Molecular markers and primers for identifying and distinguishing two species of Phalaenopsis

Abstract

The present invention relates to the field of molecular marker technology, and more particularly to molecular markers for identifying and distinguishing Phalaenopsis szechwanensis from Phalaenopsis szechwanensis. The molecular markers of the present invention can be amplified using the primer pairs shown in SEQ ID NO. 12 and SEQ ID NO. 33. The molecular markers of the present invention can effectively identify and distinguish Phalaenopsis szechwanensis from Phalaenopsis szechwanensis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to molecular markers for identifying Phalaenopsis huaxiensis and Phalaenopsis ocellaris and applications thereof. background

[0002] Phalaenopsis species in the deciduous group include Phalaenopsis wilsonii, Phalaenopsis braceana, Phalaenopsis yunnanensis, and Phalaenopsis hainanensis. Both Phalaenopsis wilsonii and Phalaenopsis braceana are deciduous and epiphytic, sharing similar morphological characteristics and habitats. These include well-developed, clustered, long, curved aerial roots with dense warty protuberances on their surfaces. Their stems are short, encased in leaf sheaths, about 1 cm long, and typically bear 4-5 leaves. The leaves are slightly fleshy, oblong or nearly elliptical, with sepals and petals varying in color from pink, green, brown, to white. The capsules are narrow and elongated. Flowering occurs from April to July, and fruiting occurs from August to September. These species grow on tree trunks or on shady, moist rocks beneath forests at altitudes of 800-2150 meters. Under natural conditions, they often shed their leaves during flowering, either being leafless or with one or two leaflets remaining. In the absence of flowers, it is difficult to distinguish between the different species based solely on morphology.

[0003] Currently, the classification and identification of Phalaenopsis still primarily relies on morphological classification and molecular biology methods. DNA markers used in Phalaenopsis classification include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), short tandem simple sequence repeat (SSR, also known as microsatellite) markers, and inter-simple sequence repeat (ISSR) markers. Each of these markers has its advantages and disadvantages. RAPD markers are the most commonly used due to their relatively low technical difficulty and cost. However, they are susceptible to experimental conditions, have poor stability and reproducibility, and are relatively unreliable. RFLP, ISSR, and SSR, which are slightly more difficult and costly, are less commonly used.

[0004] Different molecular markers have different accuracies in distinguishing species, especially between morphologically similar individuals, making the taxonomic relationships between different subgenera and species in the deciduous orchid group complicated. The morphological characteristics of deciduous Phalaenopsis species are very similar when there are no flowers after the leaves fall. The slight difference is whether there are spurs or claws at the base of the lip (Jin Xiaohua, 2019, Color Atlas of Wild Orchids in China). It is difficult to distinguish the species based on the root characteristics alone, and even professionals often make misjudgments. The most obvious feature to distinguish different species is the flower characteristics, but even in the flowering period, due to the influence of climate, geographical environment and other factors, the flower shapes and colors of Phalaenopsis huaxiensis and Phalaenopsis spicata are diverse, and some individuals (variants or variants) are often very close, especially when there are no flowers when collected in the wild, it is often difficult to distinguish, such as Figure 1 Therefore, it is very necessary to develop molecular markers to quickly and accurately distinguish deciduous Phalaenopsis species, especially to distinguish Phalaenopsis huaxiensis from Phalaenopsis occidentalis. Summary of the Invention

[0005] The present invention mainly addresses the above-mentioned technical problems and provides a simple molecular marker method to achieve the identification and differentiation of Phalaenopsis sinensis and Phalaenopsis oxysporum. Specifically, a molecular marker for identifying Phalaenopsis sinensis and Phalaenopsis oxysporum and its application are provided. The present invention utilizes a high-throughput sequencing method to assemble genomic contigs from the whole genome sequences of Phalaenopsis sinensis and Phalaenopsis oxysporum, and compares the genomic contigs of Phalaenopsis sinensis and Phalaenopsis oxysporum. There are multiple indels longer than 10bp in the genomes of Phalaenopsis sinensis and Phalaenopsis oxysporum, which are presumably used as molecular markers to identify and differentiate Phalaenopsis sinensis and Phalaenopsis oxysporum by conventional PCR and agarose gel electrophoresis, or by first-generation sequencing.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a molecular marker for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, wherein the molecular marker comprises the underlined portion of any one of the following sequences (SEQ ID NO: 1-SEQ ID NO: 11):

[0008]

[0009] Note: The dash “-” indicates the gap position in the homologous sequence alignment of two species; JN represents Phalaenopsis oxypetalum; HX represents Phalaenopsis huaxiensis.

