CN109280694B - SSR (simple sequence repeat) primers and kit for curcuma alismatifolia polymorphisms developed based on full-length transcriptome sequencing - Google Patents
SSR (simple sequence repeat) primers and kit for curcuma alismatifolia polymorphisms developed based on full-length transcriptome sequencing Download PDFInfo
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Abstract
The invention discloses a curcuma alismatifolia polymorphism SSR primer and a kit developed based on full-length transcription group sequencing. The primers comprise 20 pairs of primers, and the kit for identifying the genetic relationship of the curcuma alismatifolia comprises the primers as shown in SEQ ID NO.1-SEQ ID NO. 40. The 20 pairs of primers provided by the invention can be applied to more curcuma alismatifolia varieties and intra-variety groups for variety identification, germplasm genetic diversity and genetic relationship analysis, and can also be applied to molecular marker-assisted breeding of the curcuma alismatifolia.
Description
Technical Field
The invention relates to an SSR primer, in particular to a curcuma alismatifolia polymorphism SSR primer developed based on full-length transcription group sequencing and a kit, and belongs to the technical field of biology.
Background
The curcuma alismatifolia is originally produced in Thailand, has the characteristics of unique flower type, gorgeous flower color, long flower period and long vase service life, is called as the curcuma alismatifolia because the sterile bract is similar to the lotus and belongs to the family Zingiberaceae, and has the beauty of both the lotus and the curcuma aromatica, also called as tropical tulip (summer tulip). In recent years, the introduction of Hemerocallis zingiberensis has been gradually introduced in China, and the Hemerocallis zingiberensis has been gradually applied to cut flowers, pot flowers and garden flower mirrors. As the curcuma alismatifolia is a novel flower and has high ornamental value, the curcuma alismatifolia is popular with people, more and more foreign varieties are introduced, and a plurality of germplasm resources are domesticated in China to increasingly adapt to the domestic climate environment. The problems of synonyms of the same species, homonymous foreign matters and unclear genetic relationship among the species exist in various species resources, influence is brought to the collection, storage and utilization of the species, and the development of the subsequent new species breeding work of the curcuma alismatifolia is not facilitated. Therefore, the development of polymorphic molecular markers for deeply knowing the inter-species relationship is necessary for the subsequent development of the floral resource innovation of the curcuma alismatifolia.
With the development of molecular biology, molecular marker technology is widely applied to genetic diversity of germplasm resources and auxiliary breeding work. Dominant markers such as ISSR, RAPD and AFLP have the advantages of low cost and no need of knowing genome information, but have strong randomness and poor stability. Compared with other molecular markers, the SSR marker has the advantages of codominance and good repeatability, and is the first choice for genetic diversity analysis, genetic map construction and the like at present.
At present, the SSR application of the curcuma alismatifolia is very little, no SSR marker specially developed for the species exists, only one SSR marker is used for detecting the genetic difference between the original variety and the gamma ray mutant variety of the curcuma alismatifolia, but the used primer refers to the medicinal plant curcuma longa (turmeric) ((turmeric))Curcuma longa) In (1). Because of the species specificity of SSR markers, the markers developed for this species certainly have better stability and higher polymorphism.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a curcuma alismatifolia polymorphism SSR primer and a kit developed based on full-length transcription group sequencing.
