CN114032327B - Codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia lingua and application thereof - Google Patents
Codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia lingua and application thereof Download PDFInfo
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Abstract
The invention discloses a codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia elegans and application thereof, wherein the codominant InDel molecular marker is respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3 in nucleotide sequence. The invention discovers that a section of sequence is found in the CYC gene of a zinnia tubular flap parent A1GH by the existing PCR technology and transcriptome sequence information mining, thereby successfully obtaining the codominant InDel molecular marker of zinnia lingua type character.
Description
Technical Field
The invention relates to the field of plant molecular markers, in particular to a codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia elegans and application thereof.
Background
The zinnia elegans is an important flower bed, flower field, potted flower and cut flower material due to the long flowering phase and rich flower colors, and has important commercial value. The head-like inflorescence of zinnia is the main part of appreciation and is directly related to sensory quality. The capitate inflorescence of most compositae plants in nature consists of flat-petal lingulate flowers and tubular-petal tubular flowers, and the petal shape has very important significance for researching the interaction of plants and pollinators. The unilocular flowers of zinnia elegans which are naturally produced not only enrich the head inflorescence types of the feverfew, but also provide good materials for studying the shapes of petals.
The research finds that the shape of the petals is related to the flower symmetry, so the gene related to the flower symmetry can be used as a candidate gene for the petal tubular petal transformation. However, the development of the common separation molecular marker for the zinnia elegans tongue petal and tube petal character is slow at present, and the main reason is that due to the lack of genome information, the molecular marker is far away from a tube petal site, so that the common separation molecular marker is difficult to develop and effectively utilized.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a codominant InDel molecular marker for identifying the flat petal character and the tubular petal character of a zinnia lingua and application thereof.
In order to achieve the purpose, the invention designs a codominant InDel molecular marker for identifying the flat petal character and the tube petal character of the zinnia elegans, wherein the codominant InDel molecular marker is respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3 according to the nucleotide sequence.
The invention also provides a primer pair for obtaining the molecular marker, wherein the primer pair InDel F/R is as follows:
forward primer InDel F:5 '-AAAACCCTTGATTGGTTTGTTCAC-' 3,
reverse primer InDel R:5 '-ATGGCTTTGGAGGCTTTTTTCTT-' 3.
The invention also provides application of the molecular marker in zinnia lingua petal type related molecular marker assisted breeding.
The invention also provides application of the primer pair in auxiliary breeding of related molecular markers of the zinnia elegans lingua petal type.
The invention also provides a kit for identifying the genotype of the zinnia type separation population, which contains the primer pair InDel F/R.
The method for identifying the petal type character and the segregation population genotype of the zinnia lingua by using the kit comprises the following steps:
1) Extracting genome DNA of zinnia elegans sample to be detected
2) Taking the zinnia genetic DNA to be detected as a template, and adopting the following primer pairs:
forward primer InDel F:5 '-AACCCTTGATTGGTTTGTTCAC-' 3,
reverse primer InDel R:5 '-ATGGCTTTGGAGGCTTTTTTCTT-' 3;
performing PCR amplification to obtain a PCR product;
3) And (3) detecting a PCR product:
detecting the PCR product by 1% agarose gel electrophoresis, recording the genotype, and taking a picture under an ultraviolet lamp;
if the electrophoresis strip contains a strip of 2596bp (shown in SEQ ID NO: 2), the lingulate petal type of the head-shaped inflorescence of the zinnia elegans to be detected is a tube petal, and the zinnia elegans to be detected is a homozygous tube petal;
or, if the electrophoresis band contains a band of 442bp (shown in SEQ ID NO: 3), the lingulate petal type of the head-shaped inflorescence of the zinnia elegans to be detected is a flat petal, and the zinnia elegans to be detected is a homozygous flat petal;
or, if the electrophoresis strip contains two strips of 442bp and 2596bp at the same time, the lingulate petal type of the head-shaped inflorescence of the zinnia elegans to be detected is a flat petal; the zinnia elegans to be detected is a heterozygous flat valve.