[0010] In one embodiment, the molecular marker comprises SEQ ID NO: 1 to SEQ ID NO: 11 in Table 1 or their corresponding homologous sequences in Table 1.

[0011] Primers were further designed for the upper Indel sequence so that the upper Indel sequence was included in the primer amplification product.

[0012] Phalaenopsis huaxiensis and Phalaenopsis apex were selected, and the designed primers were used to analyze the fragment length of the PCR products. The differences between the two Phalaenopsis orchids were consistent with expectations. The PCR products can be used as molecular markers to identify and distinguish Phalaenopsis huaxiensis and Phalaenopsis apex. The sequences of the PCR products are as follows:

[0013] Primer1 PCR product

[0014] HX:

[0015] ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATCTTATACATTATTAAATTCATTATTCAATT ATTAAA---------------------AAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATATTCTATTTTCATTTTATATTATATCTATTTACTTATTTACTAATTGAATTTACTAATTGAATCTTAGAGATTCTATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAAT TAGAGTCTAAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCTAGTACTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTC-TTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA

[0016] JN:

[0017] ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATCTTATACATTATTAAATTCATTATTCAATTATTTAAAAAAATTATTCAATTATTTAAAAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATATTCTATTTTCATTTTATATTATATCTATTTACTTATTTACTAATTGAATTTACTAATTGAATCTTAGAGATTCTATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAATTAGAGTCTAAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCTAGTACTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTCTTTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA

[0018] Primer2 PCR product

[0019] JN:

[0020] GGTTCAAATCCTATTGGACGCAATTTCTTTCTATAATTTATTTCTATATATCTATTCTAGATAGAGAATAGATATATAGAAGGAACTTTATTTTAGAAAGGATAAAGGTCCTTTTTTGAATGATTCGAATCAGAAATATTTATGAAATATTTATTAAGGATTTCTATTTATTAAGATATAGAATAAAAAAAGATATAGAATAAAAATGATCCATTTACATTTATGTTTGTTCCTGAAGTAGAAAGAGTTCAATCTGTTCCTGAATAGCTTCTCTCAAAAGGGTTTCAGCTTCTTCGGT

[0021] HX:

[0022] GGTTCAAATCCTATTGGACGCAATTTCTTTCTATAATTTATTTCTATATATCTATTCTAGATAGAGAATAGATATATAGAAGGAACTTTATTTTAGAAAGGATAAAGGTCCTTTTTTGAATGATTCGAATCAGAAATATTTATGAAATATTTATTAAGGATTTCTATTTATT-----------------AAGATATAGAATAAAAATGATCCATTTACATTTATGTTTGTTCCTGAAGTAGAAAGAGTTCAATCTGTTCCTGAATAGCTTCTCTCAAAAGGGTTTCAGCTTCTTCGGT

[0023] Primer3 PCR product

[0024] HX:

[0025] TGAGCTATCCCATACTCCCATAACTATTTGATCTTAGAACTATATAGAACTCTATATTTAGATTCTATATTTATATATAGAACATAATATAAGTCTATACTATATATGTAATGAATATGAATTATGAATATGATTATGAATATGAAAATCTATTCAATTCAATAAAAATAGATTCAAATTCAATATGAATTTCCATTGAATTTTTCAATGTTTCAATAATGGAAATTCTATTCATATTGAAGTAATTTAAAGGAAGTTCAATTAAGTCAAGTTAAAAAGAAAGAAAAATAAAGTAGTATTACTTTTACTACTAGTAAAAGTAAAAGTAGAAAAAGTCTAGTCTATAAATTCTAAATATATAAAGAGTCTTAAATAGTAAGAGTATATACTTAAATAGTAATAAATAGTAATAAATAGTAAGAGTATATCATAGTATATATATATATATATATATATACTATATTACTATAATTACTATATACTATATTACTATATTATATATACTATATTACTATATTAACTATATTAATAGTAACTATATTAAACTATATTAAATAGAGTATATTTATATAAGTCAAGTAAGTCAAGTGAATCTTT

[0026] JN:

