AU2021101595A4 - SSR molecular marker primer set for identifying Cattleya Alliance and use thereof - Google Patents

SSR molecular marker primer set for identifying Cattleya Alliance and use thereof Download PDF

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AU2021101595A4
AU2021101595A4 AU2021101595A AU2021101595A AU2021101595A4 AU 2021101595 A4 AU2021101595 A4 AU 2021101595A4 AU 2021101595 A AU2021101595 A AU 2021101595A AU 2021101595 A AU2021101595 A AU 2021101595A AU 2021101595 A4 AU2021101595 A4 AU 2021101595A4
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cattleya
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ssr molecular
primers
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Heming Chen
Zuo LI
Fubing LV
Wenfang XIAO
Genfa ZHU
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Environmental Horticulture Institute of Guangdong Academy of Agricultural Sciences
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The present invention provides a SR molecular marker primer set and a fingerprint code for identifying Cattleya Alliance and use thereof in the identification of Cattleya Alliance germplasm resources, belonging to the technical field of molecular biology. The present invention provides 15 SSR molecular markers of Cattleya Alliance and amplification primer sets thereof, and utilizes TP-M13-SSR technique to analyze the genetic diversity and construct molecular ID of 26 different gennplasm resources in Cattleya Alliance, thereby identifying the Cattleya Alliance germplasm resources accurately, efficiently and stably, and laying a foundation for the identification germplasm resources, genetic relationship analysis, location of trait genes, and molecular marker-assisted breeding in Cattleya Alliance, and molecular research related to Cattleya Alliance. Cal0 Ca23 Ca07 CaO Ca9 Ca26 Cal0 Cal6 C07 Ca08 Cal8 Ca22 Ca11 Ca16 Ca18 0.72 0.77 0.82 0.87 0.92 Coefficient SampleFile Sampl Name Panel OS ISO 100 140 180 220 260 300 0{ 153.07 Fig.1 330

Description

SampleFile Sampl Name Panel OS ISO
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0{ 153.07
Fig.1
Description
SSR molecular marker primer set for identifying Cattleya Alliance and use thereof
Technical Field The present invention relates to the technical field of molecular biology, in particular to a SSR
molecular marker primer set and a fingerprint code for identifying Cattleya Alliance and use
thereof in the identification of Cattleya Alliance germplasm resources.
Background Art Cattleya, is a general name of Cattleya Alliance of Orchidaceae family and related genera, which
is native to the tropical and subtropical America and distributed from Mexico to Brazil. As
epiphytic orchids, Cattleya is very popular in the market because of the large flowers, gorgeous
colors, and different varieties blooming throughout the year. Cattleya also is a national flower of
Brazil, Columbia, and Costa Rica with very good international recognition degree, so the Cattleya
genus is considered to be the 'Queen of the Orchids'.
Cattleya in a broad-sense is a collective name, namely Cattleya Alliance, in which the most
well-known, most diverse and most concerned Cattleya species, represented by Cattleya, have
been continuously cross-bred with different genera over the past 100 years and have been
combined into a general name of a shared family. Cattleya family is one of the major species of
orchids with the most extensive cross between related genera and the most successful offspring of
cross between different genera. At present, more than 36,000 Cattleya hybrids have been
registered in the Royal Horticultural Society (RHS).
The native species of Cattleya has not been found in China, so Cattleya is introduced to China
from other countries for cultivation. However, there are a plurality of phenomena of same species
with different names and same name for different species in the introduced Cattleya, which causes
adverse effects on the crossbreeding of the Cattleya. In addition to the traditional crossbreeding,
there are few studies on Cattleya Alliance, currently mainly focusing on tissue culture and rapid
propagation, cultivation management, and distribution and identification of wild resources. Cui
Xueqiang etc. (2020) screened 6 primers by ISSR markers, and analyzed genetic diversity of 18
Cattleya germplasm resources (Cui Xueqiang, Tangxuan, Huang Changyan, et.al, Analysis of
Genetic Diversity of Cattleya Germplasms by Using ISSR Markers [J]. Journal of Southwest
Agriculture. 2020, 33 (7): 1383-1388). Fajardo et.al analyzed the genetic diversity of Cattleya granulosa, an endangered species in Brazil, and other four Brazilian native species, Brassavola tuberculata, Cattleya bicolor, Cattleya labiata, and Cattleya schofieldiana by ISSR markers; the results showed that ISSR markers could effectively detect the interspecific genetic differentiation (Fajardo C G, de Almeida Vieira F, Molina W F. Interspecific genetic analysis of orchids in Brazil using molecular markers[J].Plant Systematies and Evolution,2014,300(8):1825-1832). Microsatellites, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs), are repetitive sequences up to dozens of nucleotides, consisting of several nucleotides (2-5) as repeating units, and are widely distributed in different locations throughout the whole genome of eukaryotes, with polymorphism at each locus due to the difference in the number of repeats and the incomplete degree of repeats. A pair of specific primers is designed to correspond to the conserved sequences at the two ends of a certain microsatellite DNA to amplify the microsatellite DNA sequence at the loci; and polymorphisms of different genotype individuals at the SSR locus may be displayed through electrophoresis detection. TP-M13-SSR (simple sequence repeat with tailed primer M13) technique that combines SSR molecular marker technique and fluorescence sequencing technique has the advantages of high repeatability, and accurate results, and solves a series of problems such as low analysis flux, tedious detection process of amplification products, and large workload of data recording to a large extent (Li Huiyong et al., 2005). Therefore, using genomic sequence of Cattleya Alliance to develop new SSR molecular markers and primer set thereof, and using TP-M13-SSR technique for genetic diversity analysis and molecular identity card (ID) construction will play an important role in the identification of germplasm resources, genetic relationship analysis, location of trait-related genes, and molecular marker-assisted breeding in Cattleya Alliance, and other molecular researches related to Cattleya Alliance.
