opens holy equality, 2011).But above-mentioned mark also exists linkage distance relatively far away, and the relatively low problem of the Detection accuracy of mark, relatively low in the efficiency of selection of carrying out without in the screening of bitter taste breed cucumber.
The hereditary more complicated of cucumber fruits bitter taste, is subject to nourishing body picryl because of the impact of Bi, and recessive bi gene and Bt gene exist makes effect (Gu et al., 2007) mutually.But under the Bi genetic background that isozygotys, Bt gene is independent inheritance (Barham, 1953; Walters and Wehner, 1998; Gu et al., 2007).For get rid of cucumber nourishing body bitter taste gene pairs fruit picryl because of mutual impact, in the present invention, utilize the genotype of nourishing body bitter taste gene pure, carried out SSR molecule marker and the hereditary Position Research of Bt gene.
The method that detects at present cucumber bitter taste mostly is sense organ and tastes and chemical detection, though that chemical detection can detect bulk materials is comparatively loaded down with trivial details, its result is also not accurate enough; Sense organ is tasted and is only suitable for the detection of a small amount of material, and cannot accurately define the level (Gu Xingfang etc., 2004), so be badly in need of setting up a set of method of quick and precisely identifying bitter taste, for cultivating, without bitter taste cucumber, provides theoretical foundation.Molecular marker assisted selection (MAS) technology can be in seedling stage or directly with seed, identify because of closely linked molecule marker with picryl by finding, and simple work abridged edition, shortens breeding process greatly.
Summary of the invention
The object of the invention is to overcome prior art deficiency, provide with cucumber fruits picryl because of the closely linked SSR mark of Bt, and provide it selecting without the application on bitter taste Cucumber Germplasm, the SSR mark that adopts the present invention to obtain, can to it, carry out fruit bitter taste or fruit without the screening of bitter taste in any stage of cucumber candidate material, have efficient, restriction less, advantage accurately, for breed cucumber is selected to have improved efficiency of selection and accuracy without bitter taste material.
Cucumber fruits picryl is because of the closely linked SSR mark of Bt, and sequence is as follows:
SSR21558-F/SSR21558-R:GTGGGGGATGTGATTCAGAC/CATCATCCATTCC CCTCAAC(Seq ID No.5 and SEQ ID No.6), the feature band of its amplification is as shown in Seq ID No.1 and Seq ID No.2.Wherein the fragment of 175 Nucleotide shown in Seq ID No.1 and fruit picryl because of Bt chain, the fragment of 192 Nucleotide and fruit shown in Seq ID No.2 not picryl because of bt chain.
SSR20054-F/SSR20054-R:GTTTGTGAGGGAAACGCAAT/TCAAAAAGCTTCC TTCCTTCA(Seq ID No.7 and SEQ ID No.8), the feature band of its amplification is as shown in Seq ID No.3 and Seq ID No.4.Wherein the fragment of 127 Nucleotide shown in Seq ID No.3 and fruit picryl because of Bt chain, the fragment of 108 Nucleotide and fruit shown in Seq ID No.4 not picryl because of bt chain;
Above-mentioned SSR is marked at screening and contains without fruit picryl because of the application in the Cucumber Germplasm of Bt, and its step is as follows:
(1) adopt above-mentioned SSR to be labeled as primer pair cucumber variety and treat that the genomic dna of material selection carries out respectively pcr amplification,
(2) amplification is carried out to detected through gel electrophoresis,
(3) from the result of detected through gel electrophoresis, select the material that does not occur described fruit bitter taste feature band.
The system of described pcr amplification: Green Master Mix 5 μ L, 15ng DNA, forward, each 30nG of reverse primer, all the other are with ddH
2o complements to 10 μ L;
The program of pcr amplification is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 15s; 55 ℃ of annealing 15s; 72 ℃ are extended 30s:35 circulation; 72 ℃ of insulation 5min.
Described 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.