[0027] TGAGCTATCCCATACTCCCATAACTATTTGATCTTAGAACTATATAGAACTCTATATTTAGATTCTATATTTATATATAGAACATAATATAAGTCTATACTATATATGTAATGAATATGAATTATGAATATGATTATGAATATGAAAATCTATTCAATTCAATAAAAATAGATTCAAATTCAATATGAATTTCCATTGAATTTTTCAATGTTTCAATAATGGAAATTCTATTCATATTGAAGTAATTTAAAGGAAGTTCAATTAAGTCAAGTTAAAAAGAAAGAAAAATAAAGTAGTATTACTTTTACTACTAGTAAAAGTAAAAGTAGAAAAAGTCTAGTCTATAAATTAGAAATATATAAAGAGTCTTAAATATTAAGAGTATATACTTAA----------ATAGTAATAAATAGTAAGAGTATATCATAG----TATATATATATATATATATACTATATTACTATA--TTATATATACTATATTACTATATTA-----ACTATATTAC--TATTAACTATATTAATAGTAACTATATTAAACTATATTAAATAGAGTATATTTATATAAGTCAAGTAAGTCAAGTGAATCTTT

[0028] Primer 4 PCR product

[0029] HX:

[0030] GAGAGTTTGTTTGCTGTAGGACATACCTTGTTTCAAAATTCATCTAATGTCATCCCACTTCTCTTTTTCTTTTTTTTTTTCTCCTTTCAATTCTCTTTCTATATGTGATATGTGATGTTTAATATAGAATGCTCTTATACTTAGTTCTTTTTCTTTTCTATTCTACTTAATTATTATAACTTAATTATTATACTTATTATCTTAATACTTAATATATTTTTTATATATTTTTTTTTTTCTATTTCATATTGATCTTATATCTTAGAATATAAAGTGAAAGTAAAGAATTCCCTATCTTATACTAACTTTTCTATTAACTTAGAATAATTCTAGATAGTAATTCTAGTAATATACTATAGTAATAAGTAATAGTAATATAGTAAAGAAAAAGAGAAATTCAATTGAAAAAGAAAAAGAATAAAAAAATGATAAAGAACATATGTAATTTCTTCATGAAAGTGGATGAAGATAGATGGG

[0031] JN:

[0032] GAGAGTTTGTTTGCTGTAGGACATACCTTGTTTCAAAATTCATCTAATGTCATCCCACTTCTCTTTTTC-TTTTTTTTTTCTCCTTTCAATTCTCTTTCT ATATGTGATATGTGATGTTTAATATAGAATGCTCTTATACTTAGTTCTTTTTCTTTTCTATTCTACTTAATTATTAGAACTTAATTATTATACTTATTATCTTAATACTTAATATATATTTTTTATATA-TTTTTTTTTTCTATTTCATATTGATCTTATATCTTAGAAATATAAAGTGAAAGTAAAGAATTCCCTATCTTATACTAACT TTTCTATTAACTTAGAATAATT-------------------CTAGATAGTAATTCTAGTAATATACTATAGTAATAAGTAATAGTAATATAGTAAAGAAAAAGAA GAAATTCAATTGAAAAAGAAAAAGAATAAAAAAATGATAAAGAACATATGTAATTTCTTCATGAAAGTGGATGAAGATAGATGGG

[0033] Primer5 PCR product

[0034] HX:

[0035] CTTTCATACGGCGGGAGTCATTGGTTCAAATCCAATAGTAGGTAGAACTTATTAGATACCGGATTCCATGGTATCTAATAAGTTTTTCTACTCATCCTCTTTTTTTCGTTCTGTTCTATCATCAGATTAATCAAATTAGACTTCATTGTGGTCAATTTGTGGAATCAAGATGTAGTGTGTAGTATATAATATATATAATAAATAAATAATAAATAATAATAAATAAATCTTTTTATTTTGATTGAATGTATTGACTACTAAGAGGAAATTACTTTGATAGCTTCTACTCGTGTCCTAGC

[0036] JN:

[0037] CTTTCATACGGCGGGAGTCATTGGTTCAAATCCAATAGTAGGTAGAACTTATTAGATACCGGATTCCATGGTATCTAATAAGTTTTTCTACTCATCCTCT TTTTTTCGTTCTGTTCTATCATCAGATTAATCAAATTAGACTTCATTGTGGTCAATTTGTGGAATCAAGATGTAGTGTGTAGTATATAATATAT-----------ATAATAAATAATAATAAATAAATCTTTTTATTTTGATTGAATGTATTGACTACTAAGAGGAAATTACTTTGATAGCTTCTACTCGTGTCCTAGC