Summary of the Invention Aiming at the defects, the present invention provides a SSR molecular marker primer set and a fingerprint code for identifying Cattleya Alliance and use thereof in the identification of Cattleya Alliance germplasm resources. The present invention provides a SSR primer set for identifying Cattleya Alliance using reduced-representation genome sequencing technique, and utilizes TP-M13-SSR technique to analyze the genetic diversity and construct molecular ID of 26 different germplasm resources in Cattleya Alliance. The present invention makes the identification of Cattleya Alliance germplasm resources more accurately, efficiently and stably. The present invention also lays a foundation for the identification of germplasm resources, analysis of genetic relationship, location of trait-related genes, and molecular-marker-assisted breeding of Cattleya Alliance, and lays a foundation for related molecular researches to CattleyaAlliance. In order to achieve the aforementioned objectives, the technical solution of the present invention is as follows: In one aspect, the present invention provides a primer set of SSR molecular marker for identifying Cattleya Alliance, where the SSR molecular marker includes:1-N1006714, 2-N1204804, 10-N1060688, 11-N1995183, 12-N2010170, 22-N1388702, 25-N3457413, 36-N1012200, 39-N1014795, 47-N1036425, 212-N1001184, 215-N1003355, 216-N1006768, 220-N1009344, and 221-N10141; the primer set comprises: (1) primers for amplifying SSR molecular marker 1-N1006714:
SEQ ID NO:1 : 1-N1006714-F : 5'-AAATCACAGTCCAGGCCAAC-3'
SEQIDNO:2: 1-N1006714-R: 5'-TTAGAATTGTGGACCCAGCC-3';
(2) primers for amplifying SSR molecular marker 2-N1204804:
SEQ ID NO:3 : 2-N204804-F :5'-CAGGCCAGCATCACAAGATA-3'
SEQ ID NO:4 :2-N204804-R :5'-ACTTGGAGTTGTGGACCCAG-3';
(3) primers for amplifying SSR molecular marker 10-N1060688:
SEQ ID NO:5 : 10-N1060688-F : 5'-ACCCTTTGCTAGCTGTTGGA-3'
SEQ ID NO:6: 10-N1060688-R: 5'-TAATCAAAGGGCTACCCGTG-3'
(4) primers for amplifying SSR molecular marker 11-N1995183:
SEQIDNO:7: 11-N1995183-F: 5'-CGGTCTTGGACATGACTTGA-3'
SEQIDNO:8: 11-N1995183-R: 5'-CGGACTTAGCCTCAAGCAAC-3';
(5) primers for amplifying SSR molecular marker 12-N2010170:
SEQ ID NO:9 :12-N2010170-F : 5'-TTTTCCCCAACAACACTTCC-3'
SEQ ID NO:10 :12-N2010170-R : 5'-CTTGGTTGGATTTATGGAGGA-3';
(6) primers for amplifying SSR molecular marker 22-N1388702:
SEQIDNO:l :22-N388702-F: 5'-AGGAGGAGGTAACCCCAAAT-3'
SEQIDNO:12:22-N388702-R: 5'-TCCATCCAAGCATTGAAACC-3';
(7) primers for amplifying SSR molecular marker 25-N3457413:
SEQIDNO:13:25-N3457413-F :5'-TTGGAGGGAAGAAGAAGGGT-3'
SEQ ID NO:14 : 25-N3457413-R : 5'-TCACCCTCATATCCTCCTGG-3';
(8) primers for amplifying SSR molecular marker 36-N1012200:
100 SEQIDNO:15 :36-N1012200-F: 5'-TGGGTTACTCAGCCGTCTCT-3'
SEQ ID NO:16 : 36-N1012200-R : 5'-ACAGTCCAGGCCAACATCTC-3';
(9) primers for amplifying SSR molecular marker 39-N1014795:
SEQIDNO:17:39-N1014795-F: 5'-CGATCCACAATTCCTCCATT-3'
SEQIDNO:18:39-N1014795-R: 5'-CCCTGTTGGGACTCAGGTAA-3';
105 (10) primers for amplifying SSR molecular marker 47-N1036425:
SEQ ID NO:19: 47-N1036425-F :5'-CAAGGAGAAGCTATTGAGGTTGA-3'
SEQ ID NO:20 : 47-N1036425-R :5'-AGTGCACACTTTGCCCTTCT-3';
(11) primers for amplifying SSR molecular marker 212-N1001184:
SEQ ID NO:21 :212-N1001184-F :5'-CATCCGGTTGTTGTTTACCC-3'
110 SEQ ID NO:22 :212-N1001184-R :5'-GGCCGACAGTGGTAGGTTTA-3';
(12) primers for amplifying SSR molecular marker 215-N1003355:
SEQ ID NO:23 :215-N1003355-F :5'-CTGGCTGTAGCCAAAGCAGT-3'
SEQ ID NO:24 :215-N1003355-R :5'-GATGCTGAAGGCTTGAAAGG-3';
(13) primers for amplifying SSR molecular marker 216-N1006768:
115 SEQ ID NO:25 :216-N1006768-F :5'-ACCAAGCCAAGAAGGGATTT-3'
SEQ ID NO:26 :216-N1006768-R :5'-ATTTCGCTCGCCCTAATTCT-3';
(14) primers for amplifying SSR molecular marker 220-N1009344:
SEQ ID NO:27 : 220-N1009344-F : 5'-CCTGTCGGCCAAATTTCTTA-3'
SEQIDNO:28:220-N1009344-R: 5'-AGGGAGAATTGGCAAAGGTT-3';
120 (15) primers for amplifying SSR molecular marker 221-N10141:
SEQ ID NO:29: 221-N10141-F : 5'-GAACAATATGCAACGGGACA-3'
SEQ ID NO:30: 221-N10141-R: 5'-CGGTGACTCCATAACGAGTGT-3'.
In yet another aspect, the present invention provides a fingerprint code of SSR molecular marker for identifying Cattleya Alliance, where the fingerprint code includes a fingerprint spectrum and a 125 molecular ID, the sequence of SSR molecular marker of the molecular ID is as follows:-N1006714, 2-N1204804, 10-N1060688, 11-N1995183, 12-N2010170, 22-N388702, 25-N3457413, 36-N1012200, 39-N1014795, 47-N1036425, 212-N1001184, 215-N1003355, 216-N1006768, 220-N1009344, 221-N10141. Specifically, the molecular ID are shown in Table 1 below. 130 Table 1
Number Chinese name Latin Name Molecular ID information CattlevaVigour Ca01 Leopard-king 44334936446B243422459936002433 Leopard King Ca02 Luteola Cattleya luteola 554413331329334422469911254413 Cattlevamaxima var Ca03 Maxima 333300363418243422339966001434 alba Cattleva walkeriana Ca04 Walkeriana variant 353599333466143422009944774446 var. coerulea Ca05 Leopoldii Cattleya leopoldii 44354936446B453422449945551433
Ca06 Digbyana Rhyncholaelia 264436353346444413448834253435 dighyana Cattleva Ca07 Porphyroglossa ' 453549364416243422444955881433 porphyroglossa Ca08 Glauca Rhyncholaelia glauca 243535353344443444445533144446 Cattleya Ca09 CBC BeaufortxCattleya 3624333333AA244422445766553438 cernua CalO Fidelensis Laeliafidelensis 661549233344241422442644374426 Call Lundii Cattleya lundii 49363936348824562244578877448A Cattlevawalkeriana Ca12 Walkeriana 453539463467243422449925584434 var. alba Cattlevawalkeriana Ca13 Tokyo 1 3535AA333467243422149925554446 Tokyo1 Cal4 Nobilior Cattleya nobilior 453549445522243422449935471422 BrassocattlevaAngel Ca15 Angel Skirt ' 44373346338A243322668855473433 Skirt ProcatavolaGolden Cal6 Golden peacock Peacock 183623253322243533448A44364488
Cattleva Seagulls Tiny Ca17 Seagulls Tiny tim ' . 362433223333244422446657553499 tim BrassolaeliaSuzette Ca18 Suzette Chaney Chaney 176623333444243533463944444449
Cattleva Ca19 Lueddemanniana 353333243326243422469A56774428 lueddemanniana
Ca20 Tenebrosa Cattleya tenebrosa 333539443412243522241377594499
Ca2l Trianae Cattleya trianae 333333353377243322463856771429
Ca22 Skinneri Cattleya skinneri 333544142355463444443855224547
Ca23 Forbesii Cattleyaforbesii 44334946336C243422444936AA2433
Ca24 Brevipedunculata Cattleya 353578363312241311452655334488 brevipedunculata Ca25 Intermedia Cattleya intermedia 35133936332A242322449935793433
Ca26 Schilleriana Cattleya schilleriana 333339463312242313449944662433
Further specifically, the molecular ID is constructed according to a diploid standard, the
fingerprint data are subjected to data coding according to an SSR detection result (amplification
fragments at each locus are arranged according to the molecular weight, amplification fragments
135 (alleles) labeled with Arabic numerals 1-9 in ascending order, and alleles exceeding 9 are labeled
with capital English letters A-Z), if a locus is not amplified in a certain variety, it is recorded as 0,
and each locus occupies two positions.
Specifically, the fingerprints spectrum is shown in Table 2 below.
