CN114438250A - Iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof - Google Patents

Iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof Download PDF

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CN114438250A
CN114438250A CN202210171355.1A CN202210171355A CN114438250A CN 114438250 A CN114438250 A CN 114438250A CN 202210171355 A CN202210171355 A CN 202210171355A CN 114438250 A CN114438250 A CN 114438250A
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邵灵梅
李丹青
许曈
王小斌
张润龙
楼建华
夏宜平
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Zhejiang University ZJU
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Abstract

The invention discloses an iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof, and relates to the technical field of molecular markers. An iris SSR molecular marker group for screening two-season flowering iris varieties comprises the following 8 molecular marker groups, and the nucleotide sequences of the molecular marker groups are sequentially shown in SEQ ID NO. 17-24. The SSR molecular marker developed by the invention can simply, quickly, stably and slightly identify the iris japonica variety blooming in two seasons; the SSR marker obviously related to the two-season flowering can be discovered by the method, and an important theoretical basis is provided for assisted breeding and genetic improvement of the two-season flowering of iris; the SSR molecular marker primer group provided by the invention can well distinguish different iris varieties and is used for identifying iris varieties blooming in two seasons.

Description

Iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof
Technical Field
The invention relates to the technical field of molecular markers, in particular to an iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof.
Background
The flower is one of the most important ornamental parts of the flower. The multi-season flowering can greatly prolong the ornamental period of flowers, so that the multi-season flowering is one of key targets of flower breeding and has important application value. Two-season flowering refers to twice flowering within the annual growth cycle of the plant. In northern areas, the flowers usually bloom in spring and autumn, and in other areas, the flowers usually bloom in spring and summer. Flowers blooming in two seasons can enrich the types of ornamental flowers in gardens and forests in summer or autumn, and obviously prolong the ornamental period of the flowers.
At present, a plurality of irises capable of blooming in two seasons are cultivated abroad, and beared irises are mainly used. The Bearded Iris (Bearded Iris) is an important group in perennial root herbaceous plants of Iris (Iridaceae), has peculiar flower type and rich flower color, the flowering phase is generally 4-5 months, and the Bearded Iris (Bearded Iris) is an important garden and ground used plant. Most of the two-season flowering iris in China is introduced abroad, and due to the fact that introduction is not standard and the genetic law of the two-season flowering iris is not clear, whether the two-season flowering iris is in the non-flowering season is difficult to directly distinguish; in addition, new iris varieties created by means of crossbreeding and the like usually can be floriated in more than three years, and the flowering characters of hybrid filial generations of iris tectorum with two seasons cannot be judged at an early stage, so that great difficulty is brought to garden application and new variety cultivation of iris tectorum with two seasons.
SSR (simple Sequence repeats), also known as Microsatellite (Microsatete), is a tandem simple repeat Sequence widely distributed in eukaryotic genomes, and the length of each repeat unit is 1-6 bp. The SSR molecular marker has the advantages of good repeatability, reliable result, rich polymorphism, codominance, less required DNA and the like. The early identification of the two-season flowering iris can be realized by a molecular marker-assisted selection (MAS) method. The traditional SSR molecular marker is usually matched with polyacrylamide gel electrophoresis for polymorphism analysis, and the electrophoresis has the problems of toxicity, long electrophoresis time, difficulty in accurate identification of different allelic variation and the like. By adding a fluorescent label to the 5' end of the forward primer and a capillary electrophoresis method (Dendauw, J., et al. development of sequential tagged microsatellite sites (stms) markers in azalea: inactivation of primers and sequences and reagents and then, 546 (193-197. Dendauw, J., et al. identification of 33Chinese Rhododendron species using K sequences and AFLP data. acta Horticulture, 2002,169 and 177.), a larger number of small-phase-difference bands can be rapidly and precisely distinguished, and the experimental efficiency and accuracy can be effectively improved.
Association analysis (Association analysis) takes natural population as a research object, utilizes Linkage Disequilibrium (LD) and combines polymorphism of genes to find out loci associated with phenotypic traits (Li, Zhao Ying Qiang, whole genome Association analysis and research progress of an expansion method thereof, agricultural biotechnology bulletin, 2019, 27(01): 150-. By performing correlation analysis on the obtained high-polymorphism molecular marker loci and the flowering characters, the excellent SSR molecular marker for identifying the traits of the iris tectorum in two seasons can be effectively obtained, and the method has important significance for early identification and related research of hybrid progeny of the iris tectorum in two seasons.
