CN108707612A - A kind of and the relevant gene of late bolting character of radish and its application - Google Patents
A kind of and the relevant gene of late bolting character of radish and its application Download PDFInfo
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Abstract
The present invention provides a kind of and the relevant gene of late bolting character of radish and its applications, belong to gene engineering technology field, have nucleotide sequence shown in SEQ ID No.1.Gene provided by the invention can make radish evening bolting bloom.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to it is a kind of with the relevant gene of late bolting character of radish and its
Using.
Background technology
Radish originates from China, is the important vegetable crop in China, is urban and rural residents of China " staple vegetable " for a long time.
Year 16000000 mu of sown area accounts for the 6% of the entire vegetables production in China.Radish is long-day plant, by after low temperature vernalization
Reproductive growth is turned to by nutrient growth under long-day conditions, to which bolting is bloomed.Underdone bolting is bloomed most important to radish production
Influence be transformation of the nutrient growth to reproductive growth, inhibit expanding for fleshy root, yield and quality caused to decline.It was found that radish
Late bolting gene and cultivation radish evening bolting kind are to solve the problems, such as this fundamental way.
Based on high-throughput RNA sequencing technologies, identifies in radish and blooms the relevant gene of character with late bolting, it was found that
Key gene such as FT, CO, SOC1, FLC and LFY that a large amount of difference expression genes and control bolting are bloomed.By transcribing component
Analysis finds 3 homologous FLC genes (RsFLC1, RsFLC2 and RsFLC3), respectively position (Yi on Rs7, Rs2 and Rs3 chromosome
et al.2014； Kitashiba et al.2014).There are apparent sequence variations on RsFLC gene locis, but do not look for
To with the relevant new gene order of late bolting character of radish.
Invention content
The purpose of the present invention is to provide a kind of and relevant gene of late bolting character of radish, which can make radish late
Bolting is bloomed.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of and relevant genes of late bolting character of radish, have nucleosides shown in SEQ ID No.1
Acid sequence.
The present invention also provides application of the gene described in above-mentioned technical proposal in differentiating Radish Bolting character.
Preferably, the method for differentiating Radish Bolting character, includes the following steps：
The genomic DNA of radish to be measured is subjected to PCR amplification, obtains amplified production, the primer that the PCR amplifications use is
There is nucleotide sequence shown in SEQ ID No.2, the 1R to have nucleosides shown in SEQ ID No.3 by 1F and 1R, the 1F
Acid sequence；
When the segment of the amplified production is 2417bp, and includes the gene described in above-mentioned technical proposal, the radish
Bolting trait is Late bolting shape；
When the segment of the amplified production is 790bp, and does not include the gene described in above-mentioned technical proposal, the radish
Bolting trait is early bolting character.
Preferably, the every 30 μ l of the reaction system of the PCR amplification include：100ng genomic DNAs, 2 10 × PCR of μ l
Buffer, 1.6 0.4 0.4 μ l of μ l, a concentration of 10 μM of 1R of μ l dNTPs, a concentration of 10 μM of 1F, 1.5 μ l Taq enzymes,
24.1μl ddH2O。
Preferably, the program of the PCR amplification is：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 1min,
72 DEG C of extension 1.5min, 35 cycles；72 DEG C of extension 10min；4 DEG C of preservations.Described in above-mentioned technical proposal
Application of the gene order in radish breeding, the method for the radish breeding includes：Utilize the step described in above-mentioned technical proposal
Suddenly, the radish to be measured comprising gene described in above-mentioned technical proposal in genome is selected to carry out breeding as parent.
The present invention provides a kind of with the relevant gene of late bolting character of radish, and the gene and radish evening bolting are bloomed character
It is related.Radish is biennial crop, and bolting qualification time of blooming is longer, using the present invention as identifying radish varieties Bolting trait
With assisted selection parent in application, can bolting identification be carried out to kind to be measured in seedling stage, plant field management is saved
Cost and time.
The result of the embodiment of the present invention is shown：Gene provided by the invention can make radish evening bolting bloom.
Description of the drawings
Fig. 1 is Radish Bolting flowering time character QTL location informations, A：Bolting flowering time Primary Location is in InDel162
Between InDel170 labels；B：The radish R02 chromosomes of local cypher, candidate gene RsFLC2 be located at label InDel520 and
Between InDel535；
Fig. 2 is that agarose gel electrophoresis detects Genotyping situation.
Specific implementation mode
The present invention provides a kind of and relevant genes of late bolting character of radish, have nucleosides shown in SEQ ID No.1
Acid sequence, the nucleotide sequence are specific as follows shown：
Gene described in above-mentioned technical proposal is related to the Bolting trait of radish, the trailing plants containing gene described in above-mentioned technical proposal
The Bolting trait of fore-telling shows as Late bolting shape.
