CN107974510B - EST-SSR (expressed sequence tag-simple sequence repeat) marker primer of paphiopedilum armeniacum, development method and application thereof - Google Patents
EST-SSR (expressed sequence tag-simple sequence repeat) marker primer of paphiopedilum armeniacum, development method and application thereof Download PDFInfo
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Abstract
The invention discloses an EST-SSR labeled primer group developed based on paphiopedilum armeniacum transcriptome sequences, which comprises 13 pairs of primers, wherein the nucleotide sequences of the primers are shown as SEQ ID NO. 1-SEQ ID NO.26 of a sequence table. The invention also discloses a reagent containing the EST-SSR marker primer of paphiopedilum armeniacum. The invention further discloses a method for developing an EST-SSR labeled primer of paphiopedilum armeniacum. The EST-SSR marker primer of paphiopedilum armeniacum provided by the invention has many advantages, such as abundant polymorphism, good repeatability, stable amplification, convenience for statistics and the like, and can be used for germplasm resource genetic diversity analysis, molecular marker assisted breeding and paphiopedilum related molecular research of paphiopedilum armeniacum and other paphiopedilum species.
Description
Technical Field
The invention relates to an EST-SSR labeled primer developed based on Paphiopedilum armeniacum (paphiopediococcum armeniacum) transcriptome sequence, a development method and application thereof, and belongs to the technical field of biology.
Background
Paphiopedilum armeniacum belongs to Paphiopedilum (Orchidaceae) and is a unique original breed in China, is called orchid panda, and is rapidly reduced in population quantity and endangered to extinction due to narrow distribution area, loss of habitat and predatory digging of resources, and wild resources of Paphiopedilum armeniacum are difficult to find in an original place. All paphiopedilum plants are listed in the appendix book of International trade convention on endangered wild animal and plant species and are prohibited from trading. Therefore, the research on the genetic diversity of paphiopedilum armeniacum becomes very important, and the nursing work of paphiopedilum is more relevantly carried out.
Simple Repeat (SSR) Markers, also known as Microsatellite Markers, developed based on genome or transcriptome data have the characteristics of high polymorphism, good repeatability, strong specificity, convenient detection, codominance, uniform distribution of Markers covering the whole genome and the like, and become one of the important genetic Markers in genetic diversity analysis (Phuekvilai, pratta & Pongkam, Pradit & Peyachknagul, Surin, "Development of microscopic Markers for variant Orchid," Katsart Journal-Natural Science, 2009, 43 (3): 497-506; Kalia R K, Rai M K, Kalia S and the like, "microscopic Markers: over viral expression in" Euphyta, 309 (3): 2011334). Compared with the genome SSR marker, the EST-SSR is derived from an expression genome region, can provide an 'absolute' marker for a functional gene, and has low development cost and higher inter-species universality (Zhu Sha Dong, Jia Zhang, the 'development and application of wheat SSR marker'; inheritance, 2003, (03): 355-. The use of transcriptome sequencing (RNA-Seq) to develop SSR Markers with genetic information to analyze genetic diversity in organisms is also a simple and efficient research procedure for non-model organisms or organisms without whole genome sequencing, and has been reported in orchids many times (Tsai CC, Shih HC, Wang HV, Lin YS, Chang CH et al, "RNA-Seq SSRs of Molecular organization and sequencing for Molecular Markers across genome Phalaenopsis (Orchidaceae)," plo, 2015, 10 (11): e 0141761). Among Paphiopedilum, great songjun et al developed 10 pairs of genomic SSR markers of Paphiopedilum in 2010 (Li, l., Zeng, s., Zheng, f., Chen, z., Wu, k., Zhang, j., and Duan, j., "Isolation and Characterization of 10Polymorphic Microsatellite Loci in pathopedecium con color (Batem.) pfitter (orichidaceae) and Cross-spectra Amplification," Hortscience, 2010, 45 (8): 6-1287), and this laboratory succeeded in developing 13 pairs of EST-SSR primers with polymorphisms using the transcriptome data of Paphiopedilum armeniacum.
The EST-SSR primers developed by using the paphiopedilum armeniacum transcriptome sequence information play an important promoting role in germplasm resource genetic diversity, genetic relationship analysis, important character gene positioning, molecular marker assisted breeding, paphiopedilum related molecular research and the like of paphiopedilum and other paphiopedilum species.
