CN107365874B

CN107365874B - Huntington harlequin EST-SSR (expressed sequence tag-simple sequence repeat) labeled primer as well as development method and application thereof

Info

Publication number: CN107365874B
Application number: CN201710864768.7A
Authority: CN
Inventors: 贾瑞冬; 徐玉凤; 葛红; 周妍慧; 程浩; 杨树华; 赵鑫
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2017-09-21
Filing date: 2017-09-21
Publication date: 2020-04-07
Anticipated expiration: 2037-09-21
Also published as: CN107365874A

Abstract

The invention discloses an EST-SSR labeled primer group developed based on a paphiopedilum henryanum transcriptome sequence, which comprises 34 pairs of primers, wherein the nucleotide sequence of the primers is shown as a sequence table SEQ ID NO. 1-SEQ ID NO. 68. The invention also discloses a reagent containing the EST-SSR marker primer of the paphiopedilum henryi. The invention further discloses a method for developing the EST-SSR labeled primers of the paphiopedilum henryanum. The Huntington EST-SSR marker primer 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 the Huntington and paphiopedilum, particularly other paphiopedilum species.

Description

Huntington harlequin EST-SSR (expressed sequence tag-simple sequence repeat) labeled primer as well as development method and application thereof

Technical Field

The invention relates to an EST-SSR marker primer developed based on Paphiopedilum henryanum (paphiopediococcus henryanum) transcriptome sequence, a development method and application thereof, belonging to the technical field of biology.

Background

The Paphiopedilum henryi belongs to Paphiopedilum (Orchidaceae) plants in Orchidaceae, is originally produced in southwest part of Guangxi, southeast part of Yunnan and northern Vietnam of China, and grows in hidden gaps of limestone areas with elevation of 900-1300m or places with good rocky or drainage in evergreen broad-leaved forests and shrubs. 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. The research on genetic diversity of paphiopedilum as an endangered species becomes very important, and the nursing work of paphiopedilum is more reluctant.

Simple Repeat (SSR) Markers have been one of the important genetic Markers in genetic diversity analysis due to their high polymorphism, good reproducibility, strong specificity, convenient detection, co-dominance, uniform distribution of Markers across the entire genome, etc. (Phuekvilai, Prattana & Pongtonkam, Pradit & Peyachknagul, Surin "Development of Microtectote Markers for variant Ornda" Kaseta Journal-Natural Science, 2009, 43 (3): 497 + 506; Kalia R K, Rai M K, KaliaS, etc.; "Microtectote Markers: an overview of the recombinant expression plants," Euphica, 2011 177 (3): 309 (309): 309). 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 increase, 'development and application of wheat SSR marker', 'heredity', 2003, (03): 355-. In recent years, the sequencing technology (RNA-seq) provides opportunities for finding new transcripts and gene expression abundance and SSR sites (Zhang H, Wei L, Miao H, etc. ' Development and Identification of genetic-SSR markers in the search by RNA-seq' ' BMC genomes, 2012, 13 (1): 316- The methods are also reported in Orchid several times (Tsai CC, Shih HC, Wang HV, Lin YS, Chang CH, etc. "RNA-Seq SSRs of Molecular Markers across genome Phalaenopsis (Orchidaceae)," PLOSONE, 2015, 10 (11): e 0141761). However, no report has been reported on the EST-SSR marker of paphiopedilum.

Therefore, the development of EST-SSR primers by using the sequence information of the paphiopedilum henryi transcriptome plays an important promoting role in genetic diversity, genetic relationship analysis, important character gene positioning, molecular marker assisted breeding, paphiopedia plant related molecular research and the like of germplasm resources of paphiopedilum and other paphiopedilum, particularly paphiopedilum.

Disclosure of Invention

The invention discloses an EST-SSR labeled primer group developed based on a paphiopedilum henryanum transcriptome sequence, which comprises 34 pairs of primers, wherein the nucleotide sequence of the primers is shown as a sequence table SEQ ID NO. 1-SEQ ID NO. 68. The invention also discloses a reagent containing the EST-SSR marker primer of the paphiopedilum henryi. The invention further discloses a method for developing the EST-SSR marker primer of the paphiopedilum henryanum, which comprises the following steps: (a) designing an EST-SSR primer based on unigene sequence data obtained by sequencing and splicing paphiopedilum henryanum organ transcriptome; (b) extracting genome DNA of a paphiopedilum henryi 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 Huntington EST-SSR primer with expected characteristics. The Huntington EST-SSR marker primer 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 the Huntington and paphiopedilum, particularly other paphiopedilum.