Table 2 1-Ni 1-Ni 2-Ni 2-Ni 10-N 10-N 11-N 11-N 12-N 12-N 22-N 22-N 25-N 25-N 36-N Numb 0067 0067 2048 2048 1060 1060 1995 1995 2010 2010 1388 1388 3457 3457 1012 er 14 14 04 04 688 688 183 183 170 170 702 702 413 413 200
CaOl 154 154 143 143 123 145 136 142 183 183 231 246 160 169 184
Ca02 156 156 145 145 101 121 136 136 162 180 217 240 163 163 188
Ca03 152 152 143 147 145 145 136 142 180 183 214 238 160 169 184
Ca04 152 156 143 147 145 145 136 136 180 183 231 231 155 169 184
Ca05 154 154 143 147 123 145 136 142 183 183 231 246 169 172 184
Ca06 150 158 145 145 121 129 136 140 180 180 226 231 169 169 188
Ca07 154 156 143 147 123 145 136 142 183 183 214 231 160 169 184
CaO8 150 154 143 147 121 125 136 140 180 180 226 226 169 169 184
Ca09 152 158 141 145 121 121 136 136 180 180 243 243 160 169 188
CalO 158 158 139 147 123 145 134 136 180 180 226 226 160 169 188
Call 154 164 143 149 121 145 136 142 180 183 238 238 160 169 190
Cal2 154 156 143 147 121 145 138 142 180 183 231 234 160 169 184
Cal3 152 156 143 147 147 147 136 136 180 183 231 234 160 169 184
Cal4 154 156 143 147 123 145 138 138 186 186 217 217 160 169 184
Ca15 154 154 143 153 121 121 138 142 180 180 238 243 160 169 184
Cal6 148 162 143 149 117 121 134 140 180 180 217 217 160 169 184
Ca17 152 158 141 145 121 121 134 134 180 180 223 223 160 169 188
Cal8 148 160 149 149 117 121 136 136 180 183 226 226 160 169 184
Ca19 152 156 143 143 121 121 134 138 180 180 217 231 160 169 184
Ca20 152 152 143 147 121 145 138 138 180 183 214 217 160 169 184
Ca2l 152 152 143 143 121 121 136 140 180 180 234 234 160 169 184
Ca22 152 152 143 147 123 123 130 138 174 180 229 229 163 169 184
Ca23 154 154 143 143 123 145 138 142 180 180 231 263 160 169 184
Ca24 152 156 143 147 131 143 136 142 180 180 214 217 160 169 182
Ca25 152 156 139 143 121 145 136 142 180 180 217 243 160 169 184
Ca26 152 152 143 143 121 145 138 142 180 180 214 217 160 169 184
36-N 39-N 39-N 47-N 47-N 212- 212- 215- 215- 216- 216- 220- 220- 221- 221 Numb 1012 1014 1014 1036 1036 N100 N100 N100 N100 N100 N100 N100 N100 NIO NIO er 200 795 795 425 425 1184 1184 3355 3355 6768 6768 8344 8344 41 41
CaOl 188 145 145 201 201 210 210 276 282 - - 124 128 159 159
Ca02 188 145 145 201 207 210 210 264 264 224 230 128 128 155 159
Ca03 188 145 145 198 198 210 210 282 282 - - 122 128 159 161
Ca04 188 145 145 - - 210 210 278 278 234 234 128 128 161 167
Ca05 188 145 145 201 201 210 210 278 280 230 230 122 128 159 159
Ca06 188 144 146 201 201 207 207 276 278 224 230 126 128 159 163
Ca07 188 145 145 201 201 201 210 280 280 236 236 122 128 159 159
CaO8 188 148 148 201 201 203 203 276 276 222 228 128 128 161 167
Ca09 188 145 145 201 201 203 206 282 282 230 230 126 128 159 171
CalO 188 145 145 201 201 195 204 278 278 226 234 128 128 157 167
Call 196 145 145 201 201 203 206 290 290 234 234 128 128 171 175
Cal2 188 145 145 201 201 210 210 274 280 230 236 128 128 159 161
Cal3 188 145 145 192 201 210 210 274 280 230 230 128 128 161 167
Cal4 188 145 145 201 201 210 210 276 280 228 234 122 128 157 157
Ca15 184 145 145 207 207 207 207 280 280 228 234 126 128 159 159
Ca16 190 146 146 201 201 207 214 278 278 226 232 128 128 171 171
Ca17 188 145 145 201 201 204 204 280 284 230 230 126 128 173 173
Ca18 190 146 146 201 207 198 210 278 278 228 228 128 128 161 171
Ca19 188 145 145 201 207 210 214 280 282 234 234 128 128 157 171
Ca20 190 145 145 195 201 189 198 284 284 230 240 128 128 173 173
Ca2l 184 145 145 201 207 198 207 280 282 234 234 122 128 157 171
Ca22 188 148 148 201 201 198 207 280 280 224 224 128 130 161 169
Ca23 188 145 145 201 201 201 210 276 282 244 244 124 128 159 159
Ca24 188 144 144 201 204 195 204 280 280 226 226 128 128 171 171
Ca25 188 145 145 201 201 210 210 276 280 234 240 126 128 159 159
Ca26 188 144 146 201 201 210 210 278 278 232 232 124 128 159 159
140
In yet another aspect, the present invention provides a kit including the SSR molecular marker
primer set or the SSR molecular marker fingerprint code.
In yet another aspect, the present invention provides a use of the SSR molecular marker primer set,
the SSR molecular marker fingerprint code or the kit in the screening or identification of
145 germplasm resources of Cattleya Alliance.
In yet another aspect, the present invention provides a method for the screening of
above-mentioned SSR molecular marker, where the method includes the following steps: using
total DNA of Cattleya Alliance as a template, breaking the template into fragments of 350 bp in
length randomly by an ultrasonic processor, preparing a library by steps such as end repairing,
150 A-Tailing, adding sequencing linker, purification, and PCR amplification, performing preliminary
quantification on the library by Qubit 2.0, diluting the library to 2 ng/L, then detecting insertion
fragments in the library by Agilent 2100, and quantifying an effective concentration of the library
accurately by Q-PCR to ensure the quality of the library; performing sequencing by using Illumina
Hiseq 2500, performing quality test of the raw data by using FASTQC software, removing the
155 linker and low-quality base sequences, then splicing the double-ended reads based on the
overlapping bases using Flash software, screening the sequencing results with MISA, and
retaining sequences with microsatellite marker SSR.
In yet another aspect, the present invention provides a method for the screening of Cattleya Alliance or identification of germplasm resources, which includes the following steps: 160 (1) extracting total DNA of a plant to be detected; (2) performing PCR amplification on SSR molecular marker with the total DNA extracted in step (1) as a template; (3) detecting the PCR amplification product in step (2) by capillary electrophoresis, and collecting data; and 165 (4) performing calculation and analysis on genetic diversity index, clustering and polymorphism information content PIC according to the data obtained in step (3). Specifically, the PCR amplification system in step (2) is as follows: 10 tL of total volume of the reaction system, including 1.2 pL of DNA template (50 ng/tL), 1.0 pL of 10 x Buffer I, 0.1 pL of TAKARA HS Taq enzyme (5 U/pL), 0.6 pLof primers (5 pM), 0.8 pL of 2.5 mM dNTP, 0.5 pL of 170 TP-M13 (5 pM), complementing to 10 pL with deionized water; further specifically, the PCR amplification reaction procedure is as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; 4°C for storage. Further specifically, the primers for PCR amplification system include the sequences shown in 175 SEQ ID NO: 1-SEQ ID NO: 30. In yet another aspect, the present invention provides a use of the SSR molecular marker primer set, the SSR molecular marker fingerprint code or the kit in genetic relationship analysis , location of trait genes and molecular marker-assisted breeding in CattleyaAlliance. Compared with the prior art, the present invention has the positive and beneficial effects that: 180 1. The present invention provides SSR molecular markers of Cattleya Alliance and primer set thereof, and utilizes a TP-M13-SSR technique to analyze the genetic diversity and construct molecular ID of 26 different germplasm resources in Cattleya Alliance, thereby providing a basis for the screening of Cattleya Alliance and identification of germplasm resources. 2. By adopting the SSR molecular markers and primer set thereof for identifying Cattleya Alliance, 185 the Cattleya Alliance germplasm resources may be identified accurately, efficiently and stably, with simple and convenient operation. 3. The SSR molecular markers of Cattleya Alliance disclosed by the present invention may be used for assisted selection breeding to realize early selection at the seedling stage, thus accelerating the breeding process of Cattleya Alliance. 190
Brief Description of the Drawings FIG. 1 is a peak graph of amplification results for SSR molecular marker primer set.
FIG. 2 is a graph of genetic analysis and clustering.