Disclosure of Invention
The invention discloses an EST-SSR labeled primer group developed based on paphiopedilum armeniacum transcriptome sequences, which comprises 13 pairs of primers, wherein the nucleotide sequences of the primers are shown as SEQ ID NO. 1-SEQ ID NO.26 of a sequence table. The invention also discloses a reagent containing the EST-SSR marker primer of paphiopedilum armeniacum. The invention further discloses a method for developing EST-SSR labeled primers of paphiopedilum armeniacum, which comprises the following steps: (a) designing an EST-SSR primer based on unigene sequence data obtained by sequencing and splicing paphiopedilum armeniacum organ transcriptome; (b) extracting genome DNA of a paphiopedilum armeniacum plant sample; (c) carrying out PCR by using the EST-SSR primer obtained in the step (a) and the DNA obtained in the step (b) as a template; (d) and (c) carrying out electrophoresis on the PCR product obtained in the step (c), and analyzing the electrophoresis result, thereby screening the EST-SSR primer of paphiopedilum armeniacum with expected characteristics. The EST-SSR marker primer of paphiopedilum armeniacum provided by the invention has many advantages, such as abundant polymorphism, good repeatability, stable amplification, convenience in statistics and the like, and can be used for germplasm resource genetic diversity analysis, molecular marker assisted breeding and paphiopedilum related molecular research of paphiopedilum armeniacum and other paphiopedilum species.
In a first aspect of the invention, EST-SSR primer pairs developed based on paphiopedilum armeniacum transcriptome sequences are provided as shown in Table 1 below:
TABLE 1 paphiopedilum armeniacum EST-SSR polymorphic primer information
The sequence numbers of the primers are respectively SEQ ID NO. 1-SEQ ID NO. 26.
In one embodiment, any one of the paphiopedilum armeniacum EST-SSR primer pairs described herein may be detectably labeled. Labeling methods are well known in the art. The labels include those commonly used in the art, for example, the labels include, but are not limited to, fluorescent labels, isotopic labels, and the like. Preferably, the label is a fluorescent label. In further embodiments, the forward primer of the primer pair is labeled. In further embodiments, the reverse primer of the primer pair is labeled.
In one embodiment, the primers of the paphiopedilum armeniacum primer pair of the present invention may be modified by insertion, deletion, substitution of one or more nucleotides or modification means known to those skilled in the art, so as to improve the specificity of the primers, be used for molecular biological studies of other plants of the same genus or species, or obtain other desired properties.
In a second aspect, the invention provides a set of EST-SSR primers developed based on paphiopedilum armeniacum transcriptome sequences, wherein the primer set comprises any combination of the primer pairs disclosed in the invention.
In a third aspect, the invention provides a DNA fragment or gene product, namely an EST-SSR marker, isolated from paphiopedilum armeniacum genome by using the primer pair or primer group of the invention. The EST-SSR markers can be used for genetic diversity analysis of germplasm resources of paphiopedilum armeniacum and other paphiopedilum, molecular marker-assisted breeding and paphiopedilum related molecular research. In particular, because the EST-SSR markers are derived from a transcription region of DNA and are related to functional genes, the EST-SSR markers can be used for related researches of molecular marker assisted breeding, such as genetic linkage map construction, important trait related marker association analysis, new gene separation and identification and the like.
The invention also provides an SSR fingerprint of paphiopedilum armeniacum, other paphiopedilum plants and even orchidaceae plants which are constructed by utilizing the EST-SSR primer.
In a fourth aspect of the invention, reagents for isolating an EST-SSR marker of paphiopedilum armeniacum comprise any one pair of primers or a group of primers described in the invention.
In one embodiment, the reagent of the invention comprises a set of primers as described above, wherein each pair of primers in the set of primers is packaged separately.
A fifth aspect of the invention provides a kit comprising a reagent of the invention as described above.