The present invention also provides application of the gene described in above-mentioned technical proposal in differentiating Radish Bolting character.
In the present invention, the method for differentiating Radish Bolting character, includes the following steps：
The genomic DNA of radish to be measured is subjected to PCR amplification, obtains amplified production, the primer that the PCR amplifications use is
There is nucleotide sequence shown in SEQ ID No.2, the 1R to have nucleosides shown in SEQ ID No.3 by 1F and 1R, the 1F
Acid sequence；
When the segment of the amplified production is 2417bp, and includes gene described in claim 1, the Radish Bolting
Character is Late bolting shape；
When the segment of the amplified production is 790bp, and does not include gene described in claim 1, the radish is taken out
A kind of sedge character is early bolting character.
The present invention is not particularly limited the extracting method of the genomic DNA of the radish to be measured, using art technology
The conventional use of extracting method of personnel, such as CTAB methods.
In the present invention, the primer that the PCR amplification uses is 1F and 1R, and the 1F has shown in SEQ ID No.2
Nucleotide sequence, it is specific as follows shown：
5'-ATGGGAAGAAAAAAACTAGAGAT-3'；
The 1R has nucleotide sequence shown in SEQ ID No.3, specific as follows shown：
5’-TGCATTAATCCGTGGTAAATT-3’。
In the present invention, the every 30 μ l of the reaction system of the PCR amplification are preferably included：100ng genomic DNAs, 2 μ l 10
× PCR Buffer, 1.6 μ l dNTPs, a concentration of 10 μM of 1F 0.4 μ l, a concentration of 10 μM of 1R 0.4 μ l, 1.5 μ l Taq
Enzyme, 24.1 μ l ddH2O.The present invention is not particularly limited the source of mentioned reagent, is routinely selected using those skilled in the art
Commercial product.
In the present invention, the program of the PCR amplification is：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing
1min, 72 DEG C of extension 1.5min, 35 cycles；72 DEG C of extension 10min；4 DEG C of preservations.The present invention also provides above-mentioned technical sides
Application of the gene in radish breeding described in case, the method for the radish breeding include：Utilize the step described in above-mentioned technical proposal
Suddenly, select the breeding material comprising gene described in above-mentioned technical proposal as parent.
With reference to embodiment to it is provided by the invention it is a kind of with the relevant gene of late bolting character of radish and its application into
Row detailed description, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
With the acquisition of the relevant gene order of radish bolting resistant property shape
The present invention using conventional map based cloning method to the relevant gene of radish bolting resistant property shape carry out identification and gram
It is grand.
1, hereditary segregating population structure and phenotypic evaluation
To come from Japanese late bolting material " Ninengo " and Chinese early bolting material " Maer " as parents, structure includes
The F of 183 single plants2Group.In the spring in 2016 to parent, F1、F2Group's single plant carries out bolting and character investigation of blooming.Investigation side
Method is：Every other day investigation single plant budding and situation of blooming.The time budding for the day needed for from field planting to the visible bud of naked eyes
Number；Flowering time is that the flowers are in blossom puts required number of days from field planting to first.Late bolting material " Ninengo " budding the time and
Flowering time is respectively 91 days and 110 days, early bolting material " Maer " budding the time and flowering time is respectively 44 days and 66
It.The bolting time-variance of F2 groups single plant is 43~105 days, 81.27 days average；Flowering time variation is 70~121 days,
It is 96.7 days average；The characteristics of showing as continuously distributed Inheritance of Quantitative Characters.
2, genetic linkage maps structure and QTLs mapping (QTL)
Insertion and deletion marks the exploitation of (InDel)：Using 2500 microarray datasets of Illumina Hiseq to parent
" Ninengo " and " Maer " carries out full-length genome and resurveys sequence, with ' Aokubi DH'Reference gene group (The radish genome
and comprehensive gene expression profile of tuberous root formation and
Development, Scientific Reports (2015) 5：10835) it is with reference to the polymorphism for detecting parents.Preliminary design
500 pairs of InDel labels, it is each mark in ' the interval distance in Aokubi DH ' reference gene groups that InDel, which marks design principle,
From for 300~800kb, annealing temperature is 58~60 DEG C, and the base difference between parents is 3~8bp, and amplified fragments size is 100
~200bp, to be detected using Native-PAGE.