The SSR primers for the polymorphisms of the curcuma alismatifolia based on the sequencing development of the full-length transcription group comprise 20 pairs of primers which are respectively as follows:
P2 F: AGCAATGTTGACGAAATGGAAA (SEQ ID NO.1)，
P2 R: AAGCAATCTCTTAGGAAATCAG (SEQ ID NO.2)；
P5F: CTGGTCACCCACCTAAATCCTT (SEQ ID NO.3)，
P5R: ACGCCGTGGAGAAGAGGCTATT (SEQ ID NO.4)；
P6F: GGAGGGACTACGGGAAAGGGAA (SEQ ID NO.5)，
P6R: GCAGGACAGTTACGATTGATGA (SEQ ID NO.6)；
P7F: GAAAGCGACAAGATGCTGAGGG (SEQ ID NO.7)，
P7R: ACCTTCCTCTTCTCCACCTGAT (SEQ ID NO.8)；
P9F: GAGCCAGAAGTCACAATCAGCG (SEQ ID NO.9)，
P9R: TTGTCCTGGCTTCCTCTCTGGG (SEQ ID NO.10)；
P11F: CTGAGTGTAGGACTTGTCGGTG (SEQ ID NO.11)，
P11R: TAGCCATTGACGAACAAAAAGC (SEQ ID NO.12)；
P12F: GAAGAGCAAAAGACAGGCAGAT (SEQ ID NO.13)，
P12R: CACAGACAAAGAATGAAAACTA (SEQ ID NO.14)；
P13F: AGGGCGTCTTCAAGGTCTGGAT (SEQ ID NO.15)，
P13R: ACAACAACGGCAGCAGCAGTAT (SEQ ID NO.16)；
P14F: GCCTTTGGTATCTCTGTTCTAA (SEQ ID NO.17)，
P14R: TAATGCGGTAGGAGGGGAACAA (SEQ ID NO.18)；
P16F: TCTATGTTCTTCGCTACCTTCG (SEQ ID NO.19)，
P16R: ACCTTTATCCTCTTGGTCTCGC (SEQ ID NO.20)；
P17F: CAAAACGAGGAAAGAAACCAAA (SEQ ID NO.21)，
P17R: TATGCTTGTTGGAGGCTATCAC (SEQ ID NO.22)；
P18F: AGGGCAAAACTCAAAAGGGTAG (SEQ ID NO.23)，
P18R: CTCCTTGTAGTCCTTGTCCTGC (SEQ ID NO.24)；
P19F: AAACACAGACTGCCCTCCTTGA (SEQ ID NO.25)，
P19R: TTCCCATCAGAAGATAACAACG (SEQ ID NO.26)；
P21F: CGTTTCTCCAGACAATGACCGC (SEQ ID NO.27)，
P21R: CGTCAACTAACTCTACGCTAAC (SEQ ID NO.28)；
P23F: CCCTGGCGTAACCTCCATTTCC (SEQ ID NO.29)，
P23R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.30)；
P25F: CCTGGCGTAACCTCCATTTCCG (SEQ ID NO.31)，
P25R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.32)；
P26F: ACTCGCCAATCTCTTACACAGC (SEQ ID NO.33)，
P26R: TCGGGGTAATGGTGACGGTAAG (SEQ ID NO.34)；
P27F: TTTCACCAGGAGTAAGAGGGAT (SEQ ID NO.35)，
P27R: AAACCTCAGCCTTTTCTCTTCA (SEQ ID NO.36)；
P31F: CAGCCGTTTGTTATGCTACTTA (SEQ ID NO.37)，
P31R: TTGAAACTTTACCAAGAACAGG (SEQ ID NO.38)；
P33F: GTCAAACTTCTCAAACCACACG (SEQ ID NO.39)，
P33R: ATCATACAATACCCTCAACAAT (SEQ ID NO.40)。
a kit for identifying the genetic relationship of the curcuma alismatifolia comprises the primer.
The invention has the beneficial effects that:
the 20 pairs of SSR primers are obtained by screening 10 curcuma alismatifolia varieties and have polymorphism in the 10 curcuma alismatifolia varieties, so the primers can be used for variety identification of the curcuma alismatifolia, SSR markers are new markers which exist stably, and the 20 primers provided by the invention can be directly and correspondingly applied to more curcuma alismatifolia varieties for variety identification, genetic resource genetic relationship and genetic diversity analysis, thereby laying a good foundation for molecular marker-assisted breeding of the curcuma alismatifolia.
Drawings
FIG. 1 is a partial primer electropherogram;
FIG. 2 is a cluster map of 10 varieties based on amplified band polymorphism.