Disclosure of Invention
The invention aims to provide a group of SSR molecular marker groups related to the two-season flowering traits of iris varieties and application thereof. The invention also provides an iris transcriptome SSR specific marker primer group, and aims to assist in identifying iris two-season flowering varieties by using the primer group.
The invention provides an iris SSR molecular marker group for screening iris varieties blooming in two seasons, wherein the SSR molecular marker group comprises the following 8 SSR molecular marker groups, and the nucleotide sequence is as follows: (1) P2S 19: the sequence is shown as SEQ ID NO. 17; (2) P2S 45: the sequence is shown as SEQ ID NO. 18; (3) P2S 48: the sequence is shown as SEQ ID NO. 19; (4) P3S 12: the sequence is shown as SEQ ID NO. 20; (5) P3S 17: the sequence is shown as SEQ ID NO. 21; (6) P3S 28: the sequence is shown as SEQ ID NO. 22; (7) P3S 30: the sequence is shown as SEQ ID NO. 23; (8) P3S 41: the sequence is shown as SEQ ID NO. 24.
The invention also provides a primer group for screening the iris tectorum with two blossoms, which comprises the following 8 pairs, wherein the nucleotide sequences of the forward primer and the reverse primer are as follows:
(1) P2S 19: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; (2) P2S 45: the sequence of the forward primer is shown as SEQ ID NO.3, and the sequence of the reverse primer is shown as SEQ ID NO. 4; (3) P2S 48: the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6; (4) P3S 12: the sequence of the forward primer is shown as SEQ ID NO.7, and the sequence of the reverse primer is shown as SEQ ID NO. 8; (5) P3S 17: the sequence of the forward primer is shown as SEQ ID NO.9, and the sequence of the reverse primer is shown as SEQ ID NO. 10; (6) P3S 28: the sequence of the forward primer is shown as SEQ ID NO.11, and the sequence of the reverse primer is shown as SEQ ID NO. 12; (7) P3S 30: the sequence of the forward primer is shown as SEQ ID NO.13, and the sequence of the reverse primer is shown as SEQ ID NO. 14; (8) P3S 41: the sequence of the forward primer is shown as SEQ ID NO.15, and the sequence of the reverse primer is shown as SEQ ID NO. 16.
The invention also provides a detection reagent or a kit for screening the iris tectorum variety blooming in two seasons, which contains the primer group.
The invention also provides application of the primer group or the detection reagent or the kit in screening the two-season flowering iris variety.
The invention also provides a screening method of the iris varieties flowering in two seasons, which comprises the following steps:
(1) extracting DNA of iris tectorum genome;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer set according to claim 2;
(3) detecting the PCR product of the step (2) by capillary electrophoresis;
(4) analyzing the detection result of the step (3), and constructing an iris variety clustering dendrogram together with the detection results of the two-season and single-season flowering irises serving as standard samples;
(5) and (4) clustering dendrograms of the iris varieties constructed according to the step (4) and the iris blossoming in two seasons as a standard sample, namely the iris varieties blossoming in two seasons.
Specifically, the PCR amplification system in the step (2) is as follows: 20 μ L of 1.0 μ L each of the upstream and downstream primers, 10mM of the upstream and downstream primers, 10 μ L of 5U/. mu.L of Taq polymerase, 1.0 μ L of 20 ng/. mu.L of DNA template, ddH2O7.0 μ L; the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 93 ℃ for 30 s; annealing at 58 ℃ for 30s, extending at 72 ℃ for 1min, and performing 32 cycles; extension at 72 ℃ for 10 min.
Specifically, the capillary electrophoresis detection step in the step (3) is as follows: adding 0.5 mu L of molecular weight internal standard and 8.5 mu L of deionized formamide into the PCR product obtained in the step (2) for denaturation under the denaturation condition of 95 ℃ for 5 min; cooling on ice for more than 10min, centrifuging for 10s, and performing capillary electrophoresis detection.