Genetic linkage maps are built and qtl analysis：Take parent and F2The young leaflet tablet of each single plant of group utilizes routine
CTAB methods extract total DNA.Utilize published 626 couples of EST-SSR (Shirasawa K etc., An EST-SSR linkage map
Of Raphanus sativus and comparative genomics of the Brassicaceae, DNA
Research, (2011) 18：221-232) and 500 couples of InDel of Preliminary design of the present invention labels detect parent " Ninengo "
Polymorphism between " Maer ".By obtaining 131 pairs of polymorphisms to parents' PCR amplification and 8% polyacrylamide gel electrophoresis
Primer is used for 183 F2The label parting of group's single plant.It is opened containing 9 linkage groups using 4.0 software buildings one of Joinmap
Genetic map.Using MapQTL4.0 softwares by the radish related gene of resistance to bolting be located in radish R02 chromosomes InDel170 and
Between InDel162, the result is shown in Figure 1 A.
The relevant gene finely positioning of radish bolting resistant property shape：According to ' Aokubi DH ' reference gene group informations (The
radish genome and comprehensive gene expression profile of tuberous root
Formation and development, Scientific Reports (2015) 5：10835) and ' XYB36-2 ' refers to base
Because group information (Zhang Xiaohui etc., A de novo Genome of a Chinese Radish Cultivar,
Horticultural Plant Journal, 2015,1 (3)：155-164), to Primary Location section InDel170-
Encryption is marked in InDel162, designs 43 InDel labels altogether, is shown in Table 1.The linkage map of local cypher is constructed, as a result
See the B in Fig. 1, qtl analysis by Radish Bolting and flowering time character be located in R02 chromosomes InDel520 and InDel535 it
Between.
The sequence of table 1 43 couples of InDel labels
3, the identification of radish bolting resistant property shape related gene and sequence analysis
Radish bolting resistant property shape correlation candidate identified for genes：According to radish gene group gene annotation (The radish genome
and comprehensive gene expression profile of tuberous root formation and
Development, Scientific Reports (2015) 5：10835), in localization region InDel520 and the areas InDel535
Between, RSG31600 (Rs_scaf675：106,055-109,484) RsFLC2 genes are noted as, which, which is important, blooms
Controlling gene.
RsFLC2 gene sequencings：According to published RsFLC2 gene orders (Yi etc., Identification of
three FLOWERING LOCUS C genes responsible for vernalization response in
Radish (Raphanus sativus L.), Hort Environ Biotechnol, 2014,55：548-556) design primer
The sequence of 1F and 1R, 1F are as shown in SEQ ID No.2, the sequence of specially 5 '-ATGGGAAGAAAAAAACTAGAGAT-3 ', 1R
Row are as shown in SEQ ID No.3, specially 5 '-TGCATTAATCCGTGGTAAATT-3 ' and design primer 2F and 2R, 2F
Sequence as shown in SEQ ID No.90, the sequence such as SEQ ID of specially 5 '-AATTTACCACGGATTAATGCA-3,2R
Shown in No.91, specially 5 '-CTAATAAAGCAGTGGGAGAGTTAC-3 ' pass through conventional PCR amplification, clone and sequencing
Technology obtains late bolting parent ' Ninengo ' and early bolting parent " Maer " RsFLC2 gene orders, late bolting parent respectively
' Ninengo ' RsFLC2 gene orders are as shown in SEQ ID No.92, specially：
Early bolting parent " Maer " RsFLC2 gene orders are as shown in SEQ ID No.93, specially：
Alignment has found that in ' Ninengo ', there are one 1627bp pieces in the First Intron of RsFLC2 genes
The insertion of section, insertion point are located at the 434bp of translation initiation position.
RsFLC2 gene homologies are analyzed：Further by " Ninengo " and " Maer " RsFLC2 gene orders with
(KP027027~KP027032, KP027034, sequence is from text for the RsFLC2 gene orders for 7 parts of radish materials that NCBI is announced
It offers, Yi etc., Identification of three FLOWERING LOCUS C genes responsible for
Vernalization response in radish (Raphanus sativus L.), Hort Environ
Biotechnol, 2014,55：Multiple alignment 548-556) is carried out, does not find that there are the 1627bp to be inserted into sequence in other materials
Row.In addition to identifying the homologous sequence of 1627bp insetion sequences in ' Ninengo ' RsFLC2, BLASTn is utilized
(https：//blast.ncbi.nlm.nih.gov) compare after show, in e-100Lower 3 sequences of threshold value (from B.napus or
B.oleracea) there is 83-84% sequence similarities, but coverage is only 48.7%.
