CN106086007B - Molecular labeling and its application with tomato atropurpureus fruit Gene A TV close linkage - Google Patents
Molecular labeling and its application with tomato atropurpureus fruit Gene A TV close linkage Download PDFInfo
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Abstract
The invention belongs to tomato molecular Biotechnology in Genetic Breeding fields, and in particular to molecular labeling and its application with tomato atropurpureus fruit gene loci close linkage.The molecular labeling is HP1917, HP3217 and ATV-In, and the present invention can substitute traditional by selecting atropurpureus fruit plant to ensure that breeding material contains the method in the mutational site tomato atropurpureus fruit (atv).It detects whether breeding material contains tomato atropurpureus fruit (atv) mutation allele in plantlet stage by molecular labeling, breeding cycle can be shortened, improve breeding efficiency.
Description
Technical field
The invention belongs to tomato molecular Biotechnology in Genetic Breeding fields, and in particular to tight with tomato atropurpureus fruit gene loci
Close chain molecular labeling and its application.
Background technique
Anthocyanidin is antioxidizing substance naturally-produced in plant, has and resists inflammation, cancer, pathogen；In advance
Anti- cardiovascular disease, obesity, diabetes；Improve sleep；Enhance cutaneous immunisation power, beautifying face and moistering lotion；It delays senescence；Improve eyesight；
Etc. various biologicals activity.The intracorporal anthocyanidin of plant is presented different since type, concentration and its environment at place are different
Color, to be conducive to insect pollination, seed dispersal, resist uv damage.At present it has also been found that anthocyanidin can also extend kind
The shelf life of solanberry reality.
Tomato is Chinese or even one of most important vegetables in the world, but most cultivated tomato fruits are almost at present
Without anthocyanidin.The color of overwhelming majority purple, brown or black tamato fruit at present, is due to lycopene in ripening fruits
With the comprehensive result presented of remaining chlorophyll.In nature, the fruit of some Wild tomato species can accumulate anthocyanidin and
In atropurpureus.These Wild tomato species are separated into identification with the hybridization of common cultivation tomato, offspring respectively, it was found that multiple participation fruits
The gene loci of real epidermis anthocyanin accumulation, such as: the site Aft (Anthocyanin fruit), the Chile tomato (Solanum come
Chilense), it is a dominant locus, is located at No. 10 chromosome, thus it is speculated that it may be the equipotential base of tomato AN2 or ANT1
Cause；The site Abg (Aubergine) comes from solanum (S.lycopersicoides) and a dominant locus, is located at the 10th
Number chromosome, thus it is speculated that Abg and Aft may be allele；The site atv (Atroviolacium) comes from this Manny tomato of contract
(S.cheesmaniae), it is a recessive site, is located at No. 7 chromosome.Atv respectively with Aft and Abg there are interaction,
It is significantly higher than the tamato fruit containing only a site containing the tamato fruit anthocyanidin content there are two site simultaneously.
Since atv is a recessive mutation site, when the site is in heterozygous state, fruit color and normal plant
(wild type) fruit is similar, cannot be distinguished from normal homozygous plants.Therefore, atv is imported into excellent tomato parent, Huo Zhexuan
The excellent tomato breeding material containing atv is educated, if using traditional method by phenotype breeding, period length, low efficiency.Point
Sub- marker assisted selection breeding, is referred to and is selected using DNA molecular marker breeding material, can be shortened breeding cycle, be mentioned
High breeding efficiency.
Summary of the invention
The object of the present invention is to provide a set of and highly relevant molecular labelings of tomato atropurpureus fruit properties, and its identification
Method can be used as the molecular marker assisted selection tool of tomato atropurpureus fruit properties.
In order to achieve the object of the present invention, tomato atropurpureus fruit mutational site (atv) is positioned by finely positioning first
Into about 9.6kb.
The 9.6kb segment contains only the gene Solyc07g052490 of a prediction, it encodes a Myb transcription factor,
It therefore is MybATV by the unnamed gene.Homologous gene of the gene in petunia and arabidopsis can inhibit the life of anthocyanidin
Object synthesis.