A sixth aspect of the present invention provides a method for developing an EST-SSR primer based on paphiopedilum armeniacum transcriptome sequence, the method comprising:
(a) designing an EST-SSR primer based on unigene sequence data obtained by sequencing and splicing paphiopedilum armeniacum organ transcriptome;
(b) extracting genome DNA of a paphiopedilum armeniacum plant sample;
(c) carrying out PCR by using the EST-SSR primer obtained in the step (a) and the DNA obtained in the step (b) as a template;
(d) and (c) carrying out electrophoresis on the PCR product obtained in the step (c), and analyzing the electrophoresis result, thereby screening the EST-SSR primer of paphiopedilum armeniacum.
In one embodiment, the step (a) is performed as follows: sequencing and splicing paphiopedilum armeniacum organ transcriptome to obtain unigene sequence data; the method uses MIcroAtellite identification tool (MISA) software to search possible SSR sites in each unigene (Thiel, T., Michalek, W., Varshney, R.K. and Graner, A., "expanding EST databases for the estimation and characterization of gene-derived SSR-markers in bars (Hordeum vulgare L.)," Theoretical and Applied Genetics, 2003, 106: 411 and 422), and the search standard of SSR sites is as follows: the number of repeat sequence motifs of two, three, four, five and six nucleotides is not less than 7, 5 and 5 times, respectively; and designing SSR primers based on sequences on two wings of the SSR locus by using Primer3 software.
In one embodiment, the screening strategy for primer design is: the length is between 17 and 24bp (most suitable for 20bp), the melting temperature (Tm) is between 55 and 62 ℃, the size of a PCR amplification product is between 100 and 350bp, the GC content is between 40 and 60 percent (most suitable for 50 percent), and the others are set by default. And taking the primer combination closest to the design requirement as the primer of the SSR locus.
In one embodiment, the step (b) comprises batch extraction of the sample DNA by a modified CTAB method, which is performed as follows:
grinding leaves of paphiopedilum armeniacum into powder by using liquid nitrogen, and adding CTAB (containing beta-mercaptoethanol v/v 1%) preheated at 65 ℃; (ii) a
② water bath is carried out for 1h at 65 ℃;
taking out and cooling to room temperature (or putting into a refrigerator), adding precooled chloroform: isoamyl alcohol (24: 1), and shaking gently for 5 min;
fourthly, centrifuging, taking the supernatant, repeating twice, adding 2 times of volume of precooled absolute ethyl alcohol, and slightly reversing up and down to mix evenly;
fifthly, placing for 30min at the temperature of minus 20 ℃, separating out DNA clusters, and discarding the liquid;
sixthly, adding 70 percent ethanol to wash and precipitate, reversing the upper part and the lower part for 6 to 8 times, centrifuging, then removing the supernatant, and then washing with 90 percent ethanol;
seventhly, drying the ventilated part for 2 hours until the DNA is dried and transparent and has no alcohol taste;
adding sterile water to redissolve.
The advantages of extracting DNA by the improved CTAB method are as follows: directly abandoning liquid after DNA agglomeration, skillfully avoiding various impurity precipitates caused by a centrifugal method, and greatly improving the purity and concentration quality of the finally obtained DNA.
In one embodiment, the step (c) is performed by touchdown pcr (touch Down pcr), the reaction system of which is shown in table 2 below:
TABLE 2 PCR reaction System
The touchdown PCR method has the advantages of high amplification efficiency, less non-specific amplification and strong universality.
In one embodiment, the PCR product is electrophoresed by a fluorescence capillary electrophoresis method to identify polymorphic bands on an electropherogram, so that the paphiopedilum armeniacum EST-SSR primer with clear bands, high polymorphism and good specificity is screened out.
In a further embodiment, the method of the invention can be used to develop EST-SSR primers for other plants of paphiopedilum, and even for other plants of Orchidaceae or plants other than Orchidaceae.
The invention also provides application of the EST-SSR primer in genetic diversity analysis among different communities, different varieties, paphiopedilum and orchids of paphiopedilum.
In the present invention, "a pair of primers" and "a primer pair" are used interchangeably and refer to a pair of primers consisting of a forward primer and a reverse primer; "set of primers" is used interchangeably with "set of primers" and refers to a set of primers comprising two or more pairs of primers.