The specific steps of analyzing the detection result in the step (3) are as follows: and (4) according to the detection result of the capillary electrophoresis in the step (3), performing genetic clustering analysis on the iris varieties according to a UPGMA method: firstly, making a (0, 1) matrix of iris varieties, specifically: arranging and marking bands detected by capillary electrophoresis from small to large, wherein each variety corresponds to each band type, and the bands are marked as 1, and the bands which are not detected are marked as 0; then, a clustering dendrogram of the iris variety is made through NTSYS-pc software; the iris variety clustering dendrograms can be clustered into different groups, and are clustered with irises flowering in two seasons of a standard sample, namely the iris varieties flowering in two seasons.
The invention also provides application of the screening method in screening the iris tectorum with two flowering seasons.
The invention has the beneficial effects that: the SSR molecular marker developed by the invention is a codominant molecular marker which can identify the two-season flowering iris variety, and compared with the prior art, the SSR molecular marker has the following advantages and effects: (1) by adding the fluorescent marker and the capillary electrophoresis method, the method can simply, quickly, stably and slightly identify the two-season flowering iris variety; (2) the SSR molecular marker is associated with the two-season flowering trait, and the SSR marker which is obviously associated with the two-season flowering can be discovered by the method, so that an important theoretical basis is provided for assisted breeding and genetic improvement of iris two-season flowering.
The SSR molecular marker primer group provided by the invention can well distinguish different iris varieties and is used for identifying iris varieties blooming in two seasons.
Drawings
FIG. 1 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using a molecular marker P2S19 primer.
FIG. 2 is a SSR agarose gel electrophoresis image of DNA of 17 iris varieties amplified using a primer labeled with molecular marker P2S 45.
FIG. 3 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P2S 48.
FIG. 4 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P3S 12.
FIG. 5 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P3S 17.
FIG. 6 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P3S 28.
FIG. 7 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P3S 30.
FIG. 8 is a SSR agarose gel electrophoresis of 17 iris varieties of DNA amplified using the primer P3S 41.
FIG. 9 is a clustering dendrogram of 17 iris varieties.
FIG. 10 is a QQ chart (GLM model) of correlation analysis of the two-season flowering trait of iris varieties.
FIG. 11 is a QQ chart (MLM model) of correlation analysis of the two-season flowering trait of iris varieties.
FIG. 12 is an exemplary diagram of capillary electrophoresis peaks.
Detailed Description
Example 1
(1) Plant material
The test plant material: the plant material of the research is 17 bearded iris varieties collected from the northern area horticulture experimental farm in Ningbo city, and the species is shown in table 1. And numbering the 17 iris varieties from 1 to 17 in sequence. Wiping and shearing 17 iris varieties of leaves, putting the leaves into a self-sealing bag, putting the self-sealing bag into dry ice, returning the self-sealing bag to a laboratory, and storing the self-sealing bag in a refrigerator at the temperature of-80 ℃ for later use.
TABLE 1 Iris species information
Figure BDA0003518157410000051
(2) Primer design
Initially selecting 110 pairs of SSR primers according to an iris transcriptome, wherein the selected primers specifically comprise: PCR products have GC content of 40-60% at 200-300 bp, TM value of 58.8-60.2 ℃ and the like; wherein 55 pairs of SSR primers with two-base mutation and three-base mutation are synthesized by Shanghai biological engineering Co.
(3) Genomic DNA extraction
Extracting the genome DNA of a sample to be detected by adopting a Novozan genome DNA rapid extraction kit, determining the quality and concentration of the DNA by utilizing 1% agarose gel electrophoresis and a NanoDrop ultraviolet spectrophotometer, diluting to 20 ng/mu L, and storing in a refrigerator at the temperature of-20 ℃ for later use.
(4) Screening and detecting iris SSR molecular marker
4 iris varieties (randomly selected from 17 iris samples) were PCR amplified using the initially selected 110 primer pairs described above. 8 pairs of SSR primers with clear amplification bands, rich polymorphism and good repeatability are screened out from the SSR primers and are shown in Table 2. Fluorescent group labeled FAM is added to the 5' end of the forward primer of the 8 pairs of primers, and the sequence is as follows: tgtaaaacgacggccatt, reverse primer unchanged. All samples were PCR amplified with 8 pairs of primers with added marker sequences. The obtained PCR product is divided into two parts, one part is used for agarose gel electrophoresis separation, and the other part is used for fluorescence capillary electrophoresis detection. The PCR product sequences are shown in Table 3.
