AU2021101596A4 - SSR molecular marker primer set for identifying Rhynchostylis and use thereof - Google Patents

Abstract

The present invention provides a SSR molecular marker primer set and a fingerprint code for identifying Rhynchostylis and use thereof in the identification of Rhynchostylis germplasm resources, belonging to the technical field of molecular biology. The present invention provides 13 SSR molecular markers of Rhynchostylis and amplification primer sets thereof, and utilizes TP-M13-SSR technique to analyze the genetic diversity and construct molecular identity cards of 10 different germplasm resources in 3 species of Rhynchostylis, thereby identifying the Rhynchostylis germplasm resources accurately, efficiently and stably, and laying a foundation for the identification germplasm resources, genetic relationship analysis, location of trait associated genes, and molecular marker-assisted breeding in Rhynchostylis, and molecular research related to Rhynchostylis. RO1 R05 R06 R03 R02 R04 R07 RIO R08 R09 0.50 0.60 0.71 0.82 0.92 Coefficient 80 120 160 200 240 280 21000{ 100.18 10686 LZ--Nl1 14-R6 F1.fs LZ-5-NI0 1 14-R6 5-Ni1114 U 80 120 160 200 240 280 18000 12000 8000 0 110 0.-27 103.74 106.831 L--Ni O114-RO 7 1i a Z--N1 1-R I-N100114 A 80 120 160 200 240 280 24000{ 16000} 8000t 975 (Sample File Sample Name Panel OS so LZ-5-NlDO1614-ROB A02.fsa LZ-5-Nl001614-R08 5-NlOO1614 A B0 120 160 200 240 280 24000 16000 [9761 22

Description

80 120 160 200 240 280 21000{

100.18 10686

LZ--Nl1 14-R6 F1.fs LZ-5-NI0 1 14-R6 5-Ni1114 U

80 120 160 200 240 280 18000

12000

8000

0 110 0.-27 103.74 106.831

L--Ni O114-RO 7 1i a Z--N1 1-R I-N100114 A

80 120 160 200 240 280

24000{ 16000}

8000t

975

(Sample File Sample Name Panel OS so LZ-5-NlDO1614-ROB A02.fsa LZ-5-Nl001614-R08 5-NlOO1614 A B0 120 160 200 240 280

24000

16000

[9761

Description

SSR molecular marker primer set for identifying Rhynchostylis 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 Rhynchostylis and use thereof in the identification of Rhynchostylis germplasm resources.

Background Art The genus Rhynchostylis, which belongs to the Orchidaceae family, Epidendroideae subfamily, Vandeae tribe, and Aeridinae subtribe, is a kind of epiphytic orchid with uniaxial growth. Rhynchostylis mostly distributed in Yunnan province, Guizhou province and Hainan province in China, as well as India, Sri Lanka and Southeast Asia countries. The plants of Rhynchostylis are compact in type, dense in flower, bright in color, strong in fragrance for most of the plantsand simple in cultivation managementhave high ornamental value and development prospects. Since the middle of the 19th century, there are registration of the interspecific hybrids of Rhynchostylis, and intergeneric hybrids with closely related genera in the Royal Horticultural Society. As of November 2020, 381 F1 hybrid combinations of native species have been registered. In addition to the traditional cross breeding, there are few studies on Rhynchostylis. At present, the researches mainly focus on tissue culture and rapid propagation, cultivation management, and distribution and identification of wild resources. Mo Rao et al. (2009) found that Rhynchostylis gigantea(Rhy. gigantea) had a karyotype formula of 2n=2x=36m+2sm (Mo Rao et al., A Karyological Study of Fourteen Species in Eleven Genera of the Orchidaceae. Acta Botanica Yunnanica, 2009, 31(6):504-508). Yan Ping et al. (2014) used SRAP marker technology to analyze the genetic diversity of 144 wild resources of Rhy. gigantea from 22 populations in Hainan Island (Yan Ping et al., Genetic Diversity Analysis of Rhynchostylis gigantea (Lindl.) Ridl.'s Germplasm Resources Based on SRAP Markers, Chinese Journal of Tropical Crops, 2014, 35(9):1677-1682). Junita Kurniawati et al. (2019) used 2 RAPD primers to analyze the genetic similarity of 9 different native species of Orchidaceae including Rhy. retusa, and found that the intraspecific similarity was much higher than the interspecific similarity (Junita Kurniawati,et al.Molecular Identification of Several Orchid Species Based on OPA10 and OPA18 RAPD Marker. Journal of Physics:Conference Series, 2019, 1397(1):012042). Atsushi Matsuba et al. (2015) constructed an artificial bacterial chromosome of Rhy.coelestis containing 10,600 BAC clones (Atsushi Matsuba,et al.Molecular cytogenetic use of BAC clones in Neofinetiafalcate and Rhynchostylis coelestis.Nucleus,2015,58(3):207-210). Subhas Chandra Roy et al. (2017) used ddRAD technology to perform reduced-representation genome sequencing and genetic diversity analysis on 4 native species of orchids including Rhy. retusa (Subhas Chandra Roy, et al. Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes. Physiology and Molecular Biology of Plants,2017,23(1):169-183). 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 Rhynchostylis to develop new SSR molecular markers and primer set thereof, and using TP-M13-SSR technique for genetic diversity analysis and molecular identity card construction will play an important role in the identification of germplasm resources, genetic relationship analysis, location of trait genes, and molecular marker-assisted breeding in Rhynchostylis, and molecular research related to Rhynchostylis.