Further, it is characterized in that: in the step 2), a PCR reaction system:
| zinnia genetic group DNA to be detected | 0.5ul |
| Forward primer (0.2 nmol/ul) | 0.5ul |
| Reverse primer (0.2 nmol/ul) | 0.5ul |
| PCR mix | 5ul |
| Adding deionized water to | 10ul |
The PCR reaction conditions are as follows: performing pre-denaturation at 94 ℃ for 2min, circulating for 30 times, performing denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and performing extension at 72 ℃ for 45s; finally, extension was carried out at 72 ℃ for 2min.
The principle of the invention is as follows:
1. genetic markers, which are objective manifestations of genetic differences between individuals or populations of organisms, refer primarily to those biological characteristics that clearly reflect biological polymorphisms. With the rapid development of biotechnology, the types of genetic markers are rapidly developed and enriched, and morphological markers, cytological markers, protein markers and DNA markers appear in sequence. Compared with other labeling technologies, the DNA marker has the advantages of abundant quantity, high polymorphism, small influence by environmental conditions and development stages, simple and rapid detection means and the like. Therefore, DNA marker is an ideal molecular marker, has been widely used in molecular biology research of animals and plants, and plays an increasingly important role in gene localization and cloning, species origin and domestication. In recent years, with the development of high throughput sequencing technologies, molecular marker development techniques have also been developed rapidly. The high-throughput technology is utilized to develop the molecular marker which is based on the PCR technology, simple to operate and high in application value, and a foundation can be laid for controlling the gene localization of the important horticultural traits of the zinnia elegans and molecular-assisted selective breeding.
2. The head-shaped inflorescence of the zinnia elegans is a main ornamental part, and consists of a peripheral tongue flower and an inner wheel barrel-shaped flower, and is used for identifying a peripheral tongue petal type, wherein the flat petal tongue flower is dominant, the tubular petal tongue flower is recessive, compared with the flat petal parent S5, a 2154bp sequence is added in a molecular marker obtained from a tubular petal parent A1GH and is shown as SEQ ID NO.1, so that the sequence length polymorphism is caused, and the fragment is inserted into a coding region to cause the early termination of the coding of an amino acid sequence; therefore, primers are designed on two sides of a 2154bp sequence in the CYC gene sequence, and PCR is carried out by using the DNA of a sample to be detected as a template, so that the shape of the lingulate petal of the zinnia head-shaped inflorescence is identified.
3. The screening principle of the codominant InDel molecular marker for identifying the shape of the tongue-shaped petal of the zinnia head-shaped inflorescence is as follows:
1) A tongue-shaped petal type separation population is constructed to screen a crossover individual, transcriptome data is taken as reference, genes possibly related to petal types are screened, the genome of a homozygous flat petal (S5) and the genome of a homozygous tubular petal (A1 GH) are taken as templates for amplification, a sequence polymorphism gene segment is found and developed into a molecular marker, scanning is carried out in the crossover individual, and the result shows that the petal type and the genotype of the crossover individual are completely linked at the marker. BSA-RNA sequencing is carried out by utilizing the constructed segregation population, non-reference transcriptome analysis is carried out on RNA-seq data, SNP loci are screened according to differential expression, a plurality of pairs of primers are designed, and the multi-pair polymorphic primers are screened by utilizing parents (a homozygous flat flap gene pool and a homozygous tubular flap gene pool), F1 (a heterozygous flat flap gene pool) and an extreme mixing pool (a flat flap gene mixing pool and a tubular flap gene mixing pool in a BC1 segregation population).
2) And scanning the segregation population by using a polymorphic primer, screening and exchanging individual plants, and initially positioning the tube valve gene among a plurality of related markers with highly linked tongue-shaped tube valve characters. Taking genome of sunflower and lettuce as reference, comparing the linkage markers obtained by initial positioning to the sunflower Chr12 and lettuce Chr3 respectively by BLASTX, calling gene sequences of corresponding regions, comparing the transcription group data with the transcription group data by blast software, screening co-linear sequences with SNP loci, designing primers to perform typing identification on the exchange single plant, and further positioning the tube flap genes between related markers.