The technical scheme provided by the embodiment of the invention has the beneficial technical effects that: the EST-SSR primers of paphiopedilum armeniacum are developed for the first time, 13 pairs of the EST-SSR primers of paphiopedilum armeniacum with rich polymorphism, good repeatability, high specificity and stable amplification are provided, and a foundation is provided for genetic diversity analysis of paphiopedilum armeniacum germplasm resources, molecular marker-assisted breeding and paphiopedilum related molecular research; because the EST-SSR markers are derived from a transcription region of DNA, are related to functional genes and have good inter-species universality, the EST-SSR markers can be used for molecular biological research of congeneric plants, and particularly can be used for related research of molecular marker-assisted breeding, such as genetic linkage map construction, important character related marker association analysis, separation and identification of new genes and the like; moreover, the method provided by the invention is simple, easy and efficient, has low production cost, and can be used for developing EST-SSR primers of other paphiopedilum plants.
The foregoing is illustrative only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will be more readily understood by reference to the following detailed description.
Brief Description of Drawings
Further aspects, features of the present invention will be more readily understood by reference to the following drawings. It will be appreciated by persons skilled in the art that these drawings illustrate only some embodiments according to the invention, and should not be taken as limiting the scope of the invention.
FIG. 1 shows DNA agarose gel electrophoresis of a portion of 170 different paphiopedilum armeniacum material samples from 5 wild-populated paphiopedilum armeniacum populations.
FIGS. 2-131 show fluorescence electrophoresis patterns of the results of the polymorphic amplification of 13 pairs of primers listed in Table 1 on a part of paphiopedilum armeniacum samples, in which:
FIGS. 2-11 show the fluorescence electrophoresis patterns of the results of the polymorphic amplification of primer XH039 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 12-21 show fluorescence electrophoresis patterns of the results of polymorphic amplification of primer XH047 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 22-31 show the fluorescence electrophoresis patterns of the results of the polymorphic amplification of primer XH097 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 32-41 show fluorescence electrophoresis graphs of the results of polymorphic amplification of primer XH115 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 42-51 show fluorescence electrophoresis plots of the results of polymorphic amplification of primer XH116 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 52-61 show the fluorescence electrophoresis patterns of the results of the polymorphic amplification of primer XH119 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 62-71 show fluorescence electrophoresis plots of the results of polymorphic amplification of primer XH137 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 72-81 show fluorescence electrophoresis graphs of the results of polymorphic amplification of primer XH146 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 82-91 show the fluorescence electrophoresis patterns of the results of the polymorphic amplification of primer XH176 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 92-101 show fluorescence electrophoresis plots of the results of polymorphic amplification of primer XH179 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 102-111 show fluorescence electrophoresis graphs of the results of polymorphic amplification of primer XH206 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
FIGS. 112-121 show fluorescence electrophoresis graphs of the results of polymorphic amplification of primer XH224 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples; and
FIGS. 122-131 show fluorescence electrophoresis graphs of the results of polymorphic amplification of primer XH252 on YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14, LJ-7, LJ-27 samples;
fig. 132 shows a UPGMA clustering plot after cluster analysis of 170 different paphiopedilum armeniacum material from 5 wild populations of paphiopedilum armeniacum using the 13 primer pairs listed in table 1.
Detailed Description
In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
Example 1 development of EST-SSR primers for paphiopedilum armeniacum
EST-SSR primer design
Experimental materials: the various reagents or media components used in all examples of the invention are commercially available.
1) Construction of transcriptome libraries: extracting total RNA of paphiopedilum armeniacum organs, separating mRNA, carrying out reverse transcription synthesis and purification of cDNA, repairing the tail end, adding adenosine to connect with a sequencing joint, recovering a fragment with the size of 200-700 bp by agarose gel electrophoresis, and carrying out PCR amplification on the recovered fragment to construct a transcriptome library.
2) Acquisition of transcriptome data: sequencing the transcriptome library obtained in the step 1) to obtain transcriptome sequencing data, and splicing the sequencing data to obtain unigenes sequence data.
3) SSR locus searching: and searching SSR sites possibly existing in each unigene by using MIcrosAtellite identification tool (MISA) software. The search criteria for SSR loci are: the number of times of the repeating sequence motifs of two, three, four, five and six nucleotides is not less than 7, 5 and 5 times, respectively.