Fruit is take in the present invention the wild cucumber PI183967(Cucumis sativus var.hardwickii of bitter taste) and fruit without the cucumber 931 of the bitter taste F that contain 189 individual plants of the close thorn choosing system in Xintai City (btbt) for parent's structure that isozygoty
2colony, for examination material, carries out SSR mark genetic linkage analysis (Fig. 1,2) to it, builds the SSR linkage group (Fig. 3) of Bt gene.In this linkage group, be SSR21558 with one of linked marker of the nearest both sides of Bt gene, genetic distance is 0.5cM; Another mark SSR20054, genetic distance is 1.0cM.Through different genetic background material identifications, mark SSR21558 detected result is compared checking accuracy rate with field phenotype is: the accuracy rate that is 95%, SSR20054 mark is: 75%, and use two marks mutually to confirm the accuracy rate of verifying and can reach 95%.
Can be used for cucumber variety candidate material rapid screening with the closely linked mark SSR21558 of Bt gene and SSR20054, thereby particularly can to the material of cucumber at seedling stage or seed directly carry out Molecular Identification select without fruit picryl because of cucumber material, do not need when cucumber yields positive results by the time its fruit to taste to judge.Molecular Identification is more accurate than the artificial result of tasting, and not disturbed by other factors, and saving of work and time, is not subject to seasonal effect, has shortened widely breeding cycle.
For reach the present invention above-mentioned carry out cucumber fruits without the screening object of bitter taste material, two SSR marks can be separately for the screening of candidate material.The present invention further provides two screening methods that SSR mark confirms mutually, improved the selection accuracy of candidate material.
Accompanying drawing explanation
Fig. 1 .SSR21558 is to cucumber parents material and F
2the detected result of the random individual plant of colony
P
1: PI183967 (hardship); P
2: not 931 (not bitter);
Fig. 2 .SSR20054 is to cucumber parents material and F
2the detected result of the random individual plant of colony
P
1: PI183967 (hardship); P
2: not 931 (not bitter);
Fig. 3. cucumber Bt gene F
2colony's linkage map
Embodiment
1. materials and methods
1.1 test materials
For examination cucumber material, be PI183967(P
1), Cucumis sativus var.hardwickii, wild cucumber, by Jack professor staub of Univ Wisconsin-Madison USA, presented, for existing known kind, also on the books in the chief editors' such as Liu Shiqis in 1998 < < Vetegables Farming > > mono-book, there is preservation in this laboratory, guarantees in 20 years, to the public, to provide for confirmatory experiment from the applying date.PI183967 phenotype is that nourishing body hardship and fruit are bitter, and containing nourishing body picryl, because Bi and fruit picryl are because of Bt, genotype is BiBiBtBt.Fruit oval, deep green pericarp, tarnish, black thorn, thorn knurl are rare, have completed the 60 * order of resurveying;
931(P
2): the Xintai City of isozygotying close thorn choosing system, from the close thorn in Xintai City, through inbreeding of more generation purifying, be bred as, from cucumber seminar of Vegetable & Flower Inst., Chinese Academy of Agriculture Science, for existing known kind, also on the books in the article < < protection ground improved seeds that Zhang Fengming etc. delivered in < < north gardening > > the 7th phase in 1989---Xintai City's close thorn cucumber brief introduction > > mono-literary composition, assurance is provided for confirmatory experiment to the public in 20 years from the applying date.931 phenotypes are nourishing body hardship but fruit is not bitter, containing nourishing body picryl because of Bi and fruit without picryl because of bt, genotype is BiBibtbt.Pericarp deep green, tarnish, white thorn, thorn knurl are close.Completed the 10 * order of resurveying.Take PI183967 as maternal, and 931 is paternal hybrid, preparation F
1, selfing obtains F2 colony.