[0038] Primer6 PCR product

[0039] HX:

[0040] AGGGGCGGCAAAATTGAATCTGATGGGTCATTGTACATATGCATTTTAGTTAGTAATTCCTAATTCTAATTTCATTTCAAGCAAAAACAAAAGATCTTGAACTAAAATTAAAATATCGGATTATCTATGATCTATTTATTAGAATAGCGAACTAGGACTATATATGTATTACAATATACAATTCTTACAATAAATATACAATTCTTACAATATACAATACAAAGAAAATAAATAAGAAAAAAATATAAAATAAAAAAGAAAAAAAGAAGGAGGATTTTCAATGCAAGA <OO00086>JN:

[0042] It should be noted that there may be an error in the original text where "OO00086" in line 11 should probably be "

[0041] ". This has been corrected in the translation as much as possible while maintaining the original text's integrity.AGGGGCGGCAAAATTGAATCTGATGGGTCATTGTACATATGCATTTTAGTTAGTAATTCCTAATTCTAATTTCATTTCAAGCAAAAACAAAAGATCTTGA ACTAAAATTAAAATATCGGATTATCTATGATCTATTTATTAGAATAGCGAACTAGGACTATATATGTATTAC--------------------AATATACAATTCT TACAATATACAATACAAAGAAAATAAATAAGAAAAAAATATAAAATAAAAAAGAAAAAAAGAAGGAGGATTTTCAATGCAAGA

[0043] Primer7 PCR product

[0044] HX:

[0045] TCCAACTACAAGGAGACAGGTAGTCTAATACAAAATTCCTTTACCATCTTTTGCCTCTA-TTTTTTTATTTTAT--------------TCTAGTATTTAA TAGTATTTAAAGTATAGAAATTGAGATTTCTATTAGATTTCTATTTTATTATTTTATAATTTTCTATATTAATTCTTAATTTAATCTATTATTTATTTCATATTCAATATTACTATCTATATAGA---------------CTATATTAATCGAATAGTAAATTAGTAAATCTAAATTGATAATTTCTTTAGTAAATGATCCAAAATGAAT AGGTATGGAAGCT<​​​​TCCAACTACAAGGAGACAGGTAGTCTAATACAAAATTCCTTTACCATCTTTTGCCTCTATTTTTTTTATTTTATTCTAGTATTTAATATCTAGTATTTAATAGTATTTAAAGTATAGAAATTGAGATTTCTATTAGATTTCTATTTTATTATTTTATAATTTTCTATATTAATTCTTAATTTAATCTATTATTTATTTCATATTCAATATTACTATCTATATAGACTATATTAATATAGACTATATTAATCGAATAGTAAATTAGTAAATCTAAATTGATAATTTCTTTAGTAAATGATCCAAAATGAATAGGTATGGAAGCT

[0048] Primer8 PCR product

[0049] HX:

[0050] CGGCAATAATGTCCCTACCCATGATGAACTATAGTTATTGGTGCCCCCTAATTTTGATATAGTCAACATGCTTCTTTTTTGTTTTATTTTTTATTGAATT C-TTTTTTTTTTAATTATTATAGATTCTCAAAAATTATTAGAAATTTCAAATATATATGAGACACACACAATCTATTAATCGGATCTATTTCAGATATTCTAATA TTCTATTTTCATCTATAAGACTTCGGGTGCTAATGAAATTTCATCTATAAGACTTCGGGTGCTAATGAAACG

[0051] JN:

[0052] CGGCAATAATGTCCCTACCCATGATGAACTATAGTTATTGGTGCCCCCTAATTTTGATATAGTCAACATGCTTCTTTTTTGTTTTATTTTTTATTGAATT CTTTTTTTTTTTAATTATTATAGATTCTCAAAAATTATTAGAAATTTCAAATATATATGAGACACACACAATCTATTAATCGGATCTATTTCAGATATTCTAATATTCTAT--------------------------------TTTCATCTATAAGACTTCGGGTGCTAATGAAACG

[0053] Primer9 PCR product

[0054] JN:

[0055] TCCTCATAAGAACGTCCACGAATCTGATCAATTACTCTTCGTGCTTTGAAAACAGACATACATATATTTT---------------GAGCTAAAACTTTTG CTTCTCTATTTTCGTTCTTTATCATAAAAGTTTTCCCCCGCCAATGAATGA