Summary of the Invention Aiming at the defects, the present invention provides a SSR molecular marker primer set and a fingerprint code for identifying Rhynchostylis and use thereof in the identification of Rhynchostylis germplasm resources. The present invention provides a SSR primer set for identifying Rhynchostylis using reduced-representation genome sequencing technique, and utilizes TP-M13-SSR technique to analyze the genetic diversity and construct molecular identity cards of different germplasm resources from 3 species of Rhynchostylis. The present invention makes the identification of Rhynchostylis germplasm resources more accurately, efficiently and stably. The present invention also lays a foundation for the identification of germplasm resources, analysis of genetic relationship, molecular-marker-assisted breeding and related molecular researches to Rhynchostylis. 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 Rhynchostylis, where the SSR molecular marker includes: 5-N1001614, 7-N1001895, 14-N1007590, 25-N101022, 28-N1012378, 37-N3601419, 45-N1438823, 82-N100023, 88-N1001036, 89-N1001051, 91-N1001523, 96-N1007386 and 97-N844635. Specifically, the primer set comprises: (1) primers for amplifying SSR molecular marker 5-N1001614:

SEQIDNO:1 :5-N1001614-F: 5'-TTTGGTGGAGGATGGAGAAG-3'

SEQ ID NO:2: 5-N1001614-R: 5'-GCTTCCTTGAGGTGAAGAGC-3';

(2) primers for amplifying SSR molecular marker 7-N1001895:

SEQIDNO:3 :7-N1001895-F: 5'-ATCGCCATTTTTGTGCTCTT-3'

SEQ ID NO:4 :7-N1001895-R: 5'-ATTGAAATGATGGCTTTCGC-3';

(3) primers for amplifying SSR molecular marker 14-N1007590:

SEQIDNO:5: 14-N1007590-F: 5'-TGACATGGAAGTTATGGCATTC-3'

SEQ ID NO:6: 14-N1007590-R: 5'-TGTCGCAATTCTATGTCAGGA-3';

(4) primers for amplifying SSR molecular marker 25-N101022:

SEQ ID NO:7: 25-N101022-F : 5'-TGGACCACCTATCTCAAGGC-3'

SEQIDNO:8:25-N101022-R: 5'-ACGACATGCTCATGATTCCA-3';

(5) primers for amplifying SSR molecular marker 28-N1012378:

SEQ ID NO:9: 28-N1012378-F : 5'-GGAAGAAGGAGGAGGTGAGG-3'

SEQIDNO:10:28-N1012378-R:5'-TCCTAGAGAAAGAGCTTGGAGC -3';

(6) primers for amplifying SSR molecular marker 37-N3601419:

SEQ ID NO: I1: 37-N3601419-F :5'-AATGGAAAATCTGGCAGCAC-3'

SEQ ID NO:12 :37-N3601419-R :5'-TCTCCATCACTTCAATGGCA-3';

(7) primers for amplifying SSR molecular marker 45-N438823:

SEQIDNO:13 :45-N438823-F :5'-ATTTGGCTCAAATCCACCAA -3'

SEQIDNO:14:45-N438823-R: 5'-ATACCTGAAAGCATGGTGCC -3';

(8) primers for amplifying SSR molecular marker 82-N100023:

SEQ ID NO:15 82-N100023-F : 5'-AGGGTGGGAAGGGAGATAAA-3'

SEQ ID NO:16 :82-N100023-R: 5'-CTCTGCCTTCTCCATCTCTCA-3';

(9) primers for amplifying SSR molecular marker 88-N1001036:

SEQ ID NO:17: 88-N1001036-F: 5'-AGGGAGGGAGTGAGAGAGGA-3'

SEQ ID NO:18 : 88-N1001036-R: 5'-TCCCGCTTTCTATCGCTATC-3';

(10) primers for amplifying SSR molecular marker 89-N100105:

SEQIDNO:19:89-N100105-F: 5'-TTTCCTGCTCTCCATTCCTG-3'

SEQ ID NO:20 : 89-N100105-R: 5'-TGTCATCTTCTCACACTCCCA-3';

(11) primers for amplifying SSR molecular marker 91-N1001523:

SEQIDNO:21 :91-N1001523-F: 5'-TGATGGAATGAAGTTGAGGGA-3'

SEQ ID NO:22 : 91-N1001523-R: 5'-GGCACATATGCTTTGTGGTG-3';

(12) primers for amplifying SSR molecular marker 96-N1007386:

SEQ ID NO:23 :96-N1007386-F : 5'-AAGTCAGCCACTATGCCAGG-3'

SEQ ID NO:24 :96-N1007386-R: 5'-CAAGCGACATTTGCGTCTAA-3';

(13) primers for amplifying SSR molecular marker 97-N1844635:

SEQ ID NO:25 :97-N1844635-F : 5'-GTGGATATACCGCTTCGTCG-3'

SEQ ID NO:26 :97-N1844635-R: 5'-GAAGAAAGTGGCTGCGTCTC-3'.