SSR primer is from international Cucumber germplasm plan (CUGI), and totally 2112 to (Ren et al., 2009).The article < < An inteGrated Genetic and cytoGenetic map of the cucumber Genome > > that detailed results can be delivered in the 4th phase of PloS One2009 online referring to Ren etc.The GoTaq Green Master Mix of ShanGhai PromeGa company is used in PCR experiment; Gel electrophoresis is used and to be full of 40% non-denaturing polyacrylamide of Xinyang light company, is diluted to 6% rear use.
Embodiment 1. cucumber fruits picryls are because of the screening of the chain SSR mark of Bt
Step 1. cucumber fruits bitter taste is identified
For examination cucumber material, in seeding and seedling raising on March 14th, 2010, be colonizated in vegetable or flower institute of Chinese Academy of Agricultural Sciences greenhouse in spring on April 16.Plantation parent and F
1each 30 strains (repeating every repetition 10 strains for 3 times), F
2189 strains.Seedling stage Routine Management.After field planting, early stage night temperature control below 12 ℃, daytime in later stage temperature higher than 32 ℃, control water whole vegetative period, and enrich nitrogenous fertilizer, lures that picryl is because giving full expression to into.With reference to the method for (2004) such as Gu Xingfang, adopt the method that mouth is tasted to identify fruit bitter taste.From root melon, start until off-test (1 season of growth), if every strain has 4 fruits to occur bitter taste, this strain is decided to be fruit hardship, no longer taste, otherwise need to continue to taste, do not occur the individual plant of bitter taste, estovers is tasted all fruits on plant, and general every strain is tasted 7 more than melon.Every strain is tasted bitter taste sensitive subjects by 3 at every turn simultaneously, to guarantee the accuracy of test-results.
Result shows: at 189 strain F
2in colony, there are 126 strains that have of fruit bitter taste, not bitter have 57 strains, through the detection of the side of card, meets the Mendel heredity segregation ratio (X of 3:1
2 c=3.76<X
2 0.05.1=3.84), illustrate that cucumber fruits bitter taste is to be controlled by single-gene (Bt), bitter taste is relatively not bitter is dominant, and this proterties can be analyzed according to qualitative character.This is identical with result of study in the past.
Step 2.DNA extracts and ssr analysis
From parent, F
1and F
2on colony's individual plant, get blade, after freezing treatment, use QuickGene instrument for extracting nucleic acid, use DNA test kit to extract genomic dna.
SSR reaction system: cumulative volume 10 μ L, Green Master Mix 5 μ L, DNA(5nG μ L
-1) 3 μ L, forward, reverse primer (50nG μ L
-1) each 0.6 μ L, all the other are with ddH
2o supplies 10 μ L.PCR program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 15s; 55 ℃ of annealing 15s; 72 ℃ are extended 30s:35 circulation; 72 ℃ of insulation 5min.The permanent power electrophoretic separation of 6% non-denaturing polyacrylamide gel 160V for amplified production, silver dyes after colour developing in the enterprising line number of observation lamp house according to one's analysis.
Step 3.SSR label screening, data statistics and linkage map build
First use two parental line selection primers, then take respectively 6 fruit bitter taste individual plants and 6 fruits bitter taste group that DNA of bitter individual plant forms and not bitter group is template, be used in the primer that produces polymorphism between two parents and carry out the screening of Bt genetic marker, as long as bitter taste group has respectively the banding pattern of 5 strains or above individual plant consistent with in not bitter group, tentatively think that this mark is relevant to fruit bitter taste proterties.Screen 2112 pairs of combination of primers, selected 996 pairs of combination of primers that produce polymorphism parents, each 6 individual plants of bitter taste group and not bitter group have been tested, filtered out 24 pairs of marks, to F
2after colony verifies, 19 pairs of serviceable indicias have been obtained.