195 Detailed Description of the Invention The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention, but to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following examples and the experimental methods without specific conditions in the examples are usually carried out under 200 conventional conditions; materials, and reagents used in the following examples are commercially available unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. Example 1 Cattleya Alliance germplasm resources 205 26 Cattleya Alliance germplasm resources of different species collected and preserved in orchid resource nursery in Environmental Horticulture Research Institute of Guangdong Academy of Agricultural Sciences were adopted in the present invention, and the specific information is shown in the Table 3 below. All the experimental materials were planted in the greenhouse in Environmental Horticulture Research Institute of Guangdong Academy of Agricultural Sciences, 210 Guangdong Agricultural Modernization High-tech Demonstration Area. Table 3 Basic Information of 26 Germplasm Resources
Number Chinese name Latin Name Properties
Cattleya Vigour Leopard King Intrageneric CaO1 Leopard-king hybrid species
Ca02 Luteola Cattleya luteola Protospecies
Original Ca03 Maxima Cattleya maxima var alba .
variant
Cattleya walkerianavar coerulea Original Ca04 Walkeriana variant variant
Ca05 Leopoldii Cattleya leopoldii Protospecies
Ca06 Digbyana Rhyncholaelia digbyana Protospecies
Ca07 Porphyroglossa Cattleya porphyroglossa Protospecies
Ca08 Glauca Rhyncholaelia glauca Protospecies Intrageneric Ca09 CBC Cattleya BeaufortxCattleya cernua hybrid species
Ca10 Fidelensis Laeliafidelensis Protospecies
Call Lundii Cattleya lundii Protospecies
Cattleya walkeriana var alba Original Ca12 Walkeriana variant
Ca13 Tokyo 1 Cattleya walkeriana Tokyo 1 Bred varieties
Ca14 Nobilior Cattleya nobilior Protospecies
Ca15 Angel Skirt BrassocattleyaAngel Skirt egeec hybrid species
ProcatavolaGolden Peacock g Ca16 Golden peacock hybrid species
Ca17 Seagulls Tiny tim Cattleya Seagulls Tiny tim Bred varieties Intergeneric Ca18 Suzette Chaney BrassolaeliaSuzette Chaney hybrd spc hybrid species Cal9 Lueddemanniana Cattleya lueddemanniana Protospecies
Ca20 Tenebrosa Cattleya tenebrosa Protospecies
Ca21 Trianae Cattleya trianae Protospecies
Ca22 Skinneri Cattleya skinneri Protospecies
Ca23 Forbesii Cattleyaforbesii Protospecies
Ca24 Brevipedunculata Cattleya brevipedunculata Protospecies
Ca25 Intermedia Cattleya intermedia Protospecies
Ca26 Schilleriana Cattleya schilleriana Protospecies
Example 2 Identification of Cattleya Alliance germplasm resources 1. Extraction of total DNA A total DNA of Cattleya plants were extracted according to the operation steps of a novel plant 215 genome DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., with specific steps as follows: (1) tender root tips or tender leaf tissues of the Cattleya plants were fully milled by adding liquid nitrogen, and about 100 mg of fresh tissues of the plants were weighed; (2) to the milled powder, 400 pL of buffer GPS and 10 pL of RNase A (10 mg/mL) were added 220 quickly, followed by quickly mixing evenly by vortex oscillation, the centrifuge tube was placed in a 65°C water bath for 15 min, and the centrifuge tube was inverted during the water bath to mix the samples several times; (3) 100 pL of buffer GPA was added, followed by vortex oscillation for 1 min, and centrifugation at 12000 rpm for 5 min, supernatant was transferred to filtration column CS (filtration column CS 225 was placed in a collection tube), followed by centrifugation at 12000 rpm for 1 min, filtrate was transferred to a new centrifuge tube;
(4) an equal volume of absolute ethyl alcohol was added, followed by mixing evenly, at which time a flocculent precipitate may appear; (5) the solution and the flocculent precipitate obtained in the previous step were transferred to 230 RNase-Free adsorption column CR2 (the adsorption column CR2 was placed in a collection tube), followed by centrifugation at 12000 rpm for 1 min, waste liquid was decanted, and the RNase-Free adsorption column CR2 was placed in the collection tube; (6) to the RNase-Free adsorption column CR2 was added 550 pL of deproteinized solution RD (please check if absolute ethanol had been added before use), followed by centrifugation at 12000 235 rpm for 1 min, waste liquid was decanted, and the RNase-Free adsorption column CR2 was placed in a collection tube; (7) to the RNase-Free adsorption column CR2 was added 700 pL of rinse PW (please check if absolute ethanol had been added before use), followed by centrifugation at 12000 rpm for 1 min, waste liquid was decanted, and the RNase-Free adsorption column CR2 was placed in a collection 240 tube; (8) step (7) was repeated; (9) the RNase-Free adsorption column CR2 was replaced to the collection tube, followed by centrifugation at 12000 rpm for 2 min, the collection tube was discarded, then the RNase-Free adsorption column CR2 was transferred to a new centrifuge tube to air dry at room temperature for 245 5-10 min; (10) 50-100 pL of elution buffer TB was added to the RNase-Free adsorption column CR2, left at room temperature for 3-5 min, followed by centrifugation at 12000 rpm for 2 min, and the solution was collected into a centrifuge tube; (11) 2 pL of DNA was taken for 1.2% agarose gel electrophoresis detection, and 2 pL of DNA was 250 taken for concentration measurement with NanoDrop spectrophotometer. 2. SSR molecular marker and primer set thereof 2.1. 26 qualified Cattleya Alliance germplasm resources DNA samples were evenly mixed and broken into fragments of 350 bp in length randomly by Bioruptor ultrasonic processor, a library was prepared by steps according to NEXTFLEX@ Rapid DNA-Seq Kit (Bioo Scientific, 5144-08) 255 kit such as end trimming, A-Tailing, adding sequencing linker, purification, and PCR amplification, preliminary quantification was performed on the library by Qubit 2.0, the library was diluted to 2 ng/L, then insertion fragments in the library was detected by Agilent 2100, and an effective concentration of the library accurately was quantified by Q-PCR process to ensure the quality of the library. Sequencing was performed by using Illumina Hiseq 2500, first quality test of the raw 260 data was performed by using FASTQC software, the linker and low-quality base sequences were removed, then the double-ended reads were spliced based on the overlapping bases using Flash software, the sequencing results were screened with MISA, and sequences with microsatellite marker were retained as SSR molecular markers. 2.2. Primers were designed from SSR molecular markers, as shown in Table 4 below. 265 Table 4 SSR molecular marker primers Fluorescence SEQ ID NO Primers name SSR Sequence ( 5'-3') Labeling TP-M13