In another aspect, the present invention provides a fingerprint code of SSR molecular marker for identifying Rhynchostylis, where the fingerprint code includes a fingerprint spectrum and a molecular identity card, the sequence of SSR molecular marker of the molecular identity card is as follows: 5-N1001614, 7-N1001895, 14-N1007590, 25-N101022, 28-N1012378, 37-N3601419, -N1438823, 82-N100023, 88-N1001036, 89-N1001051, 91-N1001523, 96-N1007386, and 97-N1844635. Specifically, the molecular identity cards are shown in Table 1 below. Table 1

Latin Name Molecular identity card Number information RO1 Rhy. gigantea 'Shen's Suns' 23334514342344121112448833 R02 Rhy. gigantea 24331414242344121112440033 R03 Rhy. gigantea 14332414342344122113442613

R04 Rhy. gigantea 'JH#11' 24004414352344121112444533 R05 Rhy. gigantean var petoniana 24334444242344121111445733 R06 Rhy. gigantea f. rubra 23331444242344121111441133 R07 Rhy. coelestis 11244414121313121611443323 R08 Rhy. coelestis f. alba 11334422121215113611131322 R09 Rhy. coelestis 11123422121215114622120022 RIO Rhy. retusa 22442433332224125111443333 Further specifically, the molecular identity card 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 (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 Locus RO1 R02 R03 R04 R05 R06 R07 R08 R09 RIO 5-N1001614 100 100 98 100 100 100 98 98 98 100 5-N1001614 103 106 106 106 106 103 98 98 98 100 7-N1001895 182 182 182 - 182 182 172 182 169 192 7-N1001895 182 182 182 - 182 182 192 182 172 192 14-N1007590 124 115 117 124 124 115 124 124 120 117 14-N1007590 128 124 124 124 124 124 124 124 124 124 25-N101022 128 128 128 128 133 133 128 131 131 132 25-N101022 133 133 133 133 133 133 133 131 131 132 28-N1012378 174 171 174 174 171 171 167 167 167 174 28-N1012378 176 176 176 179 176 176 171 171 171 174 37-N3601419 187 187 187 187 187 187 181 181 181 187 37-N3601419 190 190 190 190 190 190 190 187 187 187 45-N1438823 193 193 193 193 193 193 163 163 163 182 45-N1438823 193 193 193 193 193 193 184 194 194 193 82-N100023 167 167 167 167 167 167 167 167 167 167 82-N100023 171 171 171 171 171 171 171 167 167 171 88-N1001036 143 143 135 143 143 143 143 153 157 137 88-N1001036 143 143 143 143 143 143 161 161 161 143 89-NIO01051 97 97 97 97 97 97 97 97 99 97 89-NIO01051 99 99 101 99 97 97 97 97 99 97 91-N1001523 136 136 136 136 136 136 136 116 116 136 91-N1001523 136 136 136 136 136 136 136 134 126 136

96-N1007386 150 - 111 116 124 92 113 92 - 113 96-N1007386 150 - 127 124 130 92 113 113 - 113 97-N1844635 128 128 122 128 128 128 124 124 124 128 97-N1844635 128 128 128 128 128 128 128 124 124 128

In yet another aspect, the present invention provides a kit including the SSR molecular marker amplification primer set or the SSR molecular marker fingerprint code. In yet another aspect, the present invention provides a use of the SSR molecular marker amplification primer set, the SSR molecular marker fingerprint code or the kit in the screening or identification of germplasm resources of Rhynchostylis. 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 the ten Rhynchostylis plants 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, 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. In yet another aspect, the present invention provides a method for the screening of Rhynchostylis or identification of germplasm resources, which includes 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;(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/pL), 1.0 pL of 10 x Buffer I, 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. 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 SEQ ID NO: 1-SEQ ID NO: 26. In yet another aspect, the present invention provides a use of the SSR molecular marker amplification primer set, the SSR molecular marker fingerprint code or the kit in genetic relationship analysis, location of trait associated genes and molecular marker-assisted breeding in Rhynchostylis. Compared with the prior art, the present invention has the positive and beneficial effects that: 1. The present invention provides SSR molecular markers of Rhynchostylis and primer set thereof, and utilizes a TP-M13-SSR technique to analyze the genetic diversity and construct molecular identity cards of 10 different germplasm resources in 3 species of Rhynchostylis, thereby providing a basis for the screening of Rhynchostylis and identification of germplasm resources. 2. By adopting the Rhynchostylis SSR molecular markers and primer set thereof for identifying Rhynchostylis, the Rhynchostylis germplasm resources may be identified accurately, efficiently and stably, with simple and convenient operation. 3. The SSR molecular markers of Rhynchostylis 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 Rhynchostylis.

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.

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 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 Rhynchostylis germplasm resources At present, 5 native species of Rhynchostylis had been found and identified, of which 3 native species were discovered earlier and distributed widely, had a high hybridization utilization rate, and the hybrid offspring among the 3 species had been registered; of which 2 native species were discovered later and distributed narrowly, without breeding utilization and targeted research. In addition to interspecific hybridization, Rhynchostylis had been cross-fertilized with different germplasm resources of 20 other genera such as Vanda, Phalaenopsis,Aerides, Renanthera, and Holcoglossum in Orchidaceaeto cultivate new germplasm.