There are a 4bp insertion, (Insertion is named as in the Second Exon of MybATV gene in the mutational site atv
ATV-In), protein translation terminates in advance because of frameshit.Therefore confirm that the encoded MybATV albumen in the mutational site atv loses
Inhibit the ability of anthocyanidin biosynthesis.
The physical location of insertion ATV-In are as follows: SL2.50ch07:61003572.
The present invention also provides a set of and highly relevant molecular labeling of tomato atropurpureus fruit properties, the molecular labelings
For HP1917, HP3217 and ATV-In.HP1917 is located at MybATV upstream region of gene about 21kb；HP3217 is located at MybATV gene
At the about 9.4kb of downstream；ATV-In is located at Myb gene internal.
Wherein, the specific primer sequence of each molecular labeling and its amplification target fragment length are as follows:
①HP1917
Forward primer: 5-TCACAAGTAAAGGAGCATCC-3
Reverse primer: 5-CTCTTCTTATAGTCACGTAAGTTAG-3
The Indigo Rose expanding fragment length in the site containing atv is 117bp, and common cultivation tomato (wild type) expands piece
Segment length is 138bp.
②HP3217
Forward primer: 5-ACATTGGTGGCATTGAATATGAG-3
Reverse primer: 5-ACAAGTATCACCCTATCCGTCTC-3
The Indigo Rose expanding fragment length in the site containing atv is 236bp, and common cultivation tomato (wild type) expands piece
Segment length is 200bp.
③ATV-In
Forward primer: 5-GAGGTTTCTTCGTTGGTAGTC-3
Reverse primer: 5-CTAAATAAAAGTTATTGAGTTCACG-3
The Indigo Rose expanding fragment length in the site containing atv is 89bp, common cultivation tomato (wild type) amplified fragments
Length is 85bp.
The present invention also provides a kind of methods for identifying tomato atropurpureus fruit properties, comprising the following steps:
1) genomic DNA of tomato to be measured is extracted.Genomic DNA is extracted using conventional CTAB method.
2) PCR amplification.Using tomato dna group DNA to be detected as template, the specificity for detecting InDel label is utilized
Primer carries out PCR amplification.1. PCR system (15 μ l): 1 μ l, 10 × PCR reaction buffer of 50-100ng/ μ l DNA profiling (contains Mg
Ion) 1.5 μ l, 2.5mM dNTPs 1.5 μ l, each 0.2 μ l of 0.3 μ l, 2.5U/ μ l Taq archaeal dna polymerase of 10 μM of primer pairs,
ddH2O 10.2μl.2. PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C extend
30s, 35 circulations；72 DEG C of extension 5min.
3) pcr amplification product electrophoresis detection.The pcr amplification product of molecular labeling HP1917 and HP3217 are solidifying using agarose
Gel electrophoresis detection, Ago-Gel concentration are 3%.The pcr amplification product of molecular labeling ATV-In uses polyacrylamide gel
Electrophoresis detection, polyacrylamide gel concentration are 8%.Plant containing tomato atropurpureus fruit (atv) homozygous mutation site, point
HP1917 can detecte the band to a 117bp to son label, molecular labeling HP3217 can detecte the item to a 236bp
Band, molecular labeling ATV-In can detecte the band to a 89bp；The site aa is the plant of normal homozygous (wild type), molecule
HP1917 can detecte the band to a 138bp to label, molecular labeling HP3217 can detecte the band to a 200bp,
Molecular labeling ATV-In can detecte the band to a 85bp；The site aa is that the plant of heterozygous state can be detected simultaneously by phase
Answer 2 band.
The present invention can substitute traditional by selecting atropurpureus fruit plant to ensure that it is black that breeding material contains tomato purple
The method in the mutational site color fruit (atv).In plantlet stage detecting breeding material by molecular labeling, whether to contain tomato purple black
Color fruit (atv) mutation allele can shorten breeding cycle, improve breeding efficiency.
Detailed description of the invention
Fig. 1 is the schematic diagram that tomato atropurpureus fruit Gene A TV influences fruit color and anthocyanidin content.F3 refers to F3 groups
The fruit of three kinds of genotype in body.In genotype, -/-, represent wild type；+ /+represents Mutants homozygous；It is +/-, represent heterozygosis
Body.Anthocyanidin content refers in 100 grams of fresh weights of ripening fruits exocarp containing how many milligrams of anthocyanidin.