Then, by these 19 pairs of marks to whole F
2colony analyzes, the separation case of statistics band.Codominant marker's statistical method is: from maternal P
1banding pattern be designated as a, from male parent P
2banding pattern be designated as b, the banding pattern of heterozygosis is designated as h, amplification or the ambiguous u that is designated as.Dominant marker's statistical method is: if P
1for dominant, F
2in individual plant with P
1banding pattern is consistent is designated as d, with P
2banding pattern is consistent is designated as b; If P
2for dominant, F
2in individual plant with P
1banding pattern is consistent is designated as a, with P
2banding pattern is consistent is designated as c.Finally utilizing JoinMap4.0(van Ooijen & Voorrips, 2001) mapping software builds the SSR linkage group (Fig. 3) of Bt gene, LOD threshold value 5.0, analytical data, obtains the SSR linkage map of Bt gene.The Bt assignment of genes gene mapping, in the scope of one end 1.5cM of cucumber the 5th the short arm of a chromosome, is wherein respectively to SSR21558 and SSR20054 with the nearest mark in Bt gene both sides, and genetic distance is respectively 0.5cM and 1.0cM.
SSR21558-F/SSR21558-R:
GTGGGGGATGTGATTCAGAC/CATCATCCATTCCCCTCAAC(Seq ID No.5 and SEQ IDNo.6), the feature band of its amplification is as shown in Seq ID No.1 shown in (175bp) and Seq ID No.2 (192bp).SSR20054-F/SSR20054-R:GTTTGTGAGGGAAACGCAAT/TCAAAAAGCTTCCTTCCTTCA(Seq ID No.7 and SEQ ID No.8), the feature band of its amplification is as shown in Seq ID No.3 shown in (127bp) and Seq ID No.4 (108bp).
Recovery purifying and the order-checking of step 4.PCR amplification gained differential fragment
(1) recovery of object fragment
Adopt boiling method.Concrete operations are: first target stripe is dug down to pack in the Eppendorf pipe of 1.5mL from glue, in pipe, add 100 μ L ultrapure waters, amount of water is depending on adhesive tape shade; Under normal temperature, soak 24h, proceed in 95 ℃ of water-baths (or PCR instrument) and boil after 30min, the centrifugal 3min of 5000rpm.Product is that desirable supernatant 3 μ L do template and carry out pcr amplification, and resultant product is put-20 ℃ and saved backup.
(2) purifying of object fragment
By PCR product direct purification method.In PCR product, add the dehydrated alcohol of 2 times of volumes ,-20 ℃ of placements of spending the night, the centrifugal 5min of 1,2000rpm just can obtain purified product.
(3) object fragment and carrier is connected
Reaction system is 10 μ L:PMD18-T Vector1.0 μ L; LiGation buffer I 5.0 μ L; Object fragment 4.0 μ L.
Application of sample on Bechtop, mixes reactant, of short duration centrifugal, and 16 ℃ connect about 1h, spend the night and also do not affect joint efficiency.
(4) connect the conversion of product
1) take out competent cell, SolutionA, SolutionB, is placed 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 cooling aseptic rifle head, above-mentioned suspension is divided and installs to 1.5mL centrifuge tube, every pipe adds 105 μ L, then adds the target DNA of 5 μ L, gently revolves and mixes;
4) 42 ℃ of water-bath heat shock 90s, note not rocking centrifuge tube;
5) fast pipe is transferred in ice bath, made the cooling 3 ~ 5min of cell;
6) add 500 μ L LB liquid nutrient mediums.At 37 ℃, preculture 1h on 150rpm shaking table;
7) bacterium liquid is applied to and contains 100 μ GmL
-1amp, 25 μ GmL
-1iPTG and 40 μ GmL
-1on the LB solid medium of X-GAL, with an aseptic elbow glass rod bacterium liquid is evenly coated with and is opened gently, be placed in room temperature until liquid is absorbed;
8) be inverted flat board, cultivate 12 ~ 16h for 37 ℃.
(5) the blue hickie screening of recombinant plasmid
After 37 ℃ of cultivations, on the LB flat board of coating X-Gal/IPTG, there is a small amount of blue colonies and more white colony, wherein white colony is restructuring clone.The single bacterium colony of picking white spreads upon in the LB liquid nutrient medium that pulls grid, and 37 ℃, 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, through 1.5% agarose gel electrophoresis, detect, compare detect the size of Insert Fragment with PCR Marker standard molecular weight, the clone consistent with inserting object clip size is positive colony.