Rhynchostylis germplasm resources in 3 different native species collected and preserved in Environmental Horticulture Research Institute of Guangdong Academy of Agricultural Sciences were adopted in the present invention, and the specific information was 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, Guangdong Agricultural Modernization High-tech Demonstration Area, where the root tips were collected in May 2020 and quickly frozen with liquid nitrogen for 15 minutes and then placed in an ultra-low temperature refrigerator for later use. Table 3 Basic Information of 10 Germplasm Resources in 3 species Floral Color of Number Latin Name Floral color Source scent labellum Yellow-green with RO1 Rhy. gigantea 'Shen's Suns' Strong some purplish red rpi Taia red China spots White with some R02 Rhy. gigantea Strong medium-size purplish Purplish Taiwan, red China red spots White with a little of Purplish Taiwan, R03 Rhy. gigantea Strong small-size purplish red Cin red China red spots Purplish Taiwan, R04 Rhy. gigantea 'JH#1l' Strong Pure light orange red China Rhygiganteanvar. Taiwan, R05 Strong Pure white White petoniana China

Rhy. gigantea f. rubra Strong Pure purplish red Purplish Taiwan, R06 red China Bluish purple tips Bluish R07 Rhy. coelestis Strong White, edge with a Thailand little of bluish purple R08 Rhy. coelestis f. alba Strong Pure white White Thailand White, edge with a R09 Rhy. coelestis Strong little of purplish rpi Thailand red purple RIO Rhy. retusa Faint White with a little of Purplish Myanmar small-size purplish red red spots Example 2 Identification of Rhynchostylis germplasm resources 1. Extraction of total DNA A total DNA of Rhynchostylis plants were extracted according to the operation steps of a novel plant 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 Rhynchostylis 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 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 for1 min, and centrifugation at 12000 rpm for 5 min, supernatant was transferred to filtration column CS (filtration column CS 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 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 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 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 -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 taken for concentration measurement with NanoDrop spectrophotometer. 2. SSR molecular marker and primer set thereof 2.1. 10 qualified Rhynchostylis DNA samples of germplasm resources 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) 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 inserted fragments in the library was detected by Agilent 2100, and an effective concentration of the library was accurately 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 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. Table 4 SSR molecular marker primers Fluorescent SEQ ID NO Primer name SSR Sequence ( 5'-3') label TP-M13

SEQ ID NO:1 5-N1001614-F TTTGGTGGAGGATGGAGAAG (GAA)7 5-FAM SEQ ID NO:2 5-N1001614-R GCTTCCTTGAGGTGAAGAGC

SEQ ID NO:3 7-N1001895-F ATCGCCATTTTTGTGCTCTT (TCT)8 5-FAM SEQ ID NO:4 7-N1001895-R ATTGAAATGATGGCTTTCGC

SEQ ID NO:5 14-N1007590-F TGACATGGAAGTTATGGCATTC (CT)5 5-FAM SEQ ID NO:6 14-N1007590-R TGTCGCAATTCTATGTCAGGA

SEQ ID NO:7 25-N101022-F TGGACCACCTATCTCAAGGC (ATT)5 5-FAM SEQ ID NO:8 25-N101022-R ACGACATGCTCATGATTCCA

SEQ ID NO:9 28-N1012378-F GGAAGAAGGAGGAGGTGAGG (AAG)7 5-FAM SEQ ID NO:10 28-N1012378-R TCCTAGAGAAAGAGCTTGGAGC

SEQ ID NO:11 37-N3601419-F AATGGAAAATCTGGCAGCAC (AAG)8 5-HEX SEQ ID NO:12 37-N3601419-R TCTCCATCACTTCAATGGCA

SEQ ID NO:13 45-N1438823-F ATTTGGCTCAAATCCACCAA (GAA)5 5-HEX SEQ ID NO:14 45-N1438823-R ATACCTGAAAGCATGGTGCC

SEQ ID NO:15 82-N100023-F (AG)6 AGGGTGGGAAGGGAGATAAA 5-FAM

SEQ ID NO:16 82-N100023-R CTCTGCCTTCTCCATCTCTCA

SEQ ID NO:17 88-N1001036-F AGGGAGGGAGTGAGAGAGGA (AG)5 5-FAM SEQ ID NO:18 88-N1001036-R TCCCGCTTTCTATCGCTATC

SEQ ID NO:19 89-N100105-F TTTCCTGCTCTCCATTCCTG (TA)5 5-FAM SEQ ID NO:20 89-N100105-R TGTCATCTTCTCACACTCCCA

SEQ ID NO:21 91-N1001523-F TGATGGAATGAAGTTGAGGGA (AG)8 5-FAM SEQ ID NO:22 91-N1001523-R GGCACATATGCTTTGTGGTG

SEQ ID NO:23 96-N1007386-F AAGTCAGCCACTATGCCAGG (TCC)6 5-FAM SEQ ID NO:24 96-N1007386-R CAAGCGACATTTGCGTCTAA

SEQ ID NO:25 97-N1844635-F GTGGATATACCGCTTCGTCG (GGC)5 5-FAM SEQ ID NO:26 97-N1844635-R GAAGAAAGTGGCTGCGTCTC

3. Amplification of samples PCR amplification of the samples was performed by using the primers in Table 4. pL of total volume of the reaction system included 1.2 pL of DNA template (50 ng/pL), 1.0 pL 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 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 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 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 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