SEQ ID NO:1 1-N1006714-F AAATCACAGTCCAGGCCAAC (AAAT)6 5-FAM SEQ ID NO:2 1-N1006714-R TTAGAATTGTGGACCCAGCC
SEQ ID NO:3 2-N1204804-F CAGGCCAGCATCACAAGATA (AAAT)5 5-FAM SEQ ID NO:4 2-N1204804-R ACTTGGAGTTGTGGACCCAG
SEQ ID NO:5 10-N1060688-F ACCCTTTGCTAGCTGTTGGA (AC)5 5-FAM SEQ ID NO:6 10-N1060688-R TAATCAAAGGGCTACCCGTG
SEQ ID NO:7 11-N1995183-F CGGTCTTGGACATGACTTGA (AC)6 5-FAM SEQ ID NO:8 11-N1995183-R CGGACTTAGCCTCAAGCAAC
SEQ ID NO:9 12-N2010170-F TTTTCCCCAACAACACTTCC (ACA)6 5-FAM SEQ ID NO:10 12-N2010170-R CTTGGTTGGATTTATGGAGGA
SEQ ID NO:11 22-N1388702-F AGGAGGAGGTAACCCCAAAT (ATG)11 5-HEX SEQ ID NO:12 22-N1388702-R TCCATCCAAGCATTGAAACC
SEQ ID NO:13 25-N3457413-F TTGGAGGGAAGAAGAAGGGT (CTT)6 5-HEX SEQ ID NO:14 25-N3457413-R TCACCCTCATATCCTCCTGG
SEQ ID NO:15 36-N1012200-F TGGGTTACTCAGCCGTCTCT (ATTT)5 5-HEX SEQ ID NO:16 36-N1012200-R ACAGTCCAGGCCAACATCTC
SEQ ID NO:17 39-N1014795-F CGATCCACAATTCCTCCATT (TA)6 5-FAM SEQ ID NO:18 39-N1014795-R CCCTGTTGGGACTCAGGTAA
SEQ ID NO:19 47-N1036425-F CAAGGAGAAGCTATTGAGGTTGA (AAG)5 5-FAM SEQ ID NO:20 47-N1036425-R AGTGCACACTTTGCCCTTCT
SEQ ID NO:21 212-NI001184-F CATCCGGTTGTTGTTTACCC (CCT)7 5-FAM SEQ ID NO:22 212-NI001184-R GGCCGACAGTGGTAGGTTTA
SEQ ID NO:23 215-NI003355-F CTGGCTGTAGCCAAAGCAGT (TA)5 5-FAM SEQ ID NO:24 215-NI003355-R GATGCTGAAGGCTTGAAAGG
SEQ ID NO:25 216-N1006768-F ACCAAGCCAAGAAGGGATTT (AG)8 5-FAM SEQ ID NO:26 216-N1006768-R ATTTCGCTCGCCCTAATTCT
SEQ ID NO:27 220-N1009344-F CCTGTCGGCCAAATTTCTTA (TC)5 5-FAM SEQ ID NO:28 220-N1009344-R AGGGAGAATTGGCAAAGGTT
SEQ ID NO:29 221-N10141-F GAACAATATGCAACGGGACA (AT)5 5-FAM SEQ ID NO:30 221-N10141-R CGGTGACTCCATAACGAGTGT
3. Amplification of samples PCR amplification of the samples was performed using the primers in Table 4.
10 pL of total volume of the reaction system included 1.2 pL of DNA template (50 ng/pL), 1.0 pL 270 of 10 x Buffer 1, 0.1 pL of TAKARA HS Taq enzyme (5 U/pL), 0.6 pL of primers (5 pM), 0.8 pL of 2.5 mM dNTP, 0.5 pL of TP-M13 (5 pM), complementing to 10 pL with deionized water; reaction procedure was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; 4°C for storage. 4. Detection 275 Amplification products were detected by 1.2% agarose gel electrophoresis. To each well of a 96-well plate were added 1.0 pL of amplification products and 9 pL of ROX-500 molecular weight internal standard and formamide mixture (volume ratio of 0.5:8.5), followed by denaturation for 3 min at 95°C, then detection was performed by ABI 3730XL detector, sample injection was performed for 10 s at a of voltage 1 kV, and electrophoresis was performed for 30 280 min at 15 kV. The original data file collected by Data Colletion software was imported into GeneMapper 3.2 software for analysis, the position of each peak was compared with the molecular weight internal standard in its lane, and the accurate size of the target DNA fragment was calculated. 3 replicates of the capillary electrophoresis detection were performed independently on each fluorescently labeled locus, and the 3 replicates were averaged and rounded up as the data for 285 the test material at that locus. 5. Data Analysis The genetic diversity index, clustering and polymorphism information content PIC were calculated and analyzed by NTSYS software according to the sorted data. Example 3 Primer amplification results 290 The amplification results of the SSR molecular marker primers disclosed by the present invention were shown in Table 5 below. Since there were many types of SSR molecular marker primers and Cattleya Alliance, 1-N1006714 amplification result peak graph was displayed as an example, as shown in FIG. 1. As can be seen from Table 5 and FIG. 1, the SSR molecular marker primers disclosed by the present invention had the advantages of good amplification effect, high detection 295 rate and capability of amplifying stable DNA bands. Table 5 Primer amplification results SEQ ID NO Primer Name Detection Rate Maximum Number of Peaks SEQ ID NO:1 1-N1006714-F 100.0% 4 SEQ ID NO:2 1-N1006714-R SEQ ID NO:3 2-N1204804-F 100.0% 5 SEQ ID NO:4 2-N1204804-R SEQ ID NO:5 10-N1060688-F 100.0% 8 SEQ ID NO:6 10-N1060688-R SEQ ID NO:7 11-N1995183-F 100.0% 7 SEQ ID NO:8 11-N1995183-R
SEQ ID NO:9 12-N2010170-F 100.0% 6 SEQ ID NO:10 12-N2010170-R
SEQ ID NO:11 22-N1388702-F 100.0% 11 SEQ ID NO:12 22-N1388702-R
SEQ ID NO:13 25-N3457413-F 100.0% 7 SEQ ID NO:14 25-N3457413-R
SEQ ID NO:15 36-N1012200-F 100.0% 7 SEQ ID NO:16 36-N1012200-R
SEQ ID NO:17 39-N1014795-F 100.0% 5 SEQ ID NO:18 39-N1014795-R
SEQ ID NO:19 47-N1036425-F 96.0% 4 SEQ ID NO:20 47-N1036425-R
SEQ ID NO:21 212-N1001184-F 100.0% 5 SEQ ID NO:22 212-N1001184-R
SEQ ID NO:23 215-N1003355-F 100.0% 8 SEQ ID NO:24 215-N1003355-R
SEQ ID NO:25 216-N1006768-F 92.3% 10 SEQ ID NO:26 216-N1006768-R
SEQ ID NO:27 220-N1009344-F 100.0% 5 SEQ ID NO:28 220-N1009344-R
SEQ ID NO:29 221-N10141-F 100.0% 10 SEQ ID NO:30 221-N10141-R
Example 4 Genetic analysis and clustering
The calculation and analysis results of genetic diversity index, clustering and polymorphism
300 information content PIC were shown in Table 6 and FIG. 2 below.
Table 6 Inbreeding Observed Observed Expected Polymorphis Effective Genetic Shannon coefficient in MarkerName number of heterozyg heterozyg m information number of deviation information population ine() ndxI alleles (Na) osity (Ho) osity (He) content (PIC) alleles (Ne) index (D) index (I) ______________ ________ (Fis) ____ _____
1-N1006714 9 0.57692 0.78033 0.74886 4.55219 0.2607 -0.2607 1.7329 2-N1204804 7 0.69231 0.70145 0.66063 3.34948 0.0130 -0.0130 1.4808 10-N1060688 10 0.65385 0.74160 0.70566 3.86997 0.1183 -0.1183 1.6587 11-N1995183 6 0.69231 0.73964 0.70195 3.84091 0.0640 -0.0640 1.5103 12-N2010170 5 0.34615 0.52082 0.45397 2.08688 0.3354 -0.3354 0.9545 22-N1388702 12 0.57692 0.87574 0.86356 8.04762 0.3412 -0.3412 2.2490 25-N3457413 5 0.88462 0.57060 0.48173 2.32881 -0.5503 0.5503 0.9985 36-N1012200 5 0.73077 0.61531 0.53818 2.59949 -0.1876 0.1876 1.1088 39-N1014795 4 0.07692 0.44083 0.41358 1.78836 0.8255 -0.8255 0.8730 47-N1036425 6 0.26923 0.37440 0.35339 1.59847 0.2809 -0.2809 0.8117
212-N1001184 10 0.46154 0.74482 0.72553 3.91884 0.3803 -0.3803 1.7959 215-N1003355 8 0.42308 0.80251 0.77622 5.06367 0.4728 -0.4728 1.7995 216-N1006768 10 0.38462 0.83854 0.82054 6.19355 0.5413 -0.5413 2.0350 220-N1009344 5 0.53846 0.44379 0.41978 1.79787 -0.2133 0.2133 0.9201 221-N10141 10 0.53846 0.79586 0.77339 4.89855 0.3234 -0.3234 1.8651
Example 5 Construction of molecular ID
The molecular ID was constructed according to a diploid standard, the fingerprint data were
305 subjected to data coding according to an SSR detection result (amplification fragments at each
locus were arranged according to the molecular weight, amplification fragments (alleles) labeled
with Arabic numerals 1-9 in ascending order, and alleles exceeding 9 were labeled with capital
English letters A-Z), if a locus was not amplified in a certain variety, it was recorded as 0, and
each locus occupied two positions. Where the sequence of SSR molecular marker of the molecular
310 ID was as follows: 1-N1006714, 2-N1204804, 10-N1060688, 11-N1995183, 12-N2010170, 22-N1388702, 25-N3457413, 36-N1012200, 39-N1014795, 47-N1036425, 212-N1001184, 215-N1003355, 216-N1006768, 220-N1009344, and 221-N10141. The molecularD information was shown in Table 7 below, and the fingerprint spectrum was shown in Table 8 below.