Fig. 2 is the finely positioning schematic diagram of tomato atropurpureus fruit ATV gene.The Primary Location of a ATV gene；b ATV
The finely positioning of gene；3 kinds of transcripts of MybATV gene in c ATV assignment of genes gene mapping region, in their second exon
Insertion containing a 4bp, the insertion are named as ATV-In.White box represents exon.
Fig. 3 is that molecular labeling HP1917, HP3217 and ATV-In detect Indigo Rose (mutational site containing atv),
Heinz1706 (wild type) and its electrophoretogram of filial generation.M is 50bp DNAmarker, and 1 is Heinz1706 (wild type),
2 be Indigo Rose (mutational site containing atv), and 3 be F1 (Indigo Rose × Heinz1706), and 4-11 is in F2 groups
Normal homozygosis single plant, 12-19 be the heterozygosis single plant in the mutational site atv in F2 group, and 20-27 be atv mutant homozygous in F2 group
Single plant.Molecular labeling HP1917 (above), HP3217 (middle) are 3% agarose gel electrophoresis figure piece, and ATV-In (following figure) is
8% polyacrylamide gel electrophoresis picture.
Specific embodiment
It is furtherd elucidate below by specific embodiment and illustrates the present invention, but be not intended to limit the scope of the invention.
If unspecified, embodiment is according to conventional laboratory conditions, or according to the condition of manufacturer's specification suggestion.
The determination of the InDel ATV-In of 1 tomato atropurpureus fruit gene loci of embodiment
The seed of tomato variety Indigo Rose is bought in Johnny's Selected Seeds company.Indigo
Rose contains that there are two the genetic locus Aft and atv for participating in fruit rind anthocyanin accumulation, is in atropurpureus when fruit maturation.Tomato
It takes on a red color when kind Heinz1706 fruit maturation.The two hybridization obtains F1。F1Individual plant selfing is multiplied into F2For segregating population, F2It is single
Strain self propagated is at F3For segregating population.Plantation2For 190 plants of segregating population, conventional water and fertilizer management.Sow F3For segregating population
1900 plants, after carrying out Molecular Detection to seedling with label, select exchange Plant colonization, conventional water and fertilizer management.
Fruit color identification
When tamato fruit reaches the red ripe phase, fruit color is observed.According to the presence or absence of fruit surface purple dot and how many,
Fruit color is divided into three classes: no purple dot has purple dot (but not being linked to be piece) and purple in blocks (Fig. 1).
The detection of pericarp anthocyanidin
When tamato fruit reaches the red ripe phase, 2 grams of fresh exocarp (peel) is taken, is ground into powder, adds in liquid nitrogen
10ml extracting solution (anhydrous methanol/hydrochloric acid) extracted overnight, 5000g are centrifuged 10min, take supernatant with 0.45 μm of membrane filtration, filtrate
It is detected with spectrophotometer, wavelength is respectively 535nm and 650nm.
Extracting genome DNA
Divide single plant to take cotyledon or young leaflet tablet, genomic DNA is extracted using conventional CTAB method.
Molecular labeling primer is designed, PCR amplification and electrophoresis detection are carried out
PCR system (15 μ l): 50-100ng/ μ l DNA profiling 1 μ l, 10 × PCR reaction buffer (ion containing Mg) 1.5 μ
1.5 μ l of l, 2.5mM dNTPs, 0.2 10.2 μ of μ l, ddH2O of each 0.3 μ l, 2.5U/ μ l Taq archaeal dna polymerase of 10 μM of primer pairs
l.(2) PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 are followed
Ring；72 DEG C of extension 5min.(3) electrophoresis detection is carried out using 3% Ago-Gel.