Rhynchostylis, 5-N1001614 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 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 5-N1001614-F 100% 4 SEQ ID NO:2 5-N1001614-R SEQ ID NO:3 7-N1001895-F 90% 4 SEQ ID NO:4 7-N1001895-R SEQ ID NO:5 14-N1007590-F 100% 4 SEQ ID NO:6 14-N1007590-R SEQ ID NO:7 25-N101022-F 100% 4 SEQ ID NO:8 25-N101022-R SEQ ID NO:9 28-N1012378-F 100% 8 SEQ ID NO:10 28-N1012378-R SEQ ID NO:11 37-N3601419-F 100% 4 SEQ ID NO:12 37-N3601419-R SEQ ID NO:13 45-N1438823-F 100% 7 SEQ ID NO:14 45-N1438823-R SEQ ID NO:15 82-N100023-F 100% 6 SEQ ID NO:16 82-N100023-R SEQ ID NO:17 88-N1001036-F 100% 6 SEQ ID NO:18 88-N1001036-R SEQ ID NO:19 89-N100105-F 100% 3 SEQ ID NO:20 89-N100105-R SEQ ID NO:21 91-N1001523-F 100% 4 SEQ ID NO:22 91-N1001523-R SEQ ID NO:23 96-N1007386-F 80% 4 SEQ ID NO:24 96-N1007386-R SEQ ID NO:25 97-N1844635-F 100% 3 SEQ ID NO:26 97-N1844635-R

Example 4 Genetic analysis and clustering The calculation and analysis results of genetic diversity index, clustering and polymorphism information content PIC were shown in Table 6 and FIG. 2 below. Table 6 Observed Observed Expected Polymorphis Effective Inbreeding Genetic Shannon MarkerName number of heterozyg heterozyg m information number of coefficient in deviation information alleles (Na) osity (Ho) osity (He) content (PIC) alleles (Ne) population index (D) index (I)

(Fis)

-N1001614 2 0.333 0.375 0.305 1.600 0.111 -0.111 0.562

7-N1001895 3 0.111 0.667 0.593 3.000 0.833 -0.833 1.099

14-N1007590 2 0 0.278 0.239 1.385 1 -1 0.451

-N101022 1 0 0 0 1 0 0 0

28-N1012378 5 0.667 0.729 0.682 3.689 0.085 -0.085 1.415

37-N3601419 2 0.111 0.420 0.332 1.724 0.735 -0.735 0.611

-N1438823 5 0.100 0.778 0.744 4.500 0.871 -0.871 1.561

82-N100023 6 0.800 0.670 0.612 3.030 -0.194 0.19451 1.332

88-N1001036 2 0 0.480 0.365 1.9235 1.000 -1.000 0.673

89-N1001051 2 0 0.375 0.305 1.600 1.000 -1.000 0.562

91-N1001523 2 0 0.500 0.375 2.000 1.000 -1.000 0.693

96-N1007386 4 0 0.750 0.703 4.000 1.000 -1.000 1.386

97-N1844635 1 0 0 0 1.000 0 0 0

Example 5 Construction of molecular identity cards The molecular identity card was constructed according to a diploid standard, the fingerprint data were 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 identity card was as follows: 5-N1001614, 7-N1001895, 14-N1007590, 25-N101022, 28-N1012378, 37-N3601419, 45-N1438823, 82-N100023, 88-N1001036, 89-NIO01051, 91-N1001523, 96-N1007386, and 97-N844635. The molecular identity card information was shown in Table 7 below, and the fingerprint spectrum was shown in Table 8 below. Table 7

Latin Name Molecular identity card Number information RO1 Rhy. gigantea 'Shen's Suns' 23334514342344121112448833 R02 Rhy. gigantea 24331414242344121112440033 R03 Rhy. gigantea 14332414342344122113442613 R04 Rhy. gigantea 'JH#1l' 24004414352344121112444533 R05 Rhy. gigantean var petoniana 24334444242344121111445733 R06 Rhy. gigantea f. rubra 23331444242344121111441133 R07 Rhy. coelestis 11244414121313121611443323 R08 Rhy. coelestis f. alba 11334422121215113611131322

R09 Rhy. coelestis 11123422121215114622120022 RIO Rhy. retusa 22442433332224125111443333