TABLE 28 SSR marker primer information
Figure BDA0003518157410000052
Figure BDA0003518157410000061
TABLE 38 SSR marker sequence information
Figure BDA0003518157410000062
Figure BDA0003518157410000071
SSR-labeled PCR amplification conditions
SSR marker detection PCR reaction system, see Table 4; PCR procedure, see table 5; and (3) carrying out electrophoresis on the PCR amplification product on 1% agarose gel, observing on an ultraviolet gel imager after 12 minutes, taking a picture and recording the result, wherein all SSR marker amplification bands are clear, the repeatability is good, and the size of the amplification product is 200-300 bp, which is shown in the figure 1-8.
TABLE 4 SSR marker detection PCR reaction System
Components Volume (μ L)
Upstream primer F (10mM) 1.0
Downstream primer R (10mM) 1.0
Taq polymerase(5U/μL) 10
DNA template (20 ng/. mu.L) 1.0
ddH2O 7.0
TABLE 5 PCR reaction amplification procedure
Figure BDA0003518157410000081
b. Capillary electrophoresis detection
B, adding the fluorescence-labeled amplification product obtained in the step a, the molecular weight internal standard and the deionized formamide into a special 96-well plate for a genetic analyzer, performing denaturation, placing on ice, cooling for more than 10min, and centrifuging for 10 s; the 96-well plate was then placed in a capillary electrophoresis detector (3730XL type instrument, ABI, USA);
the dosage of the internal standard is 0.5 mu L; the dosage of the deionized formamide is 8.5 mu L; denaturation conditions were 95 ℃ for 5 min.
(5) Analysis of results
analysis of genetic diversity of SSR molecular markers
Analysis of the fluorescence capillary electrophoresis data was performed using the software GENEMAPPER 4.0.0, creation of the Kit, Panel and Marker parameters required for Analysis based on site name, fluorescent label, fragment size and repeat length, opening of GeneMapper Manager from Tools menu, selection of Microatellite in the Analysis Method of GeneMapper Manager, and selection of default values for other parameters. Analyzing the data with PopGene software to obtain primer allelic factors (Na), observed heterozygosity (Ho), expected heterozygosity (He), Nei's genetic distance (Nei) and aromatic index (I); the specific parameters are as follows: the number of groups is 17, number of posts is 1, number of loci is 8, and the remaining parameters use software defaults. Polymorphism Information Content (PIC) of SSR sites was calculated using PIC _ CALC software, using default parameters. Analyzing the genetic STRUCTUREs of 17 iris varieties by using STRUCTURE software, determining the population number (K value), setting the value range to be 1-10, setting the Length of burn Period to be 30000, setting the Markov Chain Monte Carlo (MCMC) to be 100000, using the default values of other parameters, operating the software, packaging the obtained Results file folder into a zip format, and submitting the zip format to online software Structure Harvester for analyzing the optimal K value, wherein K is 4 in the embodiment.
TABLE 6 Iris variety genetic polymorphism analysis based on 8 pairs of SSR markers
Figure BDA0003518157410000082
Figure BDA0003518157410000091
According to the polymorphism analysis of the primers in Table 6, the allele number of the 8 pairs of primers has a variation range of 3-9 and an average number of 5.375; the PIC values have a variation range of 0.5072-0.8226, which is greater than 0.5, and the average value is about 0.6228, which indicates that the 8 primers have high polymorphism.
b.17 genetic relationship analysis of iris varieties
And (3) carrying out genetic clustering analysis on 17 iris varieties according to the detection result of capillary electrophoresis of the obtained PCR product by a UPGMA method. First, a (0, 1) matrix of 17 iris varieties was created (as shown in table 7), specifically: the bands detected by capillary electrophoresis were arranged from small to large and labeled, and in this example, there were 39 band types, each variety corresponded to each band type, and the band-containing ones were labeled as 1, and the band-free ones were labeled as 0. Then, a clustering dendrogram of 17 iris varieties was prepared by NTSYS-pc software using the default parameters of the software (as shown in fig. 9). At relative distance 0.43, it can be seen from the breed clustering dendrogram: 17 iris varieties can be grouped into 4 groups. Wherein 9 varieties in total, namely Antyrmn gircs, Antunn Encorc, City lights, Donhle Young, early of Essex, Gnn Rayz, I'll be back, Peach Jam and Victora Fall, are gathered into one kind and are all two-season flowering iris varieties. 5 varieties of Change of P, Broad Way Star, John Naa, Sultan's Palace, Tide's sim in total were grouped into one; black Beard, Berkeley Gold, 2 varieties in total, were grouped into one category; fufted Cloud alone is a class. The last three species are all iris varieties which can not bloom in two seasons. The result of cluster analysis shows that the SSR primer group provided by the invention can effectively divide the materials according to whether the materials have the characters of flowering in two seasons, and the SSR primer group can be used for identifying whether the iris varieties flowering in two seasons. In addition, the clustering dendrogram shows that the genetic diversity among 17 iris varieties is high.