3) Because the locating interval cannot be further reduced due to the lack of genome, and the possible inversion, ectopy and the like of chromosomes in the species evolution process are considered, all gene sequences on two sides of the locating intervals of the sunflower Chr12 and the lettuce Chr3 are taken, the transcriptome data is compared with the gene sequences by utilizing blast software, and genes with significant difference in expression quantity are screened by combining function annotation.
4) Designing a primer according to the screened differential expression gene sequence, carrying out amplification screening on a polymorphic primer by taking parental DNA (homozygous flat flap and homozygous vessel flap genomes) as a template, carrying out amplification by taking the initially positioned and screened exchanged single plant DNA as the template, counting the genotype, finally obtaining 1 phenotype and genotype complete linkage marker, and obtaining 1 marker sequence for highlighting the difference in the flat flap and vessel flap materials.
The invention has the beneficial effects that:
the invention discovers that a section of sequence is found in CYC gene of zinnia tubular flap parent A1GH by the existing PCR technology and transcriptome sequence information mining, thereby successfully obtaining the codominant InDel molecular marker of zinnia lingua type character, and the marker can be used for molecular marker diagnosis in early stage (within 2 weeks) of filial generation, while two months are needed for waiting for flowering in traditional breeding.
Drawings
FIG. 1 is a photograph of a test material in the field
In the figure, the first filial generation of A. Homozygous flat valve A1GH, B. Homozygous tubular valve S5 and C.A1GH × S5;
FIG. 2 shows the polymorphic bands found by amplification of the CYC gene of zinnia elegans of the present invention
In the figure, the genome amplification band of the homozygous flat flap (S5) is 442bp, and the genome amplification band of the homozygous tubular flap (A1 GH) is 2596bp;
FIG. 3 is a graph showing the amplification results of primer pairs in the genomic DNAs of individuals of a Boraginaceae segregating population;
in the figure, the double band (2596 bp/442 bp) is the flat-petal zinnia (heterozygous);
a single band (2596 bp/442 bp) is flat-petal zinnia (homozygous);
the single band (2596 bp) is the zinnia (homozygous) for the tube flap.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1
Method for identifying genotype of zinnia type segregation population
1. Individual preparation of genomic DNA of segregating population
Selecting 46 plant materials (including 22 flat-petal plants and 24 tubular-petal plants), and respectively extracting genome DNA of the 46 plant materials by adopting an improved CTAB method;
2. analyzing the genome DNA of all the single strains by utilizing primer pair InDel F/R amplification;
forward primer InDel F:5 '-AAAACCCTTGATTGGTTTGTTCAC-' 3,
reverse primer InDel R:5 '-ATGGCTTTGGAGGCTTTTTTCTT-' 3;
and (3) PCR reaction: the total volume is 10ul, wherein the zinnia genomic DNA template to be detected is 0.5ul, the forward primer and the reverse primer are respectively 0.2nmol/ul, the PCRmix is 5ul, and finally deionized water is added to the total volume of 10ul; the PCR reaction conditions were: performing pre-denaturation at 94 ℃ for 2min, circulating for 30 times, performing denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and performing extension at 72 ℃ for 45s; finally, extending for 2min at 72 ℃; the PCR product was detected by electrophoresis on a 1% agarose gel.