[0056] HX:

[0057] TCCTCATAAGAACGTCCACGAATCTGATCAATTACTCTTCGTGCTTTGAAAACAGACATACATATATTTTGAGTACATATATTTTGAGCTAAAACTTTTG CTTCTCTATTTTCGTTCTTTATCATAAAAGTTTTCCCCCGCCAATGAATGA

[0058] Primer10 PCR product

[0059] HX:

[0060] TGAGAAAGGACCAAGATACTTTGATTTCCATTTCAATAACCA-----------TGAAATATAAGTTTACGAATTCAATTCATGTTAATTTTACTATATGT ATATAGATATAGAATATCTAAAGTATAGAATATATAATCTAAAGTATAGAATATATAAAGATTCTAGTATTCTAGTATATAGAAATCTAATTATATAGTTATATA GGTAGAATAATTATATAAATTATATAGATAATTATAGATAATAGATAGATAGAAATAGAATTAAGGATATACAAACAAGAAGAACCAATACCAATGAAAAGGCAGJN:

[0061] TGAGAAAGGACCAAGATACTTTGATTTCCATTTCAATAACCATGAAATATAAGTGAAATATAAGTTTACGAATTCAATTCATGTTAATTTTACTATATGT ATATAGATATAGAAT---------------------ATCTAAAGTATAGAATATATAAAGATTCTAGTATTCTAGTATATAGAAATCTAATTATATAGTTATATA GGTAGAATAATTATATAAATTATATAGATAATTATAGATAATAGATAGATAGAAATAGAATTAAGGATATACAAACAAGAAGAACCAATACCAATGAAAAGGCAGPrimer11PCR product

[0062] HX:

[0063] TTTTACGTCTTTGAGCGCGCATGGATCCTTTGATTAGATTGCGACGAAAATCCGATTGTTGTGAGTAACGTATCAAAGCTACCTCTTCCGTTTGATCATC TTTTTCATCATTGTTACTAGTATTCTGAATAC---------------TAGAAAGAGAATTGGTATTCTGATCATTATCGTTATAAATTATCACACGTTTGGATCTTCTTGAATGAATCTCATAATATTCTTCCTCCAAATCTTCTTCATTTTCTTCTTCTTCTCCCAAATTCTCGGT

[0064] JN:

[0065] TTTTACGTCTTTGAGCGCGCATGGATCCTTTGATTAGATTGCGACGAAAATCCGATTGTTGTGAGTAACGTATCAAAGCTACCTCTTCCGTTTGATCATCTTTTTCATCATTGTTACTAGTATTCTGAATACTAGAAA GAGAATTGGTAGAAAGAGAATTGGTATTCTGATCATTATCGTTATAAATTATCACACGTTTGGATCTTCTTGAATGAATCTCATAATATTCTTCCTCCAAATCTTCTTCATTTTCTTCTTCTTCTCCCAAATTCTCGGT

[0066] In another embodiment, any DNA or RNA fragment containing the above PCR product sequence can be used as a molecular marker for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex.

[0067] In a second aspect, the present invention provides primers for amplifying the above-mentioned molecular markers, wherein the amplification product sequence of the primers contains the above-mentioned molecular marker sequence.

[0068] In a preferred embodiment, the primers of the present invention include at least one primer pair as shown in Primer1-Primer11, preferably including at least one primer pair sequence as shown in Primer2 or Primer3 or Primer4 or Primer6 or Primer8 or Primer9 or Primer10.

[0069]

[0070] Note: JN stands for JN-HN, and HX stands for HX-HN.

[0071] In a third aspect, the present invention provides a method for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, characterized by comprising the following steps:

[0072] 1) Extracting total DNA from the sample to be tested;

[0073] 2) sequencing the total DNA to obtain a sequencing sequence;

[0074] 3) aligning the sequence to a DNA barcode, wherein the DNA barcode is any one of SEQ ID NO: 1 to SEQ ID NO: 11;

[0075] 4) Determine the species of the sample based on the coverage of the sequencing reads on the DNA barcode.

[0076] In one embodiment, if the sequencing reads can completely cover any sequence from SEQ ID NO. 1 to SEQ ID NO: 11, the sample to be tested is determined to be the species corresponding to the barcode that can be completely covered by the sequencing reads.

[0077] In another embodiment, the DNA barcode is any of the aforementioned molecular markers.