(7) order-checking and the analysis of carrier after clone
Get 3 positive colony bacterium liquid and respectively preserve two parts in glycerine (adding 1000 μ L bacterium liquid in 330 μ L glycerine), a-20 ℃ of preservations, portion is sent to order-checking.
The checking of embodiment 2.Bt Gene Double flank SSR mark
Utilize the cucumber material of 20 parts of different ecological type that this problem collects, to verifying and determine that mark SSR21558 and SSR20054 are for the accuracy of molecular marker assisted selection with Bt gene both sides close linkage mark.The phenotype of these 20 individual plants is: 18 strain fruits are not bitter; 2 strain fruits are bitter.Utilize mark SSR21558 analytical results to show, in 20 parts of materials, all amplify object band, compare with field phenotype, the accuracy of detection is 95.0%.Another mark SSR20054 with Bt gene linkage is verified above-mentioned materials, in 20 parts of DNA, all amplify object band, compare with field phenotype, the accuracy of detection is 75.0%, uses two marks mutually to confirm the accuracy rate of verifying and can reach 95.0%.
In this research, built the SSR linkage map of cucumber fruits bitter taste Bt gene, the genetic distance of the SSR mark obtaining (SSR21558 and SSR20054) and Bt is 0.5cM and 1.0cM respectively, and these two lay a good foundation because closely linked SSR is labeled as the breeding of cucumber molecular marker assisted selection with cucumber fruits picryl.
Claims (4)
1. cucumber fruits picryl is selecting not exist described fruit picryl because of the application in the Cucumber Germplasm of Bt because of the closely linked SSR labeled primer of Bt, and its step is as follows:
(1) adopt described SSR labeled primer to treat that to cucumber the genomic dna of material selection carries out respectively pcr amplification,
(2) amplification is carried out to detected through gel electrophoresis,
(3) from the result of detected through gel electrophoresis, reject and occur and the material of cucumber fruits picryl because of the closely linked feature band of Bt,
Described SSR labeled primer is: SSR21558-F/SSR21558-R:
GTGGGGGATGTGATTCAGAC/CATCATCCATTCCCCTCAAC，
The amplification of described SSR labeled primer with cucumber fruits picryl because the nucleotide sequence of the chain feature band of Bt is as shown in Seq ID No.1,
The amplification of described SSR labeled primer with cucumber fruits not picryl because the nucleotide sequence of the closely linked feature band of bt is as shown in Seq ID No.2.
2. application according to claim 1, the system of described pcr amplification: GoTaq Green Master Mix5 μ L, 15ng DNA, forward, each 30ng of reverse primer, all the other are with ddH
2o complements to 10 μ L.
3. application according to claim 2, the program of pcr amplification is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 15s; 55 ℃ of annealing 15s; 72 ℃ are extended 30s; 35 circulations; 72 ℃ of insulation 5min.
4. application according to claim 3, described detected through gel electrophoresis refers to adopt 6% non-denaturing polyacrylamide gel, and in 160V constant voltage electrophoretic separation, last silver dyes colour developing.
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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 |
| CN103755791B (en) * | 2013-12-18 | 2019-04-05 | 中国农业科学院蔬菜花卉研究所 | Participate in transcription factor Csa5G156220 and its application of regulation cucumber cucurbitacine C synthesis |
| 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 |
| CN106916879A (en) * | 2015-12-25 | 2017-07-04 | 南京农业大学 | Mitochondrial genomes polymorphism builds cucumber finger-print and its application |
| CN108486128B (en) * | 2018-02-06 | 2021-08-24 | 华南农业大学 | Luffa fruit bitter gene LaBt and molecular marker for auxiliary selection of fruit bitter character |
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