Table 7 Number Chinese name Latin Name Molecular ID information
Ca01 Leopard-king Cattleya Vigour Leopard King 44334936446B243422459936002433
Ca02 Luteola Cattleya luteola 554413331329334422469911254413
Ca03 Maxima Cattleya maxima var. alba 333300363418243422339966001434 Walkeriana Ca04 Cattleva walkeriana var. coerulea 353599333466143422009944774446 variant 'ateaakraaa~orla 339336132094744
Ca05 Leopoldii Cattleya leopoldii 44354936446B453422449945551433
Ca06 Digbyana Rhyncholaelia digbyana 264436353346444413448834253435
Ca07 Porphyroglossa Cattleyaporphyroglossa 453549364416243422444955881433
Ca08 Glauca Rhyncholaelia glauca 243535353344443444445533144446
CattlevaBeaufortx Cattleva Ca09 CBC 3624333333AA244422445766553438 cernua
CalO Fidelensis Laeliafidelensis 661549233344241422442644374426
Call Lundii Cattleya lundii 49363936348824562244578877448A
Cal2 Walkeriana Cattleya walkeriana var. alba 453539463467243422449925584434
Cal3 Tokyo 1 Cattleya walkeriana Tokyo 1 3535AA333467243422149925554446
Ca14 Nobilior Cattleya nobilior 453549445522243422449935471422
Ca15 Angel Skirt BrassocattleyaAngel Skirt 44373346338A243322668855473433
Ca16 Golden peacock ProcatavolaGolden Peacock 183623253322243533448A44364488
Ca17 SeagullsTiny Cattleya Seagulls Tiny tim 362433223333244422446657553499 tim
Cal8 Suzette Chaney BrassolaeliaSuzette Chaney 176623333444243533463944444449
Ca19 Lueddemanniana Cattleya lueddemanniana 353333243326243422469A56774428
Ca20 Tenebrosa Cattleya tenebrosa 333539443412243522241377594499
Ca2l Trianae Cattleya trianae 333333353377243322463856771429
Ca22 Skinneri Cattleya skinneri 333544142355463444443855224547
Ca23 Forbesii Cattleyaforbesii 44334946336C243422444936AA2433
Ca24 Brevipedunculata Cattleya brevipedunculata 353578363312241311452655334488
Ca25 Intermedia Cattleya intermedia 35133936332A242322449935793433
Ca26 Schilleriana Cattleya schilleriana 333339463312242313449944662433
315
Table 8 Fingerprint spectrum 1-Ni 1-Ni 2-Ni 2-Ni 10-N 10-N 11-N 11-N 12-N 12-N 22-N 22-N 25-N 25-N 36-N Numb 0067 0067 2048 2048 1060 1060 1995 1995 2010 2010 1388 1388 3457 3457 1012 er 14 14 04 04 688 688 183 183 170 170 702 702 413 413 200
CaOl 154 154 143 143 123 145 136 142 183 183 231 246 160 169 184
Ca02 156 156 145 145 101 121 136 136 162 180 217 240 163 163 188
Ca03 152 152 143 147 145 145 136 142 180 183 214 238 160 169 184
Ca04 152 156 143 147 145 145 136 136 180 183 231 231 155 169 184
Ca05 154 154 143 147 123 145 136 142 183 183 231 246 169 172 184
Ca06 150 158 145 145 121 129 136 140 180 180 226 231 169 169 188
Ca07 154 156 143 147 123 145 136 142 183 183 214 231 160 169 184
CaO8 150 154 143 147 121 125 136 140 180 180 226 226 169 169 184
Ca09 152 158 141 145 121 121 136 136 180 180 243 243 160 169 188
CalO 158 158 139 147 123 145 134 136 180 180 226 226 160 169 188
Call 154 164 143 149 121 145 136 142 180 183 238 238 160 169 190
Cal2 154 156 143 147 121 145 138 142 180 183 231 234 160 169 184
Cal3 152 156 143 147 147 147 136 136 180 183 231 234 160 169 184
Cal4 154 156 143 147 123 145 138 138 186 186 217 217 160 169 184
Ca15 154 154 143 153 121 121 138 142 180 180 238 243 160 169 184
Cal6 148 162 143 149 117 121 134 140 180 180 217 217 160 169 184
Ca17 152 158 141 145 121 121 134 134 180 180 223 223 160 169 188
Cal8 148 160 149 149 117 121 136 136 180 183 226 226 160 169 184
Ca19 152 156 143 143 121 121 134 138 180 180 217 231 160 169 184
Ca20 152 152 143 147 121 145 138 138 180 183 214 217 160 169 184
Ca2l 152 152 143 143 121 121 136 140 180 180 234 234 160 169 184
Ca22 152 152 143 147 123 123 130 138 174 180 229 229 163 169 184
Ca23 154 154 143 143 123 145 138 142 180 180 231 263 160 169 184
Ca24 152 156 143 147 131 143 136 142 180 180 214 217 160 169 182
Ca25 152 156 139 143 121 145 136 142 180 180 217 243 160 169 184
Ca26 152 152 143 143 121 145 138 142 180 180 214 217 160 169 184
36-N 39-N 39-N 47-N 47-N 212- 212- 215- 215- 216- 216- 220- 220- 221- 221 Numb 1012 1014 1014 1036 1036 N100 N100 N100 N100 N100 N100 N100 N100 NIO NIO er 200 795 795 425 425 1184 1184 3355 3355 6768 6768 8344 8344 41 41
CaOl 188 145 145 201 201 210 210 276 282 - - 124 128 159 159
Ca02 188 145 145 201 207 210 210 264 264 224 230 128 128 155 159
Ca03 188 145 145 198 198 210 210 282 282 - - 122 128 159 161
Ca04 188 145 145 - - 210 210 278 278 234 234 128 128 161 167
Ca05 188 145 145 201 201 210 210 278 280 230 230 122 128 159 159
Ca06 188 144 146 201 201 207 207 276 278 224 230 126 128 159 163
Ca07 188 145 145 201 201 201 210 280 280 236 236 122 128 159 159
CaO8 188 148 148 201 201 203 203 276 276 222 228 128 128 161 167
Ca09 188 145 145 201 201 203 206 282 282 230 230 126 128 159 171
CalO 188 145 145 201 201 195 204 278 278 226 234 128 128 157 167
Call 196 145 145 201 201 203 206 290 290 234 234 128 128 171 175
Cal2 188 145 145 201 201 210 210 274 280 230 236 128 128 159 161
Cal3 188 145 145 192 201 210 210 274 280 230 230 128 128 161 167
Cal4 188 145 145 201 201 210 210 276 280 228 234 122 128 157 157
Ca15 184 145 145 207 207 207 207 280 280 228 234 126 128 159 159
Cal6 190 146 146 201 201 207 214 278 278 226 232 128 128 171 171
Ca17 188 145 145 201 201 204 204 280 284 230 230 126 128 173 173
Ca18 190 146 146 201 207 198 210 278 278 228 228 128 128 161 171
Ca19 188 145 145 201 207 210 214 280 282 234 234 128 128 157 171
Ca20 190 145 145 195 201 189 198 284 284 230 240 128 128 173 173
Ca2l 184 145 145 201 207 198 207 280 282 234 234 122 128 157 171
Ca22 188 148 148 201 201 198 207 280 280 224 224 128 130 161 169
Ca23 188 145 145 201 201 201 210 276 282 244 244 124 128 159 159
Ca24 188 144 144 201 204 195 204 280 280 226 226 128 128 171 171
Ca25 188 145 145 201 201 210 210 276 280 234 240 126 128 159 159
Ca26 188 144 146 201 201 210 210 278 278 232 232 124 128 159 159
Experimental Example 1 Accuracy Detection According to the primer set of SSR molecular markers and and identification method of 320 germplasm resources disclosed by the present invention, 10 samples of 26 Cattleya Alliance were selected for the identification of germplasm resources, and the detection results were shown in Table 9 below. Table 9 Accuracy detection results Cattleya Alliance Number of samples detection results Samples CaOl 10 10 Ca02 10 10 Ca03 10 10 Ca04 10 10 Ca05 10 10 Ca06 10 10 Ca07 10 10 Ca08 10 10 Ca09 10 10 CalO 10 10 Call 10 10 Ca12 10 10 Ca13 10 10 Ca14 10 10 Ca15 10 10 Ca16 10 10 Ca17 10 10
Ca18 10 10 Ca19 10 10 Ca20 10 10 Ca21 10 10 Ca22 10 10 Ca23 10 10 Ca24 10 10 Ca25 10 10 Ca26 10 10
325 As can be seen from Table 9, 26 Cattleya Alliance may be accurately identified by using the SSR
molecular marker primer set and the germplasm identification method, with an accuracy rate of
100%. The SSR molecular marker provided by the present invention may be used for assisted selection
breeding to realize early selection at the seedling stage, thus accelerating the breeding process of
330 the Cattleya Alliance.