Detailed Description
Sequencing of 'Qingmai powder' full-length transcription group of Hemerocallis zingiberensis
The sequencing material is a mixed sample which is planted in flower research center of agricultural institute of Zhejiang province and has good growth state and obtained by respectively extracting RNA from leaves, stems, green bracts, pink bracts and small flower bracts of a strain of curcuma alismatifolia. The sequencing experiment was performed by Tianjin biochip technology, Inc., and 64471 redundancy-removed gene fragments were obtained after sequencing, with a total sequence length of 132833 kb.
Second, screening of SSR primers
The SSR loci in the gene sequence are searched by using MISA software, and the parameters are set as follows: the minimum number of the repeated times of 1-6 nucleotides is respectively 10, 6, 5 and 5. The maximum number of spacer bases of the composite SSR is 100 bp. 50 pairs of primers were designed using Primer 5.0, based on the following principle: 1. the primers are positioned at two ends of the microsatellite sequence, and the length of the primers is 18 bp-25 bp; 2. the length of the expected PCR amplification fragment is 100 bp-250 bp; 3. the annealing temperature is 52-62 ℃; 4. the GC content is 40% -68%; 5. avoid the occurrence of dimer, hairpin structure and mismatching in the primer.
Thirdly, extraction of variety DNA
Genomic DNA of 10 curcuma alismatifolia varieties (Table 1) was extracted by CTAB method.
TABLE 110 varieties for SSR primer polymorphism screening
Fourthly, primary screening of agarose gel and further screening of polyacrylamide gel
50 pairs of primers are used for carrying out PCR amplification on 10 curcuma alismatifolia varieties, amplification products are firstly preliminarily screened by agarose gel electrophoresis, and target product bands are further detected by polyacrylamide gel electrophoresis. The polyacrylamide gel electrophoresis experimental procedure is as follows: (1) glue making
And (4) cleaning the glass plate, vertically placing the glass plate, and airing the glass plate for later use. The gel interlayer device is assembled by the spacing bars, the attachment plates and the back plate, horizontally placed and clamped and fixed at two sides by the clamps. Measuring 50 mL of 6% modified polyacrylamide gel solution, adding 100 mL of 20% APS and 50 mL of TEMED, slightly mixing uniformly, filling the mixture into an interlayer by using a glue filling bottle, inserting the flush end of a shark tooth comb into glue solution between two plates by 5-6 mm, and standing at room temperature for 1-1.5 h to solidify the gel.
(2) Pre-electrophoresis
After gel polymerization, the clip was removed, the gel apparatus was mounted on an electrophoresis tank, and about 1000 mL of 1 XTBE buffer was added thereto so that the liquid level did not exceed the gel level by a predetermined height. At the same time, about 400 mL of 1 XTBE buffer was added to the electrophoresis chamber. And pulling out the shark tooth comb, opening the electrophoresis apparatus, and pre-electrophoresing for 20-30 min at a constant power of 85W.
(3) Denaturation of the material
And adding the PCR amplification product into 5-10 mL of loading buffer solution, centrifuging and mixing uniformly. After denaturation at 95 ℃ for 5 min, the gel was taken out and rapidly placed on ice. Cooling for 10 min for use.
(4) Electrophoresis
And after the pre-electrophoresis is finished, suspending the electrophoresis apparatus, blowing the glue surface by using a suction pipe, removing bubbles and impurities, avoiding blocking a sample application hole, applying a proper amount of denatured sample (4.5-6.5 mL) to the sample application hole, and starting electrophoresis at a constant power of 85W. After 1.5 h, the electrophoresis was terminated.