In a first aspect of the invention, EST-SSR primer pairs developed based on the Huntington transcriptome sequences are provided as shown in Table 1 below:

TABLE 1 HENLINGROUN EST-SSR polymorphic primer information

The sequence numbers of the primers are respectively SEQ ID NO. 1-SEQ ID NO. 68.

In one embodiment, any one of the pair of paphiopedilum EST-SSR primers described herein can 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 pair of henry paphiopedilum primers 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 to improve the specificity of the primers, for molecular biological studies of other plants of the same genus or species, or to obtain other desired properties.

A second aspect of the invention provides a set of EST-SSR primers developed based on the sequence of the paphiopedilum henryi transcriptome, said primer set comprising any combination of the above primer pairs of the invention.

In a third aspect, the invention provides a DNA fragment or gene product, namely an EST-SSR marker, isolated from the genome of paphiopedilum henryi by means of a primer pair or primer set according to the invention. The EST-SSR markers can be used for genetic diversity analysis of germplasm resources of the paphiopedilum henryanum 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 other plants of paphiopedilum and even orchids, which are constructed by utilizing the EST-SSR primer.

In a fourth aspect of the invention, there is provided an agent for isolating a paphiopedilum henryi EST-SSR marker, said agent comprising any pair of primers or set of primers described herein.

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 fourth aspect of the invention provides a kit comprising a reagent of the invention as described above.

In a fourth aspect of the invention, there is provided a method for developing EST-SSR primers based on the Huntington's transcriptome sequence, the method comprising:

(a) designing an EST-SSR primer based on unigene sequence data obtained by sequencing and splicing paphiopedilum henryanum organ transcriptome;

(b) extracting genome DNA of a paphiopedilum henryi 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 the paphiopedilum henryi.

In one embodiment, the step (a) is performed as follows: sequencing and splicing a paphiopedilum henryanum 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:

① grinding the leaves of paphiopedilum henryi into powder with liquid nitrogen, adding CTAB (containing β -mercaptoethanol v/v 1%) preheated at 65 deg.C;

② water bath at 65 deg.C for 1 h;

③ cooling to room temperature (or placing in refrigerator), adding precooled chloroform isoamyl alcohol (24:1), and shaking gently for 5 min;

④ centrifuging, collecting supernatant, repeating twice, adding 2 times volume of precooled anhydrous ethanol, and slightly turning upside down to mix;

standing at ⑤ -20 deg.C for 30min to precipitate DNA cluster, and discarding the liquid;

⑥ washing the precipitate with 70% ethanol, turning upside down for 6-8 times, centrifuging, removing supernatant, and washing with 90% ethanol;

⑦ air-drying for 2h until DNA is dry and transparent and has no alcohol taste;

⑧ and adding sterile water to dissolve.

The advantages of extracting DNA by the improved CTAB method are as follows: the liquid is directly discarded after DNA agglomeration, thereby skillfully avoiding various impurity precipitates caused by a centrifugal method and greatly improving the purity and the 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, 8% polyacrylamide gel is adopted to carry out electrophoresis on the PCR product, silver staining is adopted for staining, and finally the gel is placed on a gel imaging system for photographing to identify polymorphic bands on an electropherogram, so that the Huntington 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 populations, different varieties, paphiopedilum plants 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 the paphiopedilum are developed for the first time, 34 pairs of the EST-SSR primers of the paphiopedilum, which have rich polymorphism, good repeatability, high specificity and stable amplification, are provided, and a foundation is provided for genetic diversity analysis of paphiopedilum germplasm resources, molecular marker assisted breeding and related molecular research of paphiopedilum plants; 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, new gene identification 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 33 different Henry materials from 2 wild-populated populations of Henry.

FIGS. 2-35 show electropherograms of the results of polymorphic amplification on 33 Henry paphiopedilum samples with 34 pairs of primers listed in Table 1, respectively.

Fig. 36 shows a UPGMA clustering plot after cluster analysis of 33 different huntington materials from 2 wild populations of huntington using 34 pairs of primers 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 henryanum

EST-SSR primer design

Experimental materials: the paphiopedilum henryi organ is obtained from vegetable and flower institute of Chinese academy of agricultural sciences; 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 a paphiopedilum henryi organ, separating mRNA, performing reverse transcription to synthesize and purify cDNA, repairing the tail end, adding adenosine to connect with a sequencing joint, recovering a fragment with the size of 200-700 bp through agarose gel electrophoresis, and performing 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 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 the plants of the Henry paphiopedilum are respectively taken from 8 plants of Taichonghui living group (TYC) in Shanxi county of Yunnan province and 25 plants of Dazhu living group (DN).