4) Designing an EST-SSR primer: and (3) designing SSR primers based on sequences on two wings of the SSR locus by using Primer3 software. The screening strategy for primer design was: the length is between 17 and 24bp (most suitable for 20bp), the melting temperature (Tm) is between 55 and 62 ℃, the size of a PCR amplification product is between 100 and 350bp, the GC content is between 40 and 60 percent (most suitable for 50 percent), and the others are set by default. And taking the primer combination closest to the design requirement as the primer of the SSR locus. SSR primers for PCR amplification were synthesized by Biotechnology engineering (Shanghai) GmbH.
2. Plant sample genomic DNA extraction
Experimental materials: the leaves of paphiopedilum armeniacum are respectively selected from 170 plants of Yanglan township, Fugong county, Lushui county, Laohouxiang, Longyang county, Yangxiangxiangxiangxiangxiangxiangxiangxiang and Longyang district Wamapau village in Yunnan province.
Extracting sample DNA in batches by using an improved CTAB method, and specifically operating as follows:
(1)2ml centrifuge tube (sterilization), blade installation, tube cap size two;
(2) grinding into powder with liquid nitrogen, adding 800 μ L CTAB (containing β -mercaptoethanol v/v 1%) preheated at 65 deg.C;
(3) water bath at 65 deg.C for 1h, shaking once every ten minutes;
(4) taken out and cooled to room temperature (or put into a refrigerator), and precooled chloroform is added: isoamyl alcohol (24: 1) 800. mu.L, shake gently for 5min (hand shaking);
(5)13000rpm for 10min, taking 450 mu L of supernatant to a 1.5 mu L centrifuge tube, extracting once again (24: 1, 450 mu L), sucking 300 mu L of supernatant, adding 600 mu L of precooled absolute ethyl alcohol with 2 times of volume, slightly inverting up and down, and mixing evenly;
(6) standing at-20 deg.C for 30min to precipitate DNA cluster, dragging the DNA cluster with a gun head, and discarding the liquid;
(7) adding 1ml 70% ethanol to wash the precipitate, reversing the upper part and the lower part for 6-8 times, centrifuging at 7500rpm for 5min, pouring the supernatant, washing the precipitate with 1ml 90% ethanol, centrifuging at 7500rpm, pouring the supernatant;
(8) drying the ventilated part for 2 hours until the DNA is dry and transparent and has no alcohol taste;
(9) adding 100 mu L of sterile water for redissolving.
The agarose gel electrophoresis was performed, and the results showed (FIG. 1) that the total DNA extracted was clear in electrophoresis band, indicating that the extracted DNA was of good quality.
3. PCR amplification using EST-SSR primers
The genomic DAN extracted above was used as a template, and the EST-SSR primers designed above were used for PCR amplification to identify the effectiveness of the primers. The PCR reaction system is shown in Table 2.
Analysis of PCR amplification products
And (3) carrying out electrophoresis on the PCR product by adopting a fluorescence capillary electrophoresis method. According to an electropherogram, screening EST-SSR primers of paphiopedilum armeniacum which have clear bands, high polymorphism and good specificity.
As a result, 13 pairs of primers having a good amplification effect and high polymorphism were selected, and the primer pairs are shown in Table 1. As shown in FIGS. 2-131, 96 alleles were obtained by co-detection using 13 pairs of primers, the variation range of each pair of primers allele was between 2 and 21, and the average allele factor was 7.38, indicating that the EST-SSR primers developed by the present invention can be completely used for genetic diversity analysis of paphiopedilum armeniacum.
Example 2 genetic diversity analysis Using EST-SSR primers from paphiopedilum armeniacum
Experimental materials: experimental samples of paphiopedilum armeniacum were obtained from 170 plants in the Londown township (LJ), Fugong county, Wenhouxiang (PH), Lushu county, Laohouxiang (LW), Longyang county, Yanhusuo county (YL) and Longyang county, Wama village (WM), respectively.
Analysis of the 13 EST-SSR primer amplification polymorphism information (see Table 3 below) showed that the observed and expected heterozygosity ranges from 0.1934-0.9722 and 0.0278-0.8066, respectively, and the Shannon information index ranges from 0.1709-2.2988 with an average value of 1.05, indicating that the 170 paphiopedilum armeniacum material is relatively abundant in genetic differentiation.