[0078] In a fourth aspect, the present invention provides a method for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, characterized by comprising the following steps:

[0079] 1) Extracting total DNA from the sample to be tested;

[0080] 2) performing PCR on the total DNA of the above samples using primers, wherein the primers include at least one pair of primers as shown in Primer1 to Primer11;

[0081] 3) Performing electrophoresis detection or sequencing on the PCR product and the standard, and determining the species information of the sample to be detected based on the electrophoresis or sequencing results, wherein the standard is the aforementioned PCR product.

[0082] In one embodiment, if the length of the PCR product is consistent with the length of the standard, the sample to be tested is determined to be the species corresponding to the corresponding standard.

[0083] In another embodiment, if the sequence of the PCR product is consistent with the sequence of the standard, the sample to be tested is determined to be the species corresponding to the corresponding standard.

[0084] In a fifth aspect, the present invention provides a reagent or kit containing the above-mentioned primers.

[0085] In a sixth aspect, the present invention provides any of the following applications of the above-mentioned molecular markers, primers, reagents, or kits:

[0086] (1) Application in the identification and authentication of Phalaenopsis huaxiensis and Phalaenopsis apex;

[0087] (2) Application in identification, improvement or molecular marker-assisted breeding of Phalaenopsis huaxiensis and Phalaenopsis occidentalis germplasm resources;

[0088] (3) Application in screening or creating different varieties of Phalaenopsis huaxiensis and Phalaenopsis apex;

[0089] (4) Application in constructing DNA fingerprint database of Phalaenopsis huaxiensis and Phalaenopsis apex;

[0090] (5) Application in seedling quality testing of Phalaenopsis huaxiensis and Phalaenopsis apex

[0091] The single molecular marker of the present invention can simply and effectively distinguish whether the tested species is Phalaenopsis spicata or Phalaenopsis huaxiensis, providing assurance for the identification, planting, resource utilization, and breeding of the two orchids. It is also of great significance for orchid species classification, phylogeny, and phylogeography research, as well as the protection and utilization of orchid resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Morphological photos of Phalaenopsis sinensis and Phalaenopsis aculeatus; A is Phalaenopsis sinensis and B is Phalaenopsis aculeatus.

[0093] Figure 2 A to K are the results of high-throughput sequencing assembly and comparative analysis, showing the molecular markers of Phalaenopsis huaxiensis and Phalaenopsis apex obtained.

[0094] Figure 3 This is an electrophoresis image of the PCR products of Phalaenopsis huaxiensis and Phalaenopsis aculeatus amplified using SEQ ID NO: 3-4, where M is a marker, lane A: PCR results of different primers for the same Phalaenopsis huaxiensis individual; lane B: PCR results of different primers for the same Phalaenopsis aculeatus individual.

[0095] Figure 4 The sequencing results of Primer1-F and Primer1-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0096] Figure 5 The sequencing results of Primer2-F and Primer2-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0097] Figure 6 The sequencing results of Primer3-F and Primer3-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0098] Figure 7 The sequencing results of Primer4-F and Primer4-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0099] Figure 8 The sequencing results of Primer5-F and Primer5-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0100] Figure 9The sequencing results of Primer6-F and Primer6-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0101] Figure 10 The sequencing results of Primer7-F and Primer7-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0102] Figure 11 The sequencing results of Primer8-F and Primer8-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0103] Figure 12 The sequencing results of Primer9-F and Primer9-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0104] Figure 13 The sequencing results of Primer10-F and Primer10-R amplification products were aligned with the contig sequences assembled by second-generation sequencing.

[0105] Figure 14 The sequencing results of Primer11-F and Primer11-R amplification products were aligned with the contig sequences assembled by second-generation sequencing. DETAILED DESCRIPTION

[0106] In order to make the purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the present invention.Those skilled in the art can make various modifications and replacements to the present invention without departing from the purpose and spirit of the present invention.The experimental method used in the following examples is conventional method unless otherwise specified.The materials, reagents etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0107] The plant samples of Phalaenopsis huaxiensis and Phalaenopsis apex used in the following examples can be obtained through commercial purchase or collected from the wild.

[0108] Example 1 Collection of Phalaenopsis huaxiensis and Phalaenopsis apex

[0109] The present invention Phalaenopsis huaxiensis and Phalaenopsis apex were collected from southern Yunnan ( Figure 1 ).