Table 8 Fingerprint spectrum Locus RO1 R02 R03 R04 R05 R06 R07 R08 R09 RIO

5-N1001614 100 100 98 100 100 100 98 98 98 100

5-N1001614 103 106 106 106 106 103 98 98 98 100

7-N1001895 182 182 182 - 182 182 172 182 169 192

7-N1001895 182 182 182 - 182 182 192 182 172 192

14-N1007590 124 115 117 124 124 115 124 124 120 117

14-N1007590 128 124 124 124 124 124 124 124 124 124

25-N101022 128 128 128 128 133 133 128 131 131 132

25-N101022 133 133 133 133 133 133 133 131 131 132

28-N1012378 174 171 174 174 171 171 167 167 167 174

28-N1012378 176 176 176 179 176 176 171 171 171 174

37-N3601419 187 187 187 187 187 187 181 181 181 187

37-N3601419 190 190 190 190 190 190 190 187 187 187

45-N1438823 193 193 193 193 193 193 163 163 163 182

45-N1438823 193 193 193 193 193 193 184 194 194 193

82-N100023 167 167 167 167 167 167 167 167 167 167

82-N100023 171 171 171 171 171 171 171 167 167 171

88-N1001036 143 143 135 143 143 143 143 153 157 137

88-N1001036 143 143 143 143 143 143 161 161 161 143

89-N1001051 97 97 97 97 97 97 97 97 99 97

89-N1001051 99 99 101 99 97 97 97 97 99 97

91-N1001523 136 136 136 136 136 136 136 116 116 136

91-N1001523 136 136 136 136 136 136 136 134 126 136

96-N1007386 150 - 111 116 124 92 113 92 - 113

96-N1007386 150 - 127 124 130 92 113 113 - 113

97-N1844635 128 128 122 128 128 128 124 124 124 128

97-N1844635 128 128 128 128 128 128 128 124 124 128

Experimental Example 1 Accuracy Detection According to the primer set of SSR molecular markers and and identification method of germplasm resources disclosed by the present invention, 10 samples of RO1, R02, R03, R04, R05, R06, R07, R08, R09, RI1 from each of Rhynchostylis were selected for the identification of germplasm resources, and the detection results were shown in Table 9 below. Table 9 Accuracy detection results

Rhynchostylis Number of samples detection results Plants

R01 10 10

R02 10 10

R03 10 10

R04 10 10

R05 10 10

R06 10 10

R07 10 10

R08 10 10

R09 10 10

R10 10 10

As can be seen from Table 9, 10 Rhynchostylis 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 the Rhynchostylis. The above-described embodiments illustrate only a few embodiments of the present invention, which are described in greater 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 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 Rhynchostylis, characterized in that the SSR molecular marker comprises 5-N1001614, 7-N1001895, 14-N1007590, 25-N101022, 28-N1012378, 37-N3601419, 45-N1438823, 82-N100023, 88-N1001036, 89-N1001051, 91-N1001523, 96-N1007386, and 97-N1844635; the primer set comprises: (14) primers for amplifying SSR molecular marker 5-N1001614:

SEQIDNO:1 :5-N1001614-F: 5'-TTTGGTGGAGGATGGAGAAG-3'

SEQ ID NO:2: 5-N1001614-R: 5'-GCTTCCTTGAGGTGAAGAGC-3';

(15) primers for amplifying SSR molecular marker 7-N1001895:

SEQIDNO:3 :7-N1001895-F: 5'-ATCGCCATTTTTGTGCTCTT-3'

SEQ ID NO:4 :7-N1001895-R: 5'-ATTGAAATGATGGCTTTCGC-3';

(16) primers for amplifying SSR molecular marker 14-N1007590:

SEQIDNO:5: 14-N1007590-F: 5'-TGACATGGAAGTTATGGCATTC-3'

SEQ ID NO:6: 14-N1007590-R: 5'-TGTCGCAATTCTATGTCAGGA-3'

(17) primers for amplifying SSR molecular marker 25-N101022:

SEQ ID NO:7: 25-N101022-F : 5'-TGGACCACCTATCTCAAGGC-3'

SEQIDNO:8:25-N101022-R: 5'-ACGACATGCTCATGATTCCA-3';

(18) primers for amplifying SSR molecular marker 28-N1012378:

SEQ ID NO:9: 28-N1012378-F : 5'-GGAAGAAGGAGGAGGTGAGG-3'

SEQIDNO:10:28-N1012378-R:5'-TCCTAGAGAAAGAGCTTGGAGC -3';

(19) primers for amplifying SSR molecular marker 37-N3601419:

SEQ ID NO: I1: 37-N3601419-F :5'-AATGGAAAATCTGGCAGCAC-3'

SEQ ID NO:12 :37-N3601419-R :5'-TCTCCATCACTTCAATGGCA-3';

(20) primers for amplifying SSR molecular marker 45-N1438823:

SEQIDNO:13 :45-N1438823-F :5'-ATTTGGCTCAAATCCACCAA -3'

SEQIDNO:14 :45-N1438823-R: 5'-ATACCTGAAAGCATGGTGCC -3';

(21) primers for amplifying SSR molecular marker 82-N100023:

SEQ ID NO:15 : 82-N100023-F : 5'-AGGGTGGGAAGGGAGATAAA-3'

SEQIDNO:16:82-N100023-R: 5'-CTCTGCCTTCTCCATCTCTCA-3';

(22) primers for amplifying SSR molecular marker 88-N1001036:

SEQ ID NO:17: 88-N1001036-F: 5'-AGGGAGGGAGTGAGAGAGGA-3'

SEQIDNO:18:88-N1001036-R: 5'-TCCCGCTTTCTATCGCTATC-3';