(5) Silver staining
And (3) putting the glass plate with the adhesive upwards into 2000 mL of stationary liquid, placing the glass plate on a shaking table with the rotating speed of 50-60 rpm, slightly shaking for about 20 min, taking out the gel glass plate from the stationary liquid, and rinsing for 3 min by using deionized water. And taking out the gel glass plate, placing the gel glass plate in 2000 mL of silver nitrate solution, placing the gel glass plate on a shaking table with the rotating speed of 50-60 rpm, and shaking for 30-40 min to ensure that the gel glass plate is fully dyed. And taking out the gel glass plate, putting the gel glass plate into 2000 mL of deionized water, and rinsing for 30-60 s. And taking out the gel glass plate, putting the gel glass plate into 2000 mL of color development liquid, and developing for 4-7 min on a shaking table with the rotating speed of 60 rpm until the DNA bands are clear. The gel glass plate is taken out and is placed in 2000 mL distilled water for rinsing for 3 min, and the gel glass plate is taken out and is placed in a dry place for airing. The offset plate is scanned on a scanner. The partial primer electrophoresis is shown in FIG. 1.
First, primer polymorphism analysis
The polyacrylamide gel result shows that 20 pairs of primers have good polymorphism and clear amplified bands. The results of the 20 primers on amplification polymorphisms were counted (Table 2), and 10 varieties of clusters were plotted based on the amplification polymorphisms (FIG. 2). The topological structure of the clustering tree can show the relationship of 10 varieties, C7 is the only variety of a green flower bud in green, and the clustering graph shows that the variety has the farthest relationship with other varieties. The sunset of the c2 variety and the Dutch safflower of the c3 variety which are closest to each other are the deep rosy, the white snow (c 8) and the screened variety of the violaxalin (c 10) which is naturally mutated in the white snow field also show the closest genetic relationship with each other, and the clustering result shows that the developed 20 pairs of primers not only have polymorphism, but also can reflect the genetic relationship between the varieties.
TABLE 2.20 SSR primer information for Zingiber officinale Roscoe
The polymorphic primer provided by the invention can be used for genetic diversity analysis, genetic map construction, molecular marker-assisted breeding and the like among the curcuma alismatifolia varieties.
Sequence listing
<110> research institute of Xiaoshan cotton and hemp in Zhejiang province
<120> SSR primers and kit for lotus officinalis polymorphism developed based on full-length transcriptome sequencing
<141> 2018-10-23
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agcaatgttg acgaaatgga aa 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aagcaatctc ttaggaaatc ag 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctggtcaccc acctaaatcc tt 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acgccgtgga gaagaggcta tt 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggagggacta cgggaaaggg aa 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcaggacagt tacgattgat ga 22
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaaagcgaca agatgctgag gg 22
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
accttcctct tctccacctg at 22
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gagccagaag tcacaatcag cg 22
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ttgtcctggc ttcctctctg gg 22
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctgagtgtag gacttgtcgg tg 22
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tagccattga cgaacaaaaa gc 22
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaagagcaaa agacaggcag at 22
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cacagacaaa gaatgaaaac ta 22
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agggcgtctt caaggtctgg at 22
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acaacaacgg cagcagcagt at 22