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.

Agarose gel electrophoresis was performed, and the results (FIG. 1) showed clear bands of electrophoresis of the extracted total DNA, 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 using 8% polyacrylamide gel, dyeing by using a silver staining method, and after the band is completely presented after the color development, placing the gel on a gel imaging system for photographing. According to an electropherogram, screening the EST-SSR primer of the paphiopedilum henryi with clear bands, high polymorphism and good specificity.

As a result, 34 pairs of primer sets having a good amplification effect and high polymorphism were selected, and the primer sets are shown in Table 1. As shown in FIGS. 2-35, 94 alleles were detected by 34 pairs of primers, the variation range of each pair of primers is between 2 and 5, and the average allele factor is 2.74, indicating that the ESR-SSR primers developed by the present invention can be completely used for genetic diversity analysis of Huntington.

Example 2 genetic diversity analysis Using the EST-SSR primers of paphiopedilum henryi

Experimental materials: experimental samples of huntington were obtained from 8 plants of taiyang civic (TYC) and 25 plants of dazu civic (DN) in han chestnut slope county, Yunnan, respectively.

Analysis of the 34 EST-SSR primer amplification polymorphism information (see Table 3 below) revealed observed and expected heterozygosity ranges from 0.0303 to 1.000 and 0.0597 to 0.6793, respectively, and the Shannon information index ranged from 0.14 to 1.25 with an average of 0.61, indicating that the 33 Henry materials are more genetically differentiated.

TABLE 334 analysis of EST-SSR primer amplification polymorphism information

In addition, clustering analysis was also performed on 33 different huntington materials of the above two wild populations (taiyang population and dawn population) using the 34 pairs of primers, as shown in fig. 36. The results show that 33 samples were divided into 5 large groups, the genetic similarity coefficient at 0.71 divided the material into two groups, one group in categories I, II, III, samples from the major population (DN), one group in IV, V, and samples from the solar population (TYC), indicating that the genetic similarity coefficient between different wild populations of paphiopedilum is small and the genetic basis is broad. In large population, the genetic similarity coefficient is 0.77, and the materials can be classified into 3 categories. In the solar population, the genetic similarity coefficient is 0.70, and the materials can be classified into 2 categories. Therefore, the materials of different wild populations of the paphiopedilum have difference and relatively large genetic variation, the developed 34 pairs of EST-SSR primers can be used for successfully distinguishing the populations, the genetic variation of the paphiopedilum in the same population is relatively large, and the genetic basis is wide.

Example 3 commonality analysis in paphiopedilum Using the EST-SSR primers for henry

The results (see table 4 below) of the results of the universal analysis of six other paphiopedilum plants from paphiopedilum using the henry paphiopedilum microsatellite showed that the amplification success rates of 34 pairs of EST-SSR primers in paphiopedilum majus (p.villosum), paphiopedilum schuricatum (p.venustum), paphiopedilum hirsutum (p.dianthus), paphiopedilum concolor (p.microcolor) and paphiopedium harderium (p.micranthumum) were 0.85, 0.82, 0.74, 0.68, 0.56 and 0.56, respectively, indicating that these 34 pairs of EST-SSR primers have high universal property in paphiopedia, particularly have extremely high amplification rate in paphiopedigree subgenus, and it can be seen that the closer genetic distance to paphiopedilum, the amplification efficiency of SSR primers is higher. The 34 pairs of EST-SSR primers developed by using the Huntington transcriptome sequencing are proved to be applicable to the research on genetic diversity and genetic structure of closely related species.

TABLE 434 amplification of EST-SSR primers in other six paphiopedilum plants

The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive various changes or substitutions within the technical scope of the present invention, and these should be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