As shown in fig. 2 to 3: when the amplified product is a single bright band of 442bp, the plant material is homozygous in flat petals, when the amplified product is a single bright band of 2596bp, the plant material is homozygous in tube petals, and when the amplified product is two bright bands of 442bp and 2596bp, the plant material is heterozygous in flat petals; thus, the following steps are carried out: 1 plant material to be treated is homozygous for flat petals, and 21 plant materials to be treated are heterozygous; 24 plant materials to be treated are homozygous for the pipe valves.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia elegans and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
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<211> 2154
<212> DNA
<213> Zinnia elegans
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acacatgcta aagtcttctt ctactgcatc catgcactgt ccacgtgaga ttcatgaagc 360
tttagcttat tcaccaactt ataaaacttg aagcaaccta aaaataccat ttgacgaccc 420
gataactaaa cgactaatcc aacaatcacc cccttagtcg tttattcatc acttcattta 480
caggagagtc ggttcgtcgt cctcgcaccg gctcaagttt agctcttctt ttttctccca 540
cttcggacac tccgatatgt aatgcccgaa ctcattacac ttgaagcact tcattcgtgt 600
cttgtcccgt tgtactcgac cactaccgga cccccgacct ccacctcggc cacgcccata 660
gtcttcccgg ttcgagtttc cacaaccaca atgctcacac ttgtgctcct tttcctttgc 720
cttttcatct gaagctgcca acaaaagttg attttgttcg gcttggttct cctcaatctt 780
gatcctttct tcataggctt ttagccgacc cacaacgtca tcataactga cggtcttcaa 840
atcaacaaac tgttctatag aagccaccat ataaataaaa cgtttagtca acccgttaag 900
aaatttctta accacttttt cttcttcaat aaccgagcca agtgaggcgg ctcgagaagc 960
atactcggac aacttattag caaagtcatc tatgacttct tggtccttca tcttgagatt 1020
ttcaaactct cccattagag tttgaagcct tgcctctcgg atccgttccg cacctaagtg 1080
acgggtcttg agagcatccc acatctcctt tacgtttttg agattcccta tttgcaaagt 1140
ttgttcttct gggatggctt ggaacaagag ggccattgca acattattct tcttggcatc 1200
ccgatttgtt cccggttcaa tcatctccca caccccatga acactgaaaa taacccgcat 1260
cctcatggcc cataccgtgt aattcgtgga tgacaacacc gggcactgta gtgacaaggt 1320
gttgttctcc ttaacggtaa ctagagcttg actgtccgat ttcgccatgt cccgatttac 1380
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cgactcgtct ctgataccct cttgacccgc tagaacagta tctctttacc gctgacctgc 1620
gatcacctaa gtctctccgg tcactgctga tctgcacacc ggtcactttc gatcgctgca 1680
gcctgcccct cgaatcttta tccgctctga taccaaatgt tggatatcag gccttgattt 1740
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<212> DNA
<213> Zinnia elegans
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gtgttggata tcaggccttg atttagtttt gtatgtgaat gaaaccaaac aatatggtta 180
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ctaaacgact aatccaacaa tcaccccctt agtcgtttat tcatcacttc atttacagga 600
gagtcggttc gtcgtcctcg caccggctca agtttagctc ttcttttttc tcccacttcg 660
gacactccga tatgtaatgc ccgaactcat tacacttgaa gcacttcatt cgtgtcttgt 720
cccgttgtac tcgaccacta ccggaccccc gacctccacc tcggccacgc ccatagtctt 780
cccggttcga gtttccacaa ccacaatgct cacacttgtg ctccttttcc tttgcctttt 840
catctgaagc tgccaacaaa agttgatttt gttcggcttg gttctcctca atcttgatcc 900
tttcttcata ggcttttagc cgacccacaa cgtcatcata actgacggtc ttcaaatcaa 960
caaactgttc tatagaagcc accatataaa taaaacgttt agtcaacccg ttaagaaatt 1020
tcttaaccac tttttcttct tcaataaccg agccaagtga ggcggctcga gaagcatact 1080
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tggcccatac cgtgtaattc gtggatgaca acaccgggca ctgtagtgac aaggtgttgt 1440
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ctatgttctt cgatcaacaa ctgggttaac gagccccctg atttggcctg aatcacgact 1680