The above-described embodiments illustrate only a few embodiments of the present invention,
which are described in greater detail and detail, and are not to be construed as limiting the scope of
the present invention. It should be noted that several variations and modifications may be made by
those skilled in the art without departing from the spirit of the present invention, which fall within
335 the scope of the present invention. Therefore, the patent protection scope of the present invention
should be subject to the appended claims.

Claims (10)

Claims
1. A SSR molecular marker primer set for identifying Cattleya Alliance , characterized in that the SSR molecular marker comprises 1-N1006714, 2-N1204804, 10-N1060688, 11-N1995183, 12-N2010170, 22-N1388702, 25-N3457413, 36-N1012200, 39-N1014795, 47-N1036425, 212-N1001184, 215-N1003355, 216-N1006768, 220-N1009344, and 221-N10141; the primer set comprises: (1) primers for amplifying SSR molecular marker1-N1006714:
SEQ ID NO:1 : 1-N1006714-F : 5'-AAATCACAGTCCAGGCCAAC-3'
SEQ ID NO:2: 1-N1006714-R: 5'-TTAGAATTGTGGACCCAGCC-3';
(2) primers for amplifying SSR molecular marker 2-N204804:
SEQ ID NO:3 : 2-N1204804-F : 5'-CAGGCCAGCATCACAAGATA-3'
SEQ ID NO:4 :2-N204804-R: 5'-ACTTGGAGTTGTGGACCCAG-3';
(3) primers for amplifying SSR molecular marker 10-N1060688:
SEQ ID NO:5 : 10-N1060688-F : 5'-ACCCTTTGCTAGCTGTTGGA-3'
SEQ ID NO:6: 10-N1060688-R: 5'-TAATCAAAGGGCTACCCGTG-3'
(4) primers for amplifying SSR molecular marker11-N1995183:
SEQIDNO:7: 11-N1995183-F: 5'-CGGTCTTGGACATGACTTGA-3'
SEQIDNO:8: 11-N1995183-R: 5'-CGGACTTAGCCTCAAGCAAC-3';
(5) primers for amplifying SSR molecular marker 12-N2010170:
SEQ ID NO:9 : 12-N2010170-F : 5'-TTTTCCCCAACAACACTTCC-3'
SEQ ID NO:10 : 12-N2010170-R : 5'-CTTGGTTGGATTTATGGAGGA-3';
(6) primers for amplifying SSR molecular marker 22-N1388702:
SEQIDNO:l :22-N388702-F: 5'-AGGAGGAGGTAACCCCAAAT-3'
SEQIDNO:12:22-N388702-R: 5'-TCCATCCAAGCATTGAAACC-3';
(7) primers for amplifying SSR molecular marker 25-N3457413:
SEQIDNO:13 :25-N3457413-F :5'-TTGGAGGGAAGAAGAAGGGT-3'
SEQ ID NO:14 : 25-N3457413-R : 5'-TCACCCTCATATCCTCCTGG-3';
(8) primers for amplifying SSR molecular marker 36-N1012200:
SEQIDNO:15 :36-N1012200-F: 5'-TGGGTTACTCAGCCGTCTCT-3'
SEQ ID NO:16 : 36-N1012200-R : 5'-ACAGTCCAGGCCAACATCTC-3';
(9) primers for amplifying SSR molecular marker 39-N1014795:
SEQIDNO:17:39-N1014795-F: 5'-CGATCCACAATTCCTCCATT-3'
SEQ ID NO:18 : 39-N1014795-R: 5'-CCCTGTTGGGACTCAGGTAA-3';
(10) primers for amplifying SSR molecular marker 47-N1036425:
SEQ ID NO:19: 47-N1036425-F :5'-CAAGGAGAAGCTATTGAGGTTGA-3'
SEQ ID NO:20 : 47-N1036425-R :5'-AGTGCACACTTTGCCCTTCT-3';
(11) primers for amplifying SSR molecular marker 212-N1001184:
SEQ ID NO:21 :212-N1001184-F :5'-CATCCGGTTGTTGTTTACCC-3'
SEQ ID NO:22 :212-N1001184-R :5'-GGCCGACAGTGGTAGGTTTA-3';
(12) primers for amplifying SSR molecular marker 215-N1003355:
SEQ ID NO:23 : 215-N1003355-F : 5'-CTGGCTGTAGCCAAAGCAGT-3'
SEQIDNO:24:215-N1003355-R: 5'-GATGCTGAAGGCTTGAAAGG-3';
(13) primers for amplifying SSR molecular marker 216-N1006768:
SEQ ID NO:25 : 216-N1006768-F : 5'-ACCAAGCCAAGAAGGGATTT-3'
SEQIDNO:26:216-N1006768-R: 5'-ATTTCGCTCGCCCTAATTCT-3';
(14) primers for amplifying SSR molecular marker 220-N1009344:
SEQ ID NO:27 : 220-N1009344-F : 5'-CCTGTCGGCCAAATTTCTTA-3'
SEQIDNO:28:220-N1009344-R: 5'-AGGGAGAATTGGCAAAGGTT-3';
(15) primers for amplifying SSR molecular marker 221-N10141:
SEQ ID NO:29: 221-N10141-F : 5'-GAACAATATGCAACGGGACA-3'
SEQ ID NO:30: 221-N10141-R: 5'-CGGTGACTCCATAACGAGTGT-3'.
2. A SSR molecular marker fingerprint code for identifying Cattleya Alliance, characterized in that the fingerprint code comprises a fingerprint spectrum and a molecular ID, wherein the
sequence of SSR molecular marker of the molecular ID is as follows: 1-N1006714, 2-N1204804,
-N1060688, 11-N1995183, 12-N2010170, 22-N1388702, 25-N3457413, 36-N1012200,
39-N1014795, 47-N1036425, 212-N1001184, 215-N1003355, 216-N1006768, 220-N1009344,
221-N10141.