Parent molecules mark polymorphism screening
Designed primer expands the genomic DNA of Indigo Rose, Heinz1706 and its F1, Ago-Gel electricity respectively
14 InDel molecular labelings with polymorphism of swimming detection.
The Primary Location of ATV gene
Using the AN2 gene promoter sequence difference with the site tomato Aft close linkage, an InDel molecule is developed
Mark HP1953.Using the molecular labeling to F2190 plants of plant of group carry out Molecular Detection, discovery molecular labeling HP1953 detection
It is totally 121 plants of plant of heterozygosis and homozygosis (consistent with atropurpureus fruit parent Indigo Rose), purple is all presented in fruit surface
Color.Wherein fruit surface has 97 plants of plant of purple dot, and fruit surface has 24 plants of plant of purple in blocks, card square examination table
It is bright to meet 3:1 separation.Illustrate that, in addition to Aft, there is also another recessive sites to control tamato fruit atropurpureus.For control kind
The gene of eggplant anthocyanin accumulation has carried out Molecular Detection to this 121 plants using the molecular labeling on No. 7 chromosome, in conjunction with fruit
Real coat color observes data, by ATV gene Primary Location between molecular labeling HP2675 and HP1919, the model of about 401kb
In enclosing.
The gene effect analysis of ATV gene
Select 3 plants from F2 group, the site Aft is homozygotic state (Aft (+/+)), the site atv be heterozygous state (atv
(+/-)), it carries out selfing and expands 3 F3 of numerous acquisition for group.Each F3 selects the 6 plants of site atv homozygosis such as Heinz1706 for group
Plant (atv (-/-)), the plant (atv of 5 plants of heterozygosis single plants (atv (+/-)) and the 6 plants of site atv homozygosis such as Indigo Rose
(+/+)), carry out phenotypic analysis.When fruit maturation, atv (-/-) fruits/plant epidermis presentation purple dot；Atv (+/-) fruit
Epidermis is also presented purple dot, but spot density ratio atv (-/-) somewhat higher；Atv (+/+) the sheet of purple of fruit surface presentation
Black (Fig. 1).Detect the anthocyanidin content of fresh exocarp (peel) respectively, atv (-/-) fruit exocarp (peel) every 100g
Fresh weight 26.1mg containing anthocyanidin；Atv (+/-) fruit exocarp (peel) every 100g fresh weight 35.8mg containing anthocyanidin；atv(+/+)
Fruit exocarp (peel) every 100g fresh weight 243.7mg containing anthocyanidin (Fig. 1).The above results show that atv is a recessive mutation
Site, under Aft (+/+) genetic background, when atv is homozygous mutation state, fruit surface anthocyanidin content about improves about 9
Times.
The finely positioning of ATV gene
F3 group is screened for 1900 plants using molecular labeling HP1919 and HP2675,14 plants of exchange single plants are obtained.
Further exploitation InDel, CAPS and dCAPS molecular labeling is screened in the region, Molecular Detection is carried out, in combination with fruit surface
Color and anthocyanidin measurement, finally by between the ATV assignment of genes gene mapping molecular labeling JP13 and JP17, about 9.6kb, the regional code 1
A gene Solyc07g052490 (Fig. 2), after their mutation lose vigor, anthocyanidin accumulation, the deeper disease of purple is presented in plant
Shape.Tomato atv mutant is similar, and fruit surface is purple because accumulating anthocyanidin.Therefore, Solyc07g052490 is
The unnamed gene is MybATV by ATV gene.
The determination of InDel ATV-In at ATV gene loci
According to tomato with reference to MybATV gene order design primer in genome Heinz 1706, to the tomato containing atv
Material Indigo Rose carries out PCR amplification, sequencing.Sequencing result is compared with tomato with reference to genome Heinz1706, it was found that
Multiple SNP and InDel, one of 4bp insertion are located in the Second Exon of gene coding region, can cause frameshift mutation.To kind
Eggplant Heinz1706 fruit surface carries out the transcript PCR amplification of MybATV gene, sequencing, it was found that 3 kinds of transcripts, they
1st, 2 exons are identical.Tomato Indigo Rose has also discovered these three transcripts, and still, there are one for exon 2
A 4bp insertion, leads to frameshit, protein translation terminates (Fig. 2) in advance.The 4bp insertion be named as ATV-In (ATV Insertion)。
The exploitation and verifying of embodiment 2InDel molecular labeling ATV-In
The design of primers of InDel molecular labeling ATV-In
According to the flanking sequence of ATV-In, 2 primers are devised.Forward primer is
5 '-GAGGTTTCTTCGTTGGTAGTC-3 ', reverse primer are
5’-CTAAATAAAAGTTATTGAGTTCACG-3’。
The polymorphic detection of InDel molecular labeling ATV-In
PCR amplification
With Indigo Ros, 1706 Heinz and its F1Genomic DNA be template, using for detect InDel mark
2 specific primers of ATV-In carry out PCR amplification.1. PCR system (15 μ l): 50-100ng/ μ l DNA profiling 1 μ l, 10 ×
1.5 μ l, 2.5mM dNTPs of PCR reaction buffer (ion containing Mg) 1.5 μ l, each 0.3 μ l, 2.5U/ μ l Taq of 10 μM of primer pairs
0.2 10.2 μ l of μ l, ddH2O of archaeal dna polymerase.2. PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 30s, 72 DEG C of extension 40s, 35 circulations；72 DEG C of extension 5min.