By using 13 pairs of primers to analyze the genetic diversity of 5 paphiopedilum armeniacum colonies (see table 4 below), it can be seen that the polymorphic locus percentage (PPL) of 5 paphiopedilum armeniacum colonies varied between 83.33% and 100%, with the average value of 93.33%. Wherein the percentage of the polymorphic sites of the Lushui Laolaou (LW) and Baoshan Wama (WM) population is up to 100 percent, and the percentage of the polymorphic sites of the Baoshan willow (YL) population is minimum 83.33 percent. The variation of the Nei's gene diversity index (h) of 5 colonies is 0.3978-0.5003, and the average value is 0.4564; the Shannon index (I) varied between 0.7207 and 0.9938, mean 0.8803. Of the 5 paphiopedilum armeniacum populations, the genetic diversity indices h and I were highest for the lushui old pit (LW) population, but lowest for the LJ population. Overall, the genetic diversity of the 5 populations is quite abundant.
TABLE 313 analysis of EST-SSR primer amplification polymorphism information
TABLE 4 genetic diversity of Pachyrhizus armeniaca population on both sides of Yunnan anger river
Example 3 Cluster analysis of genetic relationships between populations
Experimental materials: the experimental samples of paphiopedilum armeniacum were obtained from 170 plants of the lanhuajing town of orchids of Yunnan province, the township county of Fugong county, the old nest county of Lushui county, the Yangxian county of Longyang district, and the Wamapura county of Longyang district, respectively.
The degree of genetic differentiation between apricot yellow colonies was evaluated by the genetic distance (D) and genetic Identity (IN) of Nei's (Table 5). The genetic similarity of the 5 populations was very high, varying between 0.9188-0.9683; correspondingly, the genetic distance change range is small, and is maintained between 0.0323 and 0.0847, which shows that the genetic consistency of paphiopedilum armeniacum among groups is high, and the genetic distance is small.
Further UPMGA cluster analysis shows that the genetic distance between LY and LW is the minimum and the genetic relationship is close in 5 paphiopedilum armeniacum communities on two sides of the Yangjiang river in Yunnan province; LJ and PH are the most distant and related populations, as shown in fig. 132.
TABLE 5 genetic distance between the two sides of Yunan Yanjiang and the genetic identity between Nei's (lower triangle)
Example 4 general analysis in paphiopedilum Using EST-SSR primers of paphiopedilum Althatum
The results (see table 6 below) of the universal analysis of the other four paphiopedilum plants from paphiopedilum using the paphiopedilum armeniacum microsatellite showed that the amplification success rates of 13 pairs of EST-SSR primers in paphiopedilum mazeranum (P.malipore), paphiopedilum harderi (P.micranthum), paphiopedilum delanatium (P.delnati) and paphiopedilum concolor (P.microcolor) were 0.62, 0.54, 0.62 and 0.69, respectively, indicating that the 13 pairs of EST-SSR primers have higher universality in paphiopedihiopedilum. The 13 pairs of EST-SSR primers developed by using paphiopedilum armeniacum transcriptome sequencing are proved to be applicable to the research on genetic diversity and genetic structure of closely related species.