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gcctttggta tctctgttct aa 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
taatgcggta ggaggggaac aa 22
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tctatgttct tcgctacctt cg 22
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acctttatcc tcttggtctc gc 22
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
caaaacgagg aaagaaacca aa 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tatgcttgtt ggaggctatc ac 22
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agggcaaaac tcaaaagggt ag 22
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctccttgtag tccttgtcct gc 22
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
aaacacagac tgccctcctt ga 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ttcccatcag aagataacaa cg 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cgtttctcca gacaatgacc gc 22
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cgtcaactaa ctctacgcta ac 22
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ccctggcgta acctccattt cc 22
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gatggcgtgg agtatggcgt ct 22
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cctggcgtaa cctccatttc cg 22
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gatggcgtgg agtatggcgt ct 22
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
actcgccaat ctcttacaca gc 22
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tcggggtaat ggtgacggta ag 22
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tttcaccagg agtaagaggg at 22
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aaacctcagc cttttctctt ca 22
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cagccgtttg ttatgctact ta 22
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ttgaaacttt accaagaaca gg 22
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtcaaacttc tcaaaccaca cg 22
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
atcatacaat accctcaaca at 22
Claims (2)
1. The SSR primers for the polymorphisms of the curcuma alismatifolia based on the sequencing development of the full-length transcriptome are characterized by comprising 20 pairs of primers which are respectively as follows:
P2 F: AGCAATGTTGACGAAATGGAAA (SEQ ID NO.1)，
P2 R: AAGCAATCTCTTAGGAAATCAG (SEQ ID NO.2)；
P5F: CTGGTCACCCACCTAAATCCTT (SEQ ID NO.3)，
P5R: ACGCCGTGGAGAAGAGGCTATT (SEQ ID NO.4)；
P6F: GGAGGGACTACGGGAAAGGGAA (SEQ ID NO.5)，
P6R: GCAGGACAGTTACGATTGATGA (SEQ ID NO.6)；
P7F: GAAAGCGACAAGATGCTGAGGG (SEQ ID NO.7)，
P7R: ACCTTCCTCTTCTCCACCTGAT (SEQ ID NO.8)；
P9F: GAGCCAGAAGTCACAATCAGCG (SEQ ID NO.9)，
P9R: TTGTCCTGGCTTCCTCTCTGGG (SEQ ID NO.10)；
P11F: CTGAGTGTAGGACTTGTCGGTG (SEQ ID NO.11)，
P11R: TAGCCATTGACGAACAAAAAGC (SEQ ID NO.12)；
P12F: GAAGAGCAAAAGACAGGCAGAT (SEQ ID NO.13)，
P12R: CACAGACAAAGAATGAAAACTA (SEQ ID NO.14)；
P13F: AGGGCGTCTTCAAGGTCTGGAT (SEQ ID NO.15)，
P13R: ACAACAACGGCAGCAGCAGTAT (SEQ ID NO.16)；
P14F: GCCTTTGGTATCTCTGTTCTAA (SEQ ID NO.17)，
P14R: TAATGCGGTAGGAGGGGAACAA (SEQ ID NO.18)；
P16F: TCTATGTTCTTCGCTACCTTCG (SEQ ID NO.19)，
P16R: ACCTTTATCCTCTTGGTCTCGC (SEQ ID NO.20)；
P17F: CAAAACGAGGAAAGAAACCAAA (SEQ ID NO.21)，
P17R: TATGCTTGTTGGAGGCTATCAC (SEQ ID NO.22)；
P18F: AGGGCAAAACTCAAAAGGGTAG (SEQ ID NO.23)，
P18R: CTCCTTGTAGTCCTTGTCCTGC (SEQ ID NO.24)；
P19F: AAACACAGACTGCCCTCCTTGA (SEQ ID NO.25)，
P19R: TTCCCATCAGAAGATAACAACG (SEQ ID NO.26)；
P21F: CGTTTCTCCAGACAATGACCGC (SEQ ID NO.27)，
P21R: CGTCAACTAACTCTACGCTAAC (SEQ ID NO.28)；
P23F: CCCTGGCGTAACCTCCATTTCC (SEQ ID NO.29)，
P23R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.30)；
P25F: CCTGGCGTAACCTCCATTTCCG (SEQ ID NO.31)，
P25R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.32)；
P26F: ACTCGCCAATCTCTTACACAGC (SEQ ID NO.33)，
P26R: TCGGGGTAATGGTGACGGTAAG (SEQ ID NO.34)；
P27F: TTTCACCAGGAGTAAGAGGGAT (SEQ ID NO.35)，
P27R: AAACCTCAGCCTTTTCTCTTCA (SEQ ID NO.36)；
P31F: CAGCCGTTTGTTATGCTACTTA (SEQ ID NO.37)，
P31R: TTGAAACTTTACCAAGAACAGG (SEQ ID NO.38)；
P33F: GTCAAACTTCTCAAACCACACG (SEQ ID NO.39)，
P33R: ATCATACAATACCCTCAACAAT (SEQ ID NO.40)。
2. a kit for identifying the relatedness of curcuma alismatifolia, comprising the primer of claim 1.
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