3. The fingerprint code according to claim 2, characterized in that the molecular ID is as follows:
Number Chinese name Latin Name Molecular ID information
CaO1 Leopard-king Cattleya Vigour Leopard 44334936446B243422459936002433 King Ca02 Luteola Cattleya luteola 554413331329334422469911254413 Ca03 Maxima Cattleya maxima var alba 333300363418243422339966001434
Ca04 Walkeriana Cattleya walkeriana var 353599333466143422009944774446 variant coerulea Ca05 Leopoldii Cattleya leopoldii 44354936446B453422449945551433 Ca06 Digbyana Rhyncholaelia digbyana 264436353346444413448834253435 Ca07 Porphyroglossa Cattleya porphyroglossa 453549364416243422444955881433 Ca08 Glauca Rhyncholaelia glauca 243535353344443444445533144446 Cattleva Ca09 CBC ' 3624333333AA244422445766553438 BeaufortxCattleya cernua CalO Fidelensis Laeliafidelensis 661549233344241422442644374426 Call Lundii Cattleya lundii 49363936348824562244578877448A
Cal2 Walkeriana Cattleya walkeriana var 453539463467243422449925584434 alba Cattlevawalkeriana Cal3 Tokyo 1 3535AA333467243422149925554446 Tokyo 1 Cal4 Nobilior Cattleya nobilior 453549445522243422449935471422
Cal5 Angel Skirt BrassocattleyaAngel 44373346338A243322668855473433 Skirt ProcatavolaGolden Cal6 Golden peacock Peacock 183623253322243533448A44364488
Cal7 Seagulls Tiny Cattleya Seagulls Tiny 362433223333244422446657553499 tim tim Brassolaelia Suzette Cal8 Suzette Chaney Chaney 176623333444243533463944444449
Cal9 Lueddemanniana Cattleya lueddemanniana 353333243326243422469A56774428 Ca20 Tenebrosa Cattleya tenebrosa 333539443412243522241377594499 Ca21 Trianae Cattleya trianae 333333353377243322463856771429 Ca22 Skinneri Cattleya skinneri 333544142355463444443855224547 44334946336C243422444936AA243 Ca23 Forbesii Cattleyaforbesii 3 Brevipedunculat Cattleva Ca24 ' 353578363312241311452655334488 a brevipedunculata Ca25 Intermedia Cattleya intermedia 35133936332A242322449935793433 Ca26 Schilleriana Cattleya schilleriana 333339463312242313449944662433
4. The fingerprint code according to claim 3, characterized in that the fingerprint spectrum is as follows: 1-N 1-N 2-N 2-N 10- 10- 11- 11- 12- 12- 22- 22- 25- 25- 36 Num 100 100 120 120 N10 N10 N19 N19 N20 N20 N13 N13 N34 N34 N10 ber 671 671 480 480 606 606 951 951 101 101 887 887 574 574 122 4 4 4 4 88 88 83 83 70 70 02 02 13 13 00
CaOl 154 154 143 143 123 145 136 142 183 183 231 246 160 169 184
Ca02 156 156 145 145 101 121 136 136 162 180 217 240 163 163 188
Ca03 152 152 143 147 145 145 136 142 180 183 214 238 160 169 184
CaO4 152 156 143 147 145 145 136 136 180 183 231 231 155 169 184
Ca05 154 154 143 147 123 145 136 142 183 183 231 246 169 172 184
Ca06 150 158 145 145 121 129 136 140 180 180 226 231 169 169 188
Ca07 154 156 143 147 123 145 136 142 183 183 214 231 160 169 184
Ca08 150 154 143 147 121 125 136 140 180 180 226 226 169 169 184
Ca09 152 158 141 145 121 121 136 136 180 180 243 243 160 169 188
CalO 158 158 139 147 123 145 134 136 180 180 226 226 160 169 188
Call 154 164 143 149 121 145 136 142 180 183 238 238 160 169 190
Ca12 154 156 143 147 121 145 138 142 180 183 231 234 160 169 184
Ca13 152 156 143 147 147 147 136 136 180 183 231 234 160 169 184
Ca14 154 156 143 147 123 145 138 138 186 186 217 217 160 169 184
Ca15 154 154 143 153 121 121 138 142 180 180 238 243 160 169 184
Ca16 148 162 143 149 117 121 134 140 180 180 217 217 160 169 184
Ca17 152 158 141 145 121 121 134 134 180 180 223 223 160 169 188
Cal8 148 160 149 149 117 121 136 136 180 183 226 226 160 169 184
Ca19 152 156 143 143 121 121 134 138 180 180 217 231 160 169 184
Ca20 152 152 143 147 121 145 138 138 180 183 214 217 160 169 184
Ca2l 152 152 143 143 121 121 136 140 180 180 234 234 160 169 184
Ca22 152 152 143 147 123 123 130 138 174 180 229 229 163 169 184
Ca23 154 154 143 143 123 145 138 142 180 180 231 263 160 169 184
Ca24 152 156 143 147 131 143 136 142 180 180 214 217 160 169 182
Ca25 152 156 139 143 121 145 136 142 180 180 217 243 160 169 184
Ca26 152 152 143 143 121 145 138 142 180 180 214 217 160 169 184
36- 39- 39- 47- 47- 212- 212- 215- 215- 216- 216- 220- 220 221- 221 Num N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 N10 ber 122 147 147 364 364 011 011 033 033 067 067 083 083 141 141 00 95 95 25 25 84 84 55 55 68 68 44 44
CaOl 188 145 145 201 201 210 210 276 282 - - 124 128 159 159
Ca02 188 145 145 201 207 210 210 264 264 224 230 128 128 155 159
Ca03 188 145 145 198 198 210 210 282 282 - - 122 128 159 161
CaO4 188 145 145 - - 210 210 278 278 234 234 128 128 161 167
Ca05 188 145 145 201 201 210 210 278 280 230 230 122 128 159 159
Ca06 188 144 146 201 201 207 207 276 278 224 230 126 128 159 163
Ca07 188 145 145 201 201 201 210 280 280 236 236 122 128 159 159
Ca08 188 148 148 201 201 203 203 276 276 222 228 128 128 161 167
Ca09 188 145 145 201 201 203 206 282 282 230 230 126 128 159 171
CalO 188 145 145 201 201 195 204 278 278 226 234 128 128 157 167
Call 196 145 145 201 201 203 206 290 290 234 234 128 128 171 175
Ca12 188 145 145 201 201 210 210 274 280 230 236 128 128 159 161
Ca13 188 145 145 192 201 210 210 274 280 230 230 128 128 161 167
Ca14 188 145 145 201 201 210 210 276 280 228 234 122 128 157 157
Ca15 184 145 145 207 207 207 207 280 280 228 234 126 128 159 159
Ca16 190 146 146 201 201 207 214 278 278 226 232 128 128 171 171
Ca17 188 145 145 201 201 204 204 280 284 230 230 126 128 173 173
Ca18 190 146 146 201 207 198 210 278 278 228 228 128 128 161 171
Ca19 188 145 145 201 207 210 214 280 282 234 234 128 128 157 171
Ca20 190 145 145 195 201 189 198 284 284 230 240 128 128 173 173
Ca2l 184 145 145 201 207 198 207 280 282 234 234 122 128 157 171
Ca22 188 148 148 201 201 198 207 280 280 224 224 128 130 161 169
Ca23 188 145 145 201 201 201 210 276 282 244 244 124 128 159 159
Ca24 188 144 144 201 204 195 204 280 280 226 226 128 128 171 171
Ca25 188 145 145 201 201 210 210 276 280 234 240 126 128 159 159
Ca26 188 144 146 201 201 210 210 278 278 232 232 124 128 159 159
5. A kit for the screening of Cattleya Alliance or identification of germplasm resources,
characterized in that the kit comprises the SSR molecular marker primer set according to claim 1
or the SSR molecular marker fingerprint code according to claim 2.
6. Use of the SSR molecular marker primer set according to claim 1, the SSR molecular marker
fingerprint code according to claim 2 or the kit according to claim 5 in the screening of Cattleya
Alliance or identification of germplasm resources.
7. A method for the screening of SSR molecular marker according to claim 1, characterized in that
the method comprises the following steps: using total DNA of Cattleya Alliance as a template,
breaking the template into fragments of about 350 bp in length randomly by an ultrasonic
processor, preparing a library by steps such as end trimming, A-Tailing, adding sequencing linker,
purification, and PCR amplification, performing preliminary quantification on the library by Qubit
2.0, diluting the library to 2 ng/L, then detecting insertion fragments in the library by Agilent 2100,
and quantifying an effective concentration of the library accurately by Q-PCR to ensure the
quality of the library; performing sequencing by using Illumina Hiseq 2500, performing quality
test of the raw data by using FASTQC software, removing the linker and low-quality base
sequences, then splicing the double-ended reads based on the overlapping bases using Flash
software, screening the sequencing results with MISA, and retaining sequences with microsatellite
marker SSR.
8. A method for the screening of Cattleya Alliance or identification of germplasm resources,
characterized in that the method comprises the following steps:
(1) extracting total DNA of a plant to be detected; (2) performing PCR amplification on SSR molecular marker with the total DNA extracted in step (1) as a template; (3) detecting the PCR amplification product in step (2) by capillary electrophoresis, and collecting data; and (4) performing calculation and analysis on genetic diversity index, clustering and polymorphism information content PIC according to the data obtained in step (3).
9. The method according to claim 8, characterized in that the PCR amplification system in step (2) is as follows: 10 tL of total volume of the reaction system, comprising 1.2 tL of DNA template
, 1.0 pL of 10 x Buffer I, 0.1 pL of TAKARA HS Taq enzyme , 0.6 pL of primers, 0.8 pL of 2.5 mM dNTP, 0.5 pL of TP-M13, complementing to 10 pL with deionized water; the PCR amplification reaction procedure is as follows: 95°C for 5 min; 95°C for 30 s, 60°C for s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; 4°C for storage; the primers for PCR amplification system comprise the sequences shown in SEQ ID NO: 1-SEQ ID NO: 30.
10. Use of the SSR molecular marker primer set according to claim 1, the SSR molecular marker fingerprint code according to claim 2 or the kit according to claim 5 in analysis of genetic relationship , location of trait genes and molecular marker-assisted breeding in Cattleya Alliance.
Fig.2 Fig.1
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