TABLE 717 (0, 1) matrix of Iris species
Figure BDA0003518157410000092
Figure BDA0003518157410000101
When the iris varieties need to be identified to have the two-season flowering characters, PCR amplification and capillary electrophoresis detection can be carried out through the primer sets according to the method, the detection results are compared with the genetic relationship clustering dendrograms in the graph 9, the class groups closest to or consistent with the genetic relationship clustering dendrograms are obtained, and then whether the iris varieties to be detected are the two-season flowering varieties can be judged in an auxiliary mode. The primer set provided by the invention can provide help for breeding the two-season flowering iris.
Example 2
And (3) performing association analysis on the SSR marker and the flowering characters of the iris tectorum in two seasons.
The results of the SSR primer amplification of 8 pairs in example 1 were transformed according to the manual of software operation on the TASSEL official website. Specifically, in each pair of primers, the band appearing the most frequently is labeled "a", the band appearing the second in the frequency row is labeled "C", the band appearing the third in the frequency row is labeled "G", the band appearing the fourth in the frequency row is labeled "T", the band appearing the fifth in the frequency row is labeled "+", and the other bands are labeled "-"; and obtaining a genotype matrix according to the rule for subsequent analysis.
Assigning the flowering characters of two seasons, wherein the flowering character of the two seasons is 1, and the flowering character of the non-flowering season is 0; adding the sizes of the flower stems and the plant heights of different varieties to obtain 3 characters in total, and obtaining a character (trail) file.
And (4) performing repeated sampling analysis on repeated operation results of STRUCTURE analysis by using CLUMPP to obtain the corresponding Q value of each variety under the optimal K value. The Kinship of the genotype file was analyzed using TASSEL 5.0 software. Regression analysis was performed on 3 traits and SSR marker genotype data using the General Linear Model (GLM) and mixed model (MLM) of the TASSEL 5.0 software. In the GLM model, the Q value is taken as a covariate; in the MLM model, regression analysis is carried out on 3 phenotypic characters by adopting a K value + a Q value, and association sites are determined.
GLM analysis results show (fig. 10) that under significant (P <0.05) conditions, 1 locus was detected to be associated with the two-season flowering trait, P2S 19. In the MLM analysis results (fig. 11), no site associated with the trait was detected.
The above experimental results show that, through the embodiment 1 and the embodiment 2, the SSR molecular marker provided by the invention can well distinguish different iris varieties and is used for identifying iris two-season flowering iris varieties.