cgtctctgat accctcttga cccgctagaa cagtatctct ttaccgctga cctgcgatca 1740
cctaagtctc tccggtcact gctgatctgc acaccggtca ctttcgatcg ctgcagcctg 1800
cccctcgaat ctttatccgc tctgatacca aatgttggat atcaggcctt gatttagttt 1860
tgtatgtgaa tgaaaccaaa caatatggtt acagatacta aaaataaaga acaaaagaaa 1920
tgaagaaaac taaaaggaat actttatcat ttaacaattt aggctatata tagccatata 1980
atgacagaaa gtgggtgcta aaaagctgcg acaaaagact atttcaacta atgagccgtt 2040
gcatttgcat cttattcggt caaccatgaa ccaagtctac tctactaacc ttatcaaaat 2100
aaagcataat aaataaatag aaaatgacac atgctaaagt cttcttctac tgcatccatg 2160
cactgtccac gtgagattca tgaagcttta gcttattcac caacttataa aacttgaagc 2220
aacctaaaaa taccatttga cgacccgata actaaacgac taatccaaca aaaagggtca 2280
gattttcttg aaaaaaatgg aagaaaaaag aagaatttag tggttaataa taaaaccaac 2340
aaaaagaaga aaaaaacaag aacacaagaa cacaaatctg gtgttgatca tgtgagtctt 2400
gcaactagaa gtcagttaag agcagaagca agatcaagag ctagagaaag aacattggaa 2460
aaactgagga ttaagaagct taataatgat gtcaagactg ttgttggtga tactcattgt 2520
ttctatcttc aatcacaagg taatcatgaa gtcaaaattg gtgaaatgtt gactcaagaa 2580
aagcctccaa agccat 2596
<210> 3
<211> 442
<212> DNA
<213> Zinnia elegans
<400> 3
aaaacccttg attggttgtt cacaagatcc aagtcagcaa tcaaagattt ggttgaagaa 60
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gggtcagatt ttcttgaaaa aaatggaaga aaaaagaaga atttagtggt taataataaa 180
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ttggaaaaac tgaggattaa gaagcttaat aatgatgtca agactgttgt tggtgatact 360
cattgtttct atcttcaatc acaaggtaat catgaagtca aaattggtga aatgttgact 420
caagaaaagc ctccaaagcc at 442
<210> 4
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<212> DNA
<213> Synthetic sequences
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aaaacccttg attggttgtt cac 23
<210> 5
<211> 21
<212> DNA
<213> Synthetic sequence (Synthetic sequence)
<400> 5
atggctttgg aggcttttct t 21
Claims (2)
1. A method for identifying a zinnia type segregating population genotype, characterized in that: the method comprises the following steps:
1) Extracting genome DNA of zinnia elegans sample to be detected
2) Taking the zinnia genetic DNA to be detected as a template, and adopting the following primer pairs:
forward primer InDel F:5 'AACCCTTGATTGGTGTTCAC-3',
reverse primer InDel R:5 'ATGGCTTTGGAGGCTTTTTTCTT-containing 3';
performing PCR amplification to obtain a PCR product;
3) And (3) detecting a PCR product:
detecting the PCR product by 1% agarose gel electrophoresis, recording the genotype, and taking a picture under an ultraviolet lamp;
if the amplification product is a single bright band of 442bp, the plant material to be detected is homozygous for the flat valve,
or, if the amplification product is a single bright band of 2596bp, the plant material to be detected is homozygous for the tube flap,
or, if the amplification product is two bright bands of 442bp and 2596bp, the plant material to be detected is flat-petal heterozygous.
2. The method of claim 1, wherein: in the step 2), a PCR reaction system:
the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 2min, circulating 30 times for denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extending at 72 deg.C for 45s; finally, extension was carried out at 72 ℃ for 2min.
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| CN202111402998.4A CN114032327B (en) | 2021-11-24 | 2021-11-24 | Codominant InDel molecular marker for identifying flat petal character and tube petal character of zinnia lingua and application thereof |
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