[0110] Example 2 Development of specific molecular markers for Phalaenopsis huaxiensis and Phalaenopsis apex

[0111] The following steps were used to obtain specific molecular markers for distinguishing Phalaenopsis huaxiensis from Phalaenopsis apexii, i.e., molecular markers:

[0112] 1) Collect leaves of Phalaenopsis huaxiensis and Phalaenopsis apex;

[0113] 2) Extracting total DNA from leaves of Phalaenopsis huaxiensis and Phalaenopsis apex;

[0114] 3) performing second-generation high-throughput sequencing on the total DNA to obtain sequencing reads;

[0115] 4) Genome assembly of Phalaenopsis huaxiensis and Phalaenopsis apex was performed to obtain genome contigs sequences.

[0116] 5) Compare the genomic contigs of Phalaenopsis huaxiensis and Phalaenopsis oxysporum to obtain species-specific fragments, i.e. molecular markers.

[0117] The results are shown in Table 1 and Figure 2 There are multiple Indels longer than 10bp in the genomes of Phalaenopsis huaxiensis and Phalaenopsis oxysporum, which are speculated to be used as molecular markers to identify and distinguish Phalaenopsis huaxiensis and Phalaenopsis oxysporum through conventional PCR and agarose gel electrophoresis.

[0118]

[0119] Example 3 Primer design of specific molecular markers of Phalaenopsis huaxiensis and Phalaenopsis apex

[0120] Based on the comparison results in Example 2 and the corresponding contigs sequences, multiple pairs of PCR primers for identifying Phalaenopsis huaxiensis and Phalaenopsis apex were designed using Geneious software. The primer sequences are as follows:

[0121]

[0122] Note: JN stands for JN-HN, and HX stands for HX-HN.

[0123] Example 4 Identification of Phalaenopsis huaxiensis and Phalaenopsis apex

[0124] 1) Collecting leaves of Phalaenopsis huaxiensis and Phalaenopsis apex as samples to be tested;

[0125] 2) extracting total DNA from the sample to be tested;

[0126] 3) using the primer pairs designed in Example 3, PCR was performed on the total DNA of the above samples;

[0127] The PCR amplification reaction system consists of 25 μL, 12.5 μL of 2× Phanta Max Master Mix, 1 μL each of upstream primer (10 μM) and downstream primer (10 μM), 200-300 ng of DNA template, and ddH2O to 25 μL. 2× Phanta Max Master Mix includes Phanta Max Super-Fidelity DNA Polymerase, dNTPs, a protective agent, and a buffer system.

[0128] The PCR amplification procedure was as follows: ① pre-denaturation at 95°C for 3 min; ② denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 75 s, for 35 cycles; ③ extension at 72°C for 5 min; 4) electrophoresis detection of PCR amplification products:

[0129] The PCR products were detected by 1% agarose gel electrophoresis and stained with 10× Loading Buffer. The PCR amplification was observed under a gel imaging system, and the main bands were clear.

[0130] From the electropherogram ( Figure 3 ) It can be seen that the lengths of the PCR product fragments of primer pairs 2, 3, 4, 6, 8, 9, and 10 and the differences between the two Phalaenopsis orchids are consistent with expectations.

[0131] 5) First-generation sequencing identification of PCR amplification products:

[0132] The PCR products were subjected to first-generation Sanger sequencing, and the sequencing results were compared with the assembled contigs. Figure 4-Figure 14 .

[0133] The comparison results showed that the sequences of the amplified products of the 11 primer pairs in the two species were consistent with the expectations, and any pair of primers could distinguish the two species of Phalaenopsis.

[0134] It can be seen that the aforementioned molecular markers and corresponding primers can be used for conventional PCR gel electrophoresis or first-generation sequencing to achieve simple and effective identification and differentiation of Phalaenopsis huaxiensis and Phalaenopsis apex.

[0135] In summary, the present invention is a powerful supplement to traditional species identification. Moreover, the sample identification process is effective, can be automated and standardized, and breaks through the excessive reliance on experience. Fresh or dried plant leaves, such as specimen samples, can be used for rapid and effective identification, and an easily usable application system can be established in a relatively short period of time. Finally, the method of the present invention is of great significance for the classification, phylogeny, and phylogeographic research of orchid species, as well as the protection and utilization of Phalaenopsis resources.