Sequence listing
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<213> Bearded iris (Bearded iris)
<400> 18
cgagcttgtg tctctcttgc agtttgaaaa cctttactat atacattgtc agcttgctgt 60
ttcttttgta tgatgcatgc atgagttttc agtttattat tgttaacgtt tgtcctatgt 120
attaaaaaaa aaaatgtcca ttgtgttttc ttggtcatgt ttatgggatg atattggaaa 180
tgtcttgttc gttgtgtcac c 201
<210> 19
<211> 266
<212> DNA
<213> Bearded iris (Bearded iris)
<400> 19
agccaggcgg tttcaacata catatgtagg gcttcagaat gcctgcgcct atttacatta 60
catgcaccac cacccccttt ctgggtcaag cggcgaaccc atcgcggagc gagctccacg 120
ggttggaagg aacgctgagg caggcttggc cagcaacctc ggccatggcc tccttcctga 180
cgcctgcgtt gcacatgttt gcgagcgacc gcatgtgctt catgccgtac tgagacagcg 240
acccgcagct tgtctcgaaa gcacga 266
<210> 20
<211> 272
<212> DNA
<213> Bearded iris (Bearded iris)
<400> 20
tgacgtggac aactgcatca ggtgaacaac aagaaagtta caactcaccg atggacgatg 60
tcaacatcat cgtgagcggc gacgattcaa gtgaagacat caaagcaaat aataataata 120
ataataattc cgttgcagct gaaaaatcga ccaccactat ggataaaact ccgacgacac 180
catcttacgt gaagaagaga gtgaagaaga gaaaagaaat agatggcgta gagaagctat 240
cgcaacagat tgataggctt tgcgaggctg tt 272
<210> 21
<211> 263
<212> DNA
<213> Bearded iris (Bearded iris)
<400> 21
tctgcttcaa ggctccatgg tgtggttgca aattgtaaaa agctatagtg tggtcttcct 60
tgactcggat tcaactggac aagtctgcca gtaatcacaa tgcccatatt gtgaacttcc 120
aaagattgga ttcagctaac ttaagctccc aaatccagtg cttgaatgtt cttaactaat 180
ctaatggcac ctcgaaagaa aatggtgaat tgatacttgc taaatactac accgaattta 240
gtacgcggcc tgataaagat cct 263
<210> 22
<211> 207
<212> DNA
<213> beared iris (Bearded iris)
<400> 22
aggagccaac ccgaatgaag ggatcgctat cgttcgatgc agcgaaggag ccaatatgga 60
agatgagacc attgaaggtt tgaagaggct tgtagcttgc cttgaggaga gggaagctgc 120
gctcgagtcg cagtttcacg actactccaa tacgaaagaa caagaaaccg cattccaaaa 180
gcttcagatc atgtgccttg gcctcaa 207
<210> 23
<211> 251
<212> DNA
<213> Bearded iris (Bearded iris)
<400> 23
tagaaccacc ctcgtcacct tcgccgccgc cgctcctgca gcagcagcag cagcagccac 60
catctccgtc ggcgacaagc tccccgagtc caccctctcc tactttgacc ccgccggaga 120
gctccagacc ctgaccgtct cggacctcac caaggggaag gggaagaaag ccatcctgtt 180
cgccgttccg ggggccttca ccccgacctg ctcccagaag catctccctg gattcgttga 240
gaaggcctcg g 251
<210> 24
<211> 225
<212> DNA
<213> beared iris (Bearded iris)
<400> 24
tgctgttacg ccttcacctt taacaggtct accggagttt tctcatcttg gatggggtca 60
ctggtttacc ttgagggact tagaacttgc aaccaataga ttcacaaagg aaaatgttat 120
cggggagggc ggttatgggg ttgtttatcg tggatctctc atcaatggaa ctcctgtagc 180
tgtcaaaaag cttctgaaca atctaggcca ggctgagaag gaatt 225
<210> 25
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgtaaaacga cggccagt 18

Claims (9)

1. An iris SSR molecular marker group for screening iris varieties blooming in two seasons, which is characterized by comprising the following 8 SSR molecular marker groups, wherein the nucleotide sequence is as follows:
(1) P2S 19: the sequence is shown as SEQ ID NO. 17;
(2) P2S 45: the sequence is shown as SEQ ID NO. 18;
(3) P2S 48: the sequence is shown as SEQ ID NO. 19;
(4) P3S 12: the sequence is shown as SEQ ID NO. 20;
(5) P3S 17: the sequence is shown as SEQ ID NO. 21;
(6) P3S 28: the sequence is shown as SEQ ID NO. 22;
(7) P3S 30: the sequence is shown as SEQ ID NO. 23;
(8) P3S 41: the sequence is shown in SEQ ID NO. 24.