The polyacrylamide gel electrophoresis of pcr amplification product detects
Polyacrylamide gel concentration is 8%.The Indigo Rose in the site containing atv can detecte the item to a 89bp
Band；The site atv is that 1706 plant of Heinz of normal homozygous (wild type) can detecte the band to a 85bp；The site atv
For the F of heterozygous state1Single plant can be detected simultaneously by above-mentioned 2 band (Fig. 3).
The F of InDel molecular labeling ATV-In2Group's verifying
Using InDel molecular labeling ATV-In to F3Group plant carries out Molecular Detection.It was found that ripening fruits is presented in flakes
The plant of atropurpureus all only amplifies the band of a 89bp, shows the mutation equipotential base of InDel molecular labeling ATV-In and ATV
Because isolating.And ripening fruits is presented in the plant of atropurpureus spot, about 1/3 plant only amplifies the band of a 85bp,
Remaining plant can be detected simultaneously by above-mentioned 2 band (Fig. 3).
Although above the present invention is described in detail with a general description of the specific embodiments,
To those skilled in the art, on the basis of the present invention, it is obvious for making some modifications or improvements to it.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (2)
1. with the highly relevant molecular labeling of tomato atropurpureus fruit properties, which is characterized in that the molecular labeling be HP1917,
HP3217 and ATV-In, wherein the specific primer sequence of each molecular labeling is as follows:
① HP1917
Forward primer: 5-TCACAAGTAAAGGAGCATCC-3,
Reverse primer: 5-CTCTTCTTATAGTCACGTAAGTTAG-3；
② HP3217
Forward primer: 5-ACATTGGTGGCATTGAATATGAG-3,
Reverse primer: 5-ACAAGTATCACCCTATCCGTCTC-3；
③ ATV-In
Forward primer: 5-GAGGTTTCTTCGTTGGTAGTC-3,
Reverse primer: 5-CTAAATAAAAGTTATTGAGTTCACG-3.
2. the molecular labeling highly relevant with tomato atropurpureus fruit properties described in claim 1 is for selecting tomato purple black
Application in color fruit plant.
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| CN109423498B (en) * | 2017-08-30 | 2021-03-19 | 中国科学院遗传与发育生物学研究所 | Method for creating high-anthocyanin purple black tomato material through gene editing |
| CN107365865B (en) * | 2017-09-01 | 2020-12-08 | 中国农业大学 | Molecular marker related to tomato fruit color and application thereof |
| CN109750117B (en) * | 2017-11-08 | 2022-09-16 | 中国科学院遗传与发育生物学研究所 | Functional molecular marker of tomato anthocyanin synthesis related gene Aft and application thereof |
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| WO2003084312A2 (en) * | 2002-04-04 | 2003-10-16 | Exelixis Plant Sciences, Inc. | Identification and characterization of an anthocyanin mutant (ant1) in tomato |
| CN102703478A (en) * | 2011-12-08 | 2012-10-03 | 中国农业科学院蔬菜花卉研究所 | Application of T-GGPS (transgenic geranylgeranyl pyrophosphate synthase) gene in improvement of lycopene content of plant |
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| 《Near-isogenic lines enhancing ascorbic acid, anthocyanin and carotenoid content in tomato (Solanum lycopersicum L. cv Micro-Tom) as a tool to produce nutrient-rich fruits》;Ivan Sestari等;《Scientia horticul turae》;20141231;第175卷;摘要、第112页左栏倒数第1段 * |
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