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<212>DNA

<213> Artificial sequence

<400>54

aagaaccatc accaccaccc 20

<210>55

<211>20

<212>DNA

<213> Artificial sequence

<400>55

caaacctcgc tcggaagact 20

<210>56

<211>20

<212>DNA

<213> Artificial sequence

<400>56

agggtttcta tcgcttggcc 20

<210>57

<211>20

<212>DNA

<213> Artificial sequence

<400>57

gctgccaatt cgaatggagg 20

<210>58

<211>20

<212>DNA

<213> Artificial sequence

<400>58

gctgccgatt ctccttcctt 20

<210>59

<211>20

<212>DNA

<213> Artificial sequence

<400>59

gaaaaccgat tggcgtcgag 20

<210>60

<211>20

<212>DNA

<213> Artificial sequence

<400>60

ttcccttctc ccatttccgg 20

<210>61

<211>20

<212>DNA

<213> Artificial sequence

<400>61

agctatgagg aactgcgctg 20

<210>62

<211>20

<212>DNA

<213> Artificial sequence

<400>62

tgttgacatg atccgtggca 20

<210>63

<211>20

<212>DNA

<213> Artificial sequence

<400>63

agcttgaagt acttgggggc 20

<210>64

<211>20

<212>DNA

<213> Artificial sequence

<400>64

tccaccttct cctcctcact 20

<210>65

<211>20

<212>DNA

<213> Artificial sequence

<400>65

ccttcctcat gatccgtccg 20

<210>66

<211>20

<212>DNA

<213> Artificial sequence

<400>66

ttagctctct cccctccgag 20

<210>67

<211>20

<212>DNA

<213> Artificial sequence

<400>67

ggcgattgac ctctgctgat 20

<210>68

<211>20

<212>DNA

<213> Artificial sequence

<400>68

tcggctttgg ttgctgagat 20

Claims

1.EST-SSR primer pair combinations developed based on the Huntington's transcriptome sequences are as follows:

(1) the primer pair PH _ SSR011, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.1, and the sequence of a reverse primer is shown as SEQ ID NO. 2;

(2) the nucleotide sequence of a forward primer of the primer pair PH _ SSR025 is shown as SEQ ID NO.3, and the sequence of a reverse primer of the primer pair PH _ SSR025 is shown as SEQ ID NO. 4;

(3) the nucleotide sequence of a forward primer of the primer pair PH _ SSR026 is shown as SEQ ID NO.5, and the sequence of a reverse primer of the primer pair PH _ SSR026 is shown as SEQ ID NO. 6;

(4) the nucleotide sequence of the forward primer of the primer pair PH _ SSR041 is shown as SEQ ID NO.7, and the sequence of the reverse primer of the primer pair PH _ SSR041 is shown as SEQ ID NO. 8;

(5) the nucleotide sequence of a forward primer of the primer pair PH _ SSR060 is shown as SEQ ID NO.9, and the sequence of a reverse primer of the primer pair PH _ SSR060 is shown as SEQ ID NO. 10;

(6) the nucleotide sequence of a forward primer of the primer pair PH _ SSR172 is shown as SEQ ID NO.11, and the sequence of a reverse primer of the primer pair PH _ SSR172 is shown as SEQ ID NO. 12;

(7) the nucleotide sequence of a forward primer of the primer pair PH _ SSR272 is shown as SEQ ID NO.13, and the sequence of a reverse primer of the primer pair PH _ SSR272 is shown as SEQ ID NO. 14;

(8) the nucleotide sequence of a forward primer of the primer pair PH _ SSR334 is shown as SEQ ID NO.15, and the sequence of a reverse primer of the primer pair PH _ SSR334 is shown as SEQ ID NO. 16;

(9) a primer pair PH _ SSR343, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.17, and the sequence of a reverse primer is shown as SEQ ID NO. 18;

(10) the nucleotide sequence of a forward primer of the primer pair PH _ SSR351 is shown as SEQ ID NO.19, and the sequence of a reverse primer of the primer pair PH _ SSR351 is shown as SEQ ID NO. 20;

(11) a primer pair PH _ SSR356, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.21, and the sequence of a reverse primer is shown as SEQ ID NO. 22;

(12) the nucleotide sequence of a forward primer of the primer pair PH _ SSR372 is shown as SEQ ID NO.23, and the sequence of a reverse primer of the primer pair PH _ SSR372 is shown as SEQ ID NO. 24;

(13) a primer pair PH _ SSR376, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.25, and the sequence of a reverse primer is shown as SEQ ID NO. 26;

(14) the nucleotide sequence of a forward primer of the primer pair PH _ SSR383 is shown as SEQ ID NO.27, and the sequence of a reverse primer of the primer pair PH _ SSR383 is shown as SEQ ID NO. 28;

(15) the nucleotide sequence of a forward primer of the primer pair PH _ SSR413 is shown as SEQ ID NO.29, and the sequence of a reverse primer of the primer pair PH _ SSR413 is shown as SEQ ID NO. 30;

(16) a primer pair PH _ SSR416, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.31, and the sequence of a reverse primer is shown as SEQ ID NO. 32;

(17) a primer pair PH _ SSR425, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.33, and the sequence of a reverse primer is shown as SEQ ID NO. 34;

(18) the nucleotide sequence of a forward primer of the primer pair PH _ SSR426 is shown as SEQ ID NO.35, and the sequence of a reverse primer of the primer pair PH _ SSR426 is shown as SEQ ID NO. 36;