TABLE 613 EST-SSR primers amplification in the other four paphiopedilum plants
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
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Claims (8)
1. The EST-SSR primer group of paphiopedilum armeniacum comprises the following primer pairs:
(1) the nucleotide sequence of a forward primer of the primer pair PA _ SSR039 is shown in SEQ ID NO.1, and the sequence of a reverse primer of the primer pair PA _ SSR039 is shown in SEQ ID NO. 2;
(2) the nucleotide sequence of a forward primer of the primer pair PH _ SSR047 is shown as SEQ ID NO.3, and the sequence of a reverse primer of the primer pair PH _ SSR047 is shown as SEQ ID NO. 4;
(3) the nucleotide sequence of a forward primer of the primer pair PH _ SSR097 is shown as SEQ ID NO.5, and the sequence of a reverse primer of the primer pair PH _ SSR 096 is shown as SEQ ID NO. 6;
(4) the nucleotide sequence of a forward primer of the primer pair PH _ SSR115 is shown as SEQ ID NO.7, and the sequence of a reverse primer of the primer pair PH _ SSR115 is shown as SEQ ID NO. 8;
(5) the nucleotide sequence of a forward primer of the primer pair PH _ SSR116 is shown as SEQ ID NO.9, and the sequence of a reverse primer of the primer pair PH _ SSR116 is shown as SEQ ID NO. 10;
(6) the nucleotide sequence of a forward primer of the primer pair PH _ SSR119 is shown as SEQ ID NO.11, and the sequence of a reverse primer of the primer pair PH _ SSR119 is shown as SEQ ID NO. 12;
(7) the nucleotide sequence of a forward primer of the primer pair PH _ SSR137 is shown as SEQ ID NO.13, and the sequence of a reverse primer of the primer pair PH _ SSR137 is shown as SEQ ID NO. 14;
(8) the nucleotide sequence of a forward primer of the primer pair PH _ SSR146 is shown as SEQ ID NO.15, and the sequence of a reverse primer of the primer pair PH _ SSR146 is shown as SEQ ID NO. 16;
(9) the nucleotide sequence of a forward primer of the primer pair PH _ SSR176 is shown as SEQ ID NO.17, and the sequence of a reverse primer of the primer pair PH _ SSR176 is shown as SEQ ID NO. 18;
(10) the nucleotide sequence of a forward primer of the primer pair PH _ SSR179 is shown as SEQ ID NO.19, and the sequence of a reverse primer of the primer pair PH _ SSR179 is shown as SEQ ID NO. 20;
(11) the nucleotide sequence of a forward primer of the primer pair PH _ SSR206 is shown in SEQ ID NO.21, and the sequence of a reverse primer of the primer pair PH _ SSR206 is shown in SEQ ID NO. 22;
(12) the nucleotide sequence of a forward primer of the primer pair PH _ SSR224 is shown as SEQ ID NO.23, and the sequence of a reverse primer of the primer pair PH _ SSR224 is shown as SEQ ID NO. 24; and
(13) the nucleotide sequence of the forward primer of the primer pair PH _ SSR252 is shown in SEQ ID NO.25, and the sequence of the reverse primer is shown in SEQ ID NO. 26.
2. The paphiopedilum armeniacum EST-SSR primer set of claim 1, wherein one primer of said primer pair is labeled.
3. The paphiopedilum armeniacum EST-SSR primer set of claim 2, wherein the marker is a fluorescent marker.
4. A reagent for isolating an EST-SSR marker of paphiopedilum armeniacum, the reagent comprising the primer set according to any one of claims 1-3.
5. The reagent of claim 4, wherein the reagent comprises a primer set according to any one of claims 1-3, wherein each pair of primers in the primer set is packaged separately.
6. A kit comprising the reagent of claim 4 or 5.
7. Use of the Paphiopedilum EST-SSR primer set according to claim 1 for determining genetic diversity of Paphiopedilum, wherein the Paphiopedilum is Paphiopedilum armeniacum (Paphiopedilum armeniacum), Paphiopedilum malipidicum (paphiopediiopedilum micrantum), Paphiopedilum micranthum (paphiopediiopediiobium micranthum), Paphiopedilum delenii (paphiopedihiopediiopediococcum), or Paphiopedilum concolor.
8. Use of the Paphiopedilum EST-SSR primer set according to claim 1 for determining the genetic relationship between populations of Paphiopedilum, wherein the Paphiopedilum is Paphiopedilum armeniacum (Paphiopedilum armeniacum), Paphiopedilum malipidicum (paphiopedihiopedilum micranthum), Paphiopedilum micranthum (paphiopedihiopedihiopediococcum), Paphiopedilum deleniacum (paphiopediococcum delenii) or Paphiopedilum concolor.
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| CN110423747B (en) * | 2019-07-22 | 2023-01-31 | 西南大学 | Cymbidium SSR primer and application thereof in population genetic diversity analysis |
| CN110564722A (en) * | 2019-10-18 | 2019-12-13 | 西南林业大学 | Method for extracting paphiopedilum genome DNA by using improved CTAB method |
| CN110699480B (en) * | 2019-11-18 | 2023-10-24 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group for hybridization of EST-SSR (expressed sequence tag-simple sequence repeat) markers of cymbidium kanran and screening method |
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