2. A primer group for screening iris tectorum with two-season blooming is characterized by comprising the following 8 pairs of primers, wherein the nucleotide sequences of a forward primer and a reverse primer are as follows:
(1) P2S 19: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;
(2) P2S 45: the sequence of the forward primer is shown as SEQ ID NO.3, and the sequence of the reverse primer is shown as SEQ ID NO. 4;
(3) P2S 48: the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6;
(4) P3S 12: the sequence of the forward primer is shown as SEQ ID NO.7, and the sequence of the reverse primer is shown as SEQ ID NO. 8;
(5) P3S 17: the sequence of the forward primer is shown as SEQ ID NO.9, and the sequence of the reverse primer is shown as SEQ ID NO. 10;
(6) P3S 28: the sequence of the forward primer is shown as SEQ ID NO.11, and the sequence of the reverse primer is shown as SEQ ID NO. 12;
(7) P3S 30: the sequence of the forward primer is shown as SEQ ID NO.13, and the sequence of the reverse primer is shown as SEQ ID NO. 14;
(8) P3S 41: the sequence of the forward primer is shown as SEQ ID NO.15, and the sequence of the reverse primer is shown as SEQ ID NO. 16.
3. An assay reagent or kit for screening a two-season flowering iris variety, comprising the primer set of claim 2.
4. The primer set according to claim 2, or the detection reagent or the kit according to claim 3, for use in screening iris varieties having two flowering season.
5. A screening method of iris varieties flowering in two seasons is characterized by comprising the following steps:
(1) extracting DNA of iris tectorum genome;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer set according to claim 2;
(3) detecting the PCR product of the step (2) by capillary electrophoresis;
(4) analyzing the detection result of the step (3), and constructing an iris variety clustering dendrogram together with the detection results of the two-season and single-season flowering irises serving as standard samples;
(5) and (4) clustering dendrograms of the iris varieties constructed according to the step (4) and the iris blossoming in two seasons as a standard sample, namely the iris varieties blossoming in two seasons.
6. The screening method according to claim 5, wherein the PCR amplification system in step (2) is: 20 μ L of 1.0 μ L each of the upstream and downstream primers, 10mM of the upstream and downstream primers, 5U/. mu.L of Taq polymerase10 μ L, 20 ng/. mu.L of DNA template 1.0 μ L, ddH2O7.0 μ L; the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 93 ℃ for 30 s; annealing at 58 ℃ for 30s, extending at 72 ℃ for 1min, and performing 32 cycles; extension at 72 ℃ for 10 min.
7. The screening method according to claim 5, wherein the capillary electrophoresis detection step in the step (3) is: adding 0.5 mu L of molecular weight internal standard and 8.5 mu L of deionized formamide into the PCR product obtained in the step (2) for denaturation under the denaturation condition of 95 ℃ for 5 min; cooling on ice for more than 10min, centrifuging for 10s, and performing capillary electrophoresis detection.
8. The screening method according to claim 5, wherein in the step (4), the specific steps of analyzing the detection result of the step (3) are as follows: and (4) according to the detection result of the capillary electrophoresis in the step (3), performing genetic clustering analysis on the iris varieties according to a UPGMA method: firstly, making a (0, 1) matrix of iris varieties, specifically: arranging and marking bands detected by capillary electrophoresis from small to large, wherein each variety corresponds to each band type, and the bands are marked as 1, and the bands which are not detected are marked as 0; then, a clustering dendrogram of the iris variety is made through NTSYS-pc software; the iris variety clustering dendrograms can be clustered into different groups, and are clustered with irises flowering in two seasons of a standard sample, namely the iris varieties flowering in two seasons.
9. Use of the screening method according to any one of claims 5 to 8 for screening a two-season flowering iris variety.
CN202210171355.1A 2022-02-24 2022-02-24 Iris SSR molecular marker group for screening iris varieties blooming in two seasons and application thereof Pending CN114438250A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130033897A (en) * 2011-09-27 2013-04-04 대한민국(환경부 국립생물자원관장) Chloroplast dna molecular marker and method of identifying the geographical origin of korean iris dichotoma by using thereof
CN111642351A (en) * 2020-06-12 2020-09-11 江苏省中国科学院植物研究所 Method for promoting early flowering of German iris and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130033897A (en) * 2011-09-27 2013-04-04 대한민국(환경부 국립생물자원관장) Chloroplast dna molecular marker and method of identifying the geographical origin of korean iris dichotoma by using thereof
CN111642351A (en) * 2020-06-12 2020-09-11 江苏省中国科学院植物研究所 Method for promoting early flowering of German iris and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHUNXUE TANG等: "EST and EST-SSR marker resources for Iris", BMC PLANT BIOLOGY, vol. 9, no. 72, pages 1 - 11 *

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Application publication date: 20220506