(19) the nucleotide sequence of a forward primer of the primer pair PH _ SSR430 is shown as SEQ ID NO.37, and the sequence of a reverse primer of the primer pair PH _ SSR430 is shown as SEQ ID NO. 38;

(20) a primer pair PH _ SSR447, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.39, and the sequence of a reverse primer is shown as SEQ ID NO. 40;

(21) a primer pair PH _ SSR535, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.41, and the sequence of a reverse primer is shown as SEQ ID NO. 42;

(22) the nucleotide sequence of a forward primer of the primer pair PH _ SSR547 is shown as SEQ ID NO.43, and the sequence of a reverse primer of the primer pair PH _ SSR547 is shown as SEQ ID NO. 44;

(23) the nucleotide sequence of a forward primer of the primer pair PH _ SSR548 is shown as SEQ ID NO.45, and the sequence of a reverse primer of the primer pair PH _ SSR548 is shown as SEQ ID NO. 46;

(24) a primer pair PH _ SSR592, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.47, and the sequence of a reverse primer is shown as SEQ ID NO. 48;

(25) the nucleotide sequence of a forward primer of the primer pair PH _ SSR645 is shown in SEQ ID NO.49, and the sequence of a reverse primer of the primer pair PH _ SSR645 is shown in SEQ ID NO. 50;

(26) a primer pair PH _ SSR651, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.51, and the sequence of a reverse primer is shown as SEQ ID NO. 52;

(27) a primer pair PH _ SSR658, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.53, and the sequence of a reverse primer is shown as SEQ ID NO. 54;

(28) the nucleotide sequence of a forward primer of the primer pair PH _ SSR669 is shown in SEQ ID NO.55, and the sequence of a reverse primer of the primer pair PH _ SSR669 is shown in SEQ ID NO. 56;

(29) the nucleotide sequence of a forward primer of the primer pair PH _ SSR687 is shown as SEQ ID NO.57, and the sequence of a reverse primer of the primer pair PH _ SSR687 is shown as SEQ ID NO. 58;

(30) the nucleotide sequence of a forward primer of the primer pair PH _ SSR698 is shown in SEQ ID NO.59, and the sequence of a reverse primer of the primer pair PH _ SSR698 is shown in SEQ ID NO. 60;

(31) the nucleotide sequence of a forward primer of the primer pair PH _ SSR716 is shown as SEQ ID NO.61, and the sequence of a reverse primer of the primer pair PH _ SSR716 is shown as SEQ ID NO. 62;

(32) the nucleotide sequence of a forward primer of the primer pair PH _ SSR800 is shown as SEQ ID NO.63, and the sequence of a reverse primer of the primer pair PH _ SSR800 is shown as SEQ ID NO. 64;

(33) a primer pair PH _ SSR809, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.65, and the sequence of a reverse primer is shown as SEQ ID NO. 66; and

(34) the nucleotide sequence of the forward primer of the primer pair PH _ SSR877 is shown in SEQ ID NO.67, and the sequence of the reverse primer is shown in SEQ ID NO. 68.

2. The paphiopedilum EST-SSR primer pair combination of claim 1, wherein one primer of the primer pair is detectably labeled.

3. The paphiopedilum EST-SSR primer pair combination of claim 2, wherein the label is a fluorescent label.

4. An agent for isolating a paphiopedilum EST-SSR marker, said agent comprising the primer pair combination of any one of claims 1-3.

5. The reagent of claim 4, wherein each primer in the primer pair combination is packaged separately.

6. A kit comprising the reagent of claim 4 or 5.

7. A method for developing EST-SSR primers based on paphiopedilum henryi transcriptome sequences, the method comprising:

(b) extracting genome DNA of a paphiopedilum henryi plant sample;

(d) carrying out electrophoresis on the PCR product obtained in the step (c), and analyzing the electrophoresis result, thereby screening the EST-SSR primer of the paphiopedilum henryanum;

wherein the primer is as described in claim 1.

8. The method of claim 7, wherein step (b) comprises batch extraction of sample DNA by a modified CTAB method, as follows:

① grinding the leaves of paphiopedilum henryi into powder with liquid nitrogen, adding CTAB preheated at 65 deg.C;

② water bath at 65 deg.C for 1 h;

⑧ and adding sterile water to dissolve.

9. The method of claim 7 or 8, wherein the step (c) is performed by touchdown PCR with the reaction program: denaturation at 94 deg.C for 5 min; circulating for 10 times at 94 ℃ for 30s, 65 ℃ for 30s and 72 ℃ for 40 s; circulating for 15 times at 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 40 s; circulating for 10 times at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 40 s; then extension at 72 ℃ for 10 min.