Embodiment 2
Application of the 1627bp insetion sequences in identification radish evening bolting and early bolting material in RsFLC2
1, identification of the 1627bp insetion sequences in late bolting and early bolting radish in RsFLC2
1) DNA is extracted
Conventional CTAB methods extract the genomic DNA of 183 kinds of radish materials to be measured in table 2 respectively, and 183 kinds in table 2 are to be measured
Radish material is to obtain F1 generation, the F2 single plants that F1 selfings obtain using parent's Ninengo and Maer hybridization.
2) PCR amplification and detection
Utilize 1F (5'- ATGGGAAGAAAAAAACTAGAGAT-3 ') and 1R (5 '-TGCATTAATCCGTGGTAAATT-
3 ') primer pair radish material to be measured carries out PCR amplification.
PCR reaction systems：Including 100ng genomic DNAs, 2 μ 10 × PCR of l Buffer, 1.6 μ l dNTPs, it is above-mentioned on
Each 0.4 μ l of downstream primer (10 μM), 1.5UTaq enzymes add ddH2O to 30 μ l.PCR amplification program is：94 DEG C of pre-degeneration 4min；
94 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C of extension 1.5min, 35 recycle；72 DEG C of extension 10min；4 DEG C of preservations.Amplification
After, Genotyping situation is detected using 1.5% agarose gel electrophoresis, as a result sees Fig. 2
As can be drawn from Figure 2, late bolting material expands the segment for obtaining 2417bp in F2 groups, wherein including
The Insert Fragment of 1627bp, amplification obtains the segment of 790bp in early bolting material.
2, Radish Bolting Characters Identification
Using the index for time as Radish Bolting morning and evening of buddingging, investigation standard is needed for from field planting to the visible bud of naked eyes
Number of days the results are shown in Table 2.
The grade scale of investigation is as follows：
Early bolting：Budding the time be 43 days to 69 days；
Middle bolting：Budding the time be 70 days to 91 days；
Late bolting：Budding the time be 92 days to 105 days.
2 183 parts of material RsFLC2 Genotypings of table and disease index statistical form
As can be drawn from Table 2, in 31 parts of Fields detections are early bolting material, molecular markers for identification is homozygosis 790bp
Amplified fragments；In 31 parts of Fields detections are late bolting material, molecular markers for identification is homozygosis 2417bp amplified fragments, this hair
Bright method identifies that accuracy is 100%.
As seen from the above embodiment, provided by the invention radish evening bolting to be made to open with the relevant gene of radish evening bolting
Flower.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of and relevant gene of late bolting character of radish has nucleotide sequence shown in SEQ ID No.1.
2. application of the gene described in claim 1 in differentiating Radish Bolting character.
3. application according to claim 2, which is characterized in that the method for differentiating Radish Bolting character, including following step
Suddenly：
The genomic DNA of radish to be measured is subjected to PCR amplification, obtains amplified production, the primer that the PCR amplification uses be 1F and
There is nucleotide sequence shown in SEQ ID No.2, the 1R to have nucleotides sequence shown in SEQ ID No.3 by 1R, the 1F
Row；
When the segment of the amplified production is 2417bp, and includes gene described in claim 1, the Radish Bolting character
For Late bolting shape；
When the segment of the amplified production is 790bp, and does not include gene described in claim 1, the Radish Bolting character
For early bolting character.
4. application according to claim 3, which is characterized in that the every 30 μ l of reaction system of the PCR amplification include：
100ng genomic DNAs, 2 μ 10 × PCR of l Buffer, 1.6 μ l dNTPs, a concentration of 10 μM of 1F 0.4 μ l, a concentration of 10 μM
0.4 μ l of 1R, 1.5 μ l Taq enzymes, 24.1 μ l ddH2O。
5. application according to claim 3 or 4, which is characterized in that the program of the PCR amplification is：94 DEG C of pre-degenerations
4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C of extension 1.5min, 35 recycle；72 DEG C of extension 10min；4 DEG C of preservations.
6. the method for application of the gene described in claim 1 in radish breeding, the radish breeding includes：Utilize claim
Step described in 3 selects the radish to be measured comprising gene described in claim 1 in genome to carry out breeding as parent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251536A (en) * | 2020-11-17 | 2021-01-22 | 中国农业科学院麻类研究所 | SNP molecular marker closely linked with bolting property of radish and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251536A (en) * | 2020-11-17 | 2021-01-22 | 中国农业科学院麻类研究所 | SNP molecular marker closely linked with bolting property of radish and application thereof |
| CN112251536B (en) * | 2020-11-17 | 2022-07-19 | 中国农业科学院麻类研究所 | SNP molecular marker closely linked with bolting property of radish and application thereof |
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| CN108707612B (en) | 2020-08-18 |
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