(23) primers for amplifying SSR molecular marker 89-N100105:

SEQIDNO:19:89-N100105-F: 5'-TTTCCTGCTCTCCATTCCTG-3'

SEQIDNO:20:89-N100105-R: 5'-TGTCATCTTCTCACACTCCCA-3';

(24) primers for amplifying SSR molecular marker 91-N1001523:

SEQIDNO:21 :91-N1001523-F: 5'-TGATGGAATGAAGTTGAGGGA-3'

SEQIDNO:22:91-N1001523-R: 5'-GGCACATATGCTTTGTGGTG-3';

(25) primers for amplifying SSR molecular marker 96-N1007386:

SEQ ID NO:23 : 96-N1007386-F : 5'-AAGTCAGCCACTATGCCAGG-3'

SEQIDNO:24:96-N1007386-R: 5'-CAAGCGACATTTGCGTCTAA-3';

(26) primers for amplifying SSR molecular marker 97-N1844635:

SEQ ID NO:25 :97-N1844635-F : 5'-GTGGATATACCGCTTCGTCG-3'

SEQ ID NO:26 :97-N1844635-R: 5'-GAAGAAAGTGGCTGCGTCTC-3'.

2. A SSR molecular marker fingerprint code for identifying Rhynchostylis, characterized in that the fingerprint code comprises a fingerprint spectrum and a molecular identity card, wherein the sequence of SSR molecular marker of the molecular identity card is as follows: 5-N1001614, 7-N1001895, 14-N1007590, 25-N101022, 28-N1012378, 37-N3601419, 45-N1438823, 82-N100023, 88-N1001036, 89-NIO01051, 91-N1001523, 96-N1007386, and 97-N844635. 3. The fingerprint code according to claim 2, characterized in that the molecular identity card is as follows:

Latin Name Molecular identity card Number information RO1 Rhy. gigantea 'Shen's Suns' 23334514342344121112448833 R02 Rhy. gigantea 24331414242344121112440033 R03 Rhy. gigantea 14332414342344122113442613 R04 Rhy. gigantea 'JH#11' 24004414352344121112444533 R05 Rhy. gigantean var petoniana 24334444242344121111445733 R06 Rhy. gigantea f. rubra 23331444242344121111441133 R07 Rhy. coelestis 11244414121313121611443323 R08 Rhy. coelestis f. alba 11334422121215113611131322 R09 Rhy. coelestis 11123422121215114622120022 RIO Rhy. retusa 22442433332224125111443333

4. The fingerprint code according to claim 3, characterized in that the fingerprint spectrum is as

follows:

Locus R01 R02 R03 R04 R05 R06 R07 R08 R09 R10

5-N1001614 100 100 98 100 100 100 98 98 98 100

5-N1001614 103 106 106 106 106 103 98 98 98 100

7-N1001895 182 182 182 - 182 182 172 182 169 192

7-N1001895 182 182 182 - 182 182 192 182 172 192

14-N1007590 124 115 117 124 124 115 124 124 120 117 14-N1007590 128 124 124 124 124 124 124 124 124 124

25-N101022 128 128 128 128 133 133 128 131 131 132

25-N101022 133 133 133 133 133 133 133 131 131 132

28-N1012378 174 171 174 174 171 171 167 167 167 174

28-N1012378 176 176 176 179 176 176 171 171 171 174

37-N3601419 187 187 187 187 187 187 181 181 181 187 37-N3601419 190 190 190 190 190 190 190 187 187 187 45-N1438823 193 193 193 193 193 193 163 163 163 182

45-N1438823 193 193 193 193 193 193 184 194 194 193

82-N100023 167 167 167 167 167 167 167 167 167 167 82-N100023 171 171 171 171 171 171 171 167 167 171 88-N1001036 143 143 135 143 143 143 143 153 157 137 88-N1001036 143 143 143 143 143 143 161 161 161 143

89-N1001051 97 97 97 97 97 97 97 97 99 97 89-N1001051 99 99 101 99 97 97 97 97 99 97

91-N1001523 136 136 136 136 136 136 136 116 116 136

91-N1001523 136 136 136 136 136 136 136 134 126 136

96-N1007386 150 - 111 116 124 92 113 92 - 113

96-N1007386 150 - 127 124 130 92 113 113 - 113

97-N1844635 128 128 122 128 128 128 124 124 124 128

97-N1844635 128 128 128 128 128 128 128 124 124 128

5. A kit for the screening of Rhynchostylis 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

Rhynchostylis 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 the ten Rhynchostylis plantsas 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 Rhynchostylis 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 pL of total volume of the reaction system, comprising 1.2 pL of DNA template

(50 ng/pL), 1.0 pL of 10 x Buffer I, 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; 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: 26.

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 Rhynchostylis.