[0136] Based on the disclosure and teachings of the above description, those skilled in the art may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and any modifications and variations of the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although certain specific terms are used in this description, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. A molecular marker for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, characterized in that: The molecular marker is an indel marker, and the sequence of the indel in Phalaenopsis huaxiensis is: ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATTCTTATACATTATTAAATTCATTATTCAATTATTAAAAAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATATTCTATTTTCATTTTATAATTATATCTATTTACTATTTACTAATTGAATTTACTAATTGAATCTTAGAGATTC TATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAATTAGAGTCTAAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCTAGTACTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTC-TTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA; The sequence of the indel in Phalaenopsis spicata is: ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATCTTATACATTATTAAATTCATTATTCAATTATTTAAAAAAATTATTCAATTATTTAAAAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATTCTATTTTCATTTTATATTATATCTATTTACTTATTTACTAATTGAATTTAC TAATTGAATCTTAGAGATTCTATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAATTAGAGTCTAAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCT AGTACTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTCTTTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA.

2. Primers for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, the primers being a primer pair Primer1, the primer pair consisting of Primer1-F: ACGAGAAGAATAATTTTCATTGCCA and Primer1-R: TATTTGTTCCACCGCGGGA; The sequence of the PCR product of the primer pair Primer1 in Phalaenopsis huaxiensis is as follows: ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATCTTATACATTATTAAATTCATTATTCAATTATTAAAAAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATTCTATTTTCATTTTATATTATATCTATTTACTTATTTACTAATTGAATTTACTAAT TGAATCTTAGAGATTCTATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAATTAGAGTCTAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCTAG TACTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTC-TTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA; The sequence of the PCR product of the primer pair Primer1 in Phalaenopsis spicata is as follows: ACGAGAAGAATAATTTTCATTGCCATTCCTAATTATTATAAATTAGATAAGATTCTAATAGATAGATTATCTTATACATTATTAAATTCATTATTCAATTATTTAAAAAAATTATTCAATTATTTAAAAAAAAATTATCTATTATCTATATCTAACTATATCTAAATTAAGAATTAATAGAATGAATATATTCTATTTTCATTTTATATTATATCTATTTACTTATTTACTAATTGAATTTACTAATTGAAT CTTAGAGATTCTATTTCTATTCTCAGTATATTTAGTATATTCTAGTAAATTAGAGTCTAAATTAGAAGATTTAATCTTTATAGCTTTATAGTAAAAATAGTAAAAAAGAGTAAAAGTGTTCTAGTA CTCTTATCTAGTACTCTTACTAGTATATAGTATATACTAATATAGTACTAATATTGTACTAATGTACTAAGGTTTTTCGTCTTTATTCTTTTTTTTTTTCAAGTTTTCCCGCGGTGGAACAAAATA.

3. A kit comprising the primer according to claim 2.

4. A method for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, characterized in that The following steps are involved: 1) Extract total DNA from the sample to be tested; 2) Sequencing the total DNA from step 1) to obtain a sequencing sequence; 3) aligning the sequence to the DNA barcode, wherein the DNA barcode is the sequence of the indel marker in Huaxi Phalaenopsis and Phalaenopsis cuspidatus as shown in claim 1; 4) Determine the species of the sample based on the coverage of the DNA barcode by the sequencing reads. If the sequencing reads can completely cover the DNA barcode in step 3), then the sample to be tested is determined to be the species corresponding to the DNA barcode that can be completely covered by the sequencing reads.

5. A method for identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex, characterized in that The following steps are involved: 1) Extract total DNA from the sample to be tested; 2) performing PCR on the total DNA obtained in step 1) using primers, wherein the primers are the primers according to claim 2; 3) Sequencing the PCR product and the standard, and determining the species information of the sample to be tested based on the sequencing results. If the PCR product sequence is consistent with the standard sequence, the sample to be tested is determined to be the species corresponding to the corresponding standard; the standard sequence is the sequence of the indel marker in Phalaenopsis huaxiensis and Phalaenopsis aculeatus as shown in claim 1.

6. Any of the following uses of the molecular marker according to claim 1: (1) Application in identifying and distinguishing Phalaenopsis huaxiensis and Phalaenopsis apex; (2) Application in identification, improvement or molecular marker-assisted breeding of Phalaenopsis huaxiensis and Phalaenopsis occidentalis germplasm resources; (3) Application in constructing DNA fingerprint database of Phalaenopsis huaxiensis and Phalaenopsis apex.

7. Use of the primers according to claim 2 or the kit according to claim 3 in identifying and differentiating Phalaenopsis huaxiensis and Phalaenopsis apex.

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