FIG. 2 FIG. 1

ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ12342562ÿ781985 ÿÿ ÿÿ ÿÿ2ÿÿÿ8ÿÿ !ÿ"!#ÿÿ" ÿ1ÿÿ ÿÿÿ

$ ÿÿ11ÿÿ%ÿ&ÿÿÿ!#ÿ##ÿ!ÿÿÿÿ ÿÿ

' ÿÿ$ $

$ÿÿ ÿÿ

( ÿÿ$(ÿÿÿÿÿÿ ÿÿ

) ÿÿ*8ÿÿ'+,ÿÿ 2021101596

ÿÿ $ ÿÿ ÿÿ $ ÿÿ$ ÿÿ $

$ÿÿ-5"ÿÿ $

'ÿÿÿ.ÿÿ ÿÿ / ÿÿ ÿÿ ÿ ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ$ ÿÿ ÿÿ ÿÿ $ ÿÿ$ÿÿ $ ÿÿ$ ÿÿ $

$ÿÿ-5"ÿÿ $

'ÿÿÿ.ÿÿ ÿÿ / ÿÿ$ÿÿ ÿ ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ$ ÿÿ ÿÿ ÿÿ $ ÿÿ'ÿÿ $ ÿÿ$ ÿÿ $

$ÿÿ-5"ÿÿ $

'ÿÿÿ.ÿÿ ÿÿ / ÿÿ'ÿÿ ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ$ ÿÿ ÿÿ ÿÿ $ ÿÿ/ÿÿ $ ÿÿ$ ÿÿ $

$ÿÿ-5"ÿÿ $

'ÿÿÿ.ÿÿ ÿÿ / ÿÿ/ÿÿ ÿ ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ$ ÿÿ ÿÿ ÿÿ $ ÿÿ,ÿÿ $ ÿÿ$$ÿÿ $

$ÿÿ-5"ÿÿ $

'ÿÿÿ.ÿÿ ÿÿ / ÿÿ,ÿÿ

0123201122ÿ1002011320ÿ03ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ55ÿÿ ÿÿ ÿÿ 65789ÿÿ ÿÿ 65779ÿÿ57ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿ ÿÿ 0103132200ÿ3020103211ÿ2ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ57ÿÿ ÿÿ 2021101596 ÿÿ 65789ÿÿÿÿ 65779ÿÿ58ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿÿÿ 0112332330ÿ2030322113ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ58ÿÿ ÿÿ ÿÿ 65789ÿÿÿÿ 65779ÿÿ58ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿÿÿ 2312320130ÿ3201200332ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ58ÿÿ ÿÿ ÿÿ 65789ÿÿÿÿ 65779ÿÿ58ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿÿÿ 1122122112ÿ1121101211ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ58ÿÿ ÿÿ ÿÿ 65789ÿÿ78ÿÿ 65779ÿÿ55ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿ78ÿÿ 0330212122ÿ2121300112ÿ13ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ55ÿÿ ÿÿ ÿÿ 65789ÿÿ77ÿÿ 65779ÿÿ58ÿÿ 65759ÿÿ ÿÿ 6579ÿÿ2032ÿ3ÿÿ ÿÿ 6889ÿÿ77ÿÿ

0012200001ÿ4122402404ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ85ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ85ÿÿ 1414401404ÿ1140012240ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ 2021101596 ÿÿ 75869ÿÿ8 ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ8 ÿÿ 0111224140ÿ0014404400ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ8ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ8ÿÿ 0104412000ÿ2401221244ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ8ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ8ÿÿ 0222122200ÿ2220201000ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ8ÿÿ 75889ÿÿ58ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ8ÿÿ 4141244114ÿ1440141414ÿ0ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ58ÿÿ ÿÿ ÿÿ 75869ÿÿ8ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 758 9ÿÿ0140ÿ4ÿÿ ÿÿ 7669ÿÿ8ÿÿ

0111011101ÿ3101010110ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ45ÿÿ ÿÿ ÿÿ 64758ÿÿ79ÿÿ 64778ÿÿ45ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ79ÿÿ 31333ÿ3031303ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ45ÿÿ ÿÿ 2021101596 ÿÿ 64758ÿÿ7ÿÿ 64778ÿÿ45ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ7ÿÿ 333313ÿ303331ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ45ÿÿ ÿÿ ÿÿ 64758ÿÿ45ÿÿ 64778ÿÿ47ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ45ÿÿ 3130333ÿ3003ÿ0ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ47ÿÿ ÿÿ ÿÿ 64758ÿÿ47ÿÿ 64778ÿÿ47ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ47ÿÿ 3103110031ÿ0013310111ÿ0ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ47ÿÿ ÿÿ ÿÿ 64758ÿÿ44ÿÿ 64778ÿÿ45ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ44ÿÿ 11003031ÿ333131131ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ45ÿÿ ÿÿ ÿÿ 64758ÿÿ4 ÿÿ 64778ÿÿ45ÿÿ 64748ÿÿ ÿÿ 647 8ÿÿ030ÿÿÿ ÿÿ 6558ÿÿ4 ÿÿ

0012301330ÿ3202133011ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ5 ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 7589ÿÿ0230ÿ3ÿÿ ÿÿ 7 669ÿÿ5 ÿÿ 3001310302ÿ2213123200ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ 2021101596 ÿÿ 75869ÿÿ5ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 7589ÿÿ0230ÿ3ÿÿ ÿÿ 7 669ÿÿ5ÿÿ 1211020203ÿ3132231231ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ 75869ÿÿ5ÿÿ 75889ÿÿ56ÿÿ 75859ÿÿ ÿÿ 7589ÿÿ0230ÿ3ÿÿ ÿÿ 7 669ÿÿ5ÿÿ 1001000121ÿ1321312323ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ56ÿÿ ÿÿ ÿÿ