NL2032552B1 - Phylogenetic classification method of allium vegetable genetic resources based on different molecular markers and use thereof - Google Patents
Phylogenetic classification method of allium vegetable genetic resources based on different molecular markers and use thereof Download PDFInfo
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- 241000234282 Allium Species 0.000 title claims abstract description 108
- 235000013311 vegetables Nutrition 0.000 title claims abstract description 63
- 230000002068 genetic effect Effects 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000003321 amplification Effects 0.000 claims abstract description 29
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 29
- 108091023242 Internal transcribed spacer Proteins 0.000 claims abstract description 27
- 108020004414 DNA Proteins 0.000 claims abstract description 26
- 241000196324 Embryophyta Species 0.000 claims abstract description 17
- 239000000463 material Substances 0.000 claims abstract description 17
- 101150027061 RPS16 gene Proteins 0.000 claims abstract description 15
- 101100200593 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) rps19 gene Proteins 0.000 claims abstract description 12
- 108091029795 Intergenic region Proteins 0.000 claims abstract description 12
- 101100418504 Schizosaccharomyces pombe (strain 972 / ATCC 24843) rps1601 gene Proteins 0.000 claims abstract description 12
- 101150116648 rpsP gene Proteins 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 238000004458 analytical method Methods 0.000 claims abstract description 8
- 241000894007 species Species 0.000 claims description 13
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 1
- 238000012797 qualification Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 5
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 108700031407 Chloroplast Genes Proteins 0.000 abstract description 3
- 238000010276 construction Methods 0.000 abstract description 3
- 238000013398 bayesian method Methods 0.000 abstract 1
- 240000002234 Allium sativum Species 0.000 description 22
- 240000006108 Allium ampeloprasum Species 0.000 description 17
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 17
- 244000291564 Allium cepa Species 0.000 description 11
- 240000008654 Allium ramosum Species 0.000 description 10
- 244000257727 Allium fistulosum Species 0.000 description 9
- 235000008553 Allium fistulosum Nutrition 0.000 description 9
- 241001275743 Allium ascalonicum Species 0.000 description 8
- 235000010167 Allium cepa var aggregatum Nutrition 0.000 description 8
- 235000002545 Allium longicuspis Nutrition 0.000 description 8
- 241000383620 Allium mongolicum Species 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 235000004611 garlic Nutrition 0.000 description 8
- 235000005255 Allium cepa Nutrition 0.000 description 6
- 244000003377 Allium tuberosum Species 0.000 description 6
- 235000005338 Allium tuberosum Nutrition 0.000 description 6
- NOIRDLRUNWIUMX-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.O=C1NC(N)=NC2=C1NC=N2 NOIRDLRUNWIUMX-UHFFFAOYSA-N 0.000 description 5
- 235000018645 Allium odorum Nutrition 0.000 description 5
- 241000383692 Allium sikkimense Species 0.000 description 4
- 210000003763 chloroplast Anatomy 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 244000066882 Allium wallichii Species 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 229930191988 viviparum Natural products 0.000 description 3
- 241000981179 Allium caeruleum Species 0.000 description 2
- 241001513212 Allium hookeri Species 0.000 description 2
- 241000383737 Allium tenuissimum Species 0.000 description 2
- 244000161286 Allium victorialis Species 0.000 description 2
- 101100418717 Rhodobacter capsulatus (strain ATCC BAA-309 / NBRC 16581 / SB1003) rbsR gene Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000010835 comparative analysis Methods 0.000 description 2
- 101150085492 rpsF gene Proteins 0.000 description 2
- 101150006569 rpsR gene Proteins 0.000 description 2
- 101150029588 rpsR1 gene Proteins 0.000 description 2
- 101150039504 6 gene Proteins 0.000 description 1
- 241000383510 Allium anisopodium Species 0.000 description 1
- 241001193913 Allium chrysanthum Species 0.000 description 1
- 241001513208 Allium cyathophorum Species 0.000 description 1
- 241000304531 Allium macrostemon Species 0.000 description 1
- 241001194534 Allium plurifoliatum Species 0.000 description 1
- 241001513194 Allium polyrhizum Species 0.000 description 1
- 241000383704 Allium strictum Species 0.000 description 1
- 235000011644 Allium victorialis Nutrition 0.000 description 1
- 235000018640 Allium wallichii Nutrition 0.000 description 1
- 241000410046 Cyathophorum Species 0.000 description 1
- 101150034814 F gene Proteins 0.000 description 1
- 241000353097 Molva molva Species 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
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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
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- G16B10/00—ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
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Abstract
The present disclosure belongs to the technical field of phylogenetic classification for resources, and discloses a phylogenetic classification method of Allium vegetable genetic resources based on different molecular markers and use thereof. The method includes the following steps: selection of test materials; extraction and detection of a plant genomic DNA; amplification and sequencing of a sequence; sequence assembly and feature analysis; and construction of a phylogenetic tree. In the present disclosure; classification; identification and phylogenetic evolution of the Allium vegetable genetic resources is analyzed by the phylogenetic tree constructed by a Bayesian method; a phylogenetic tree of 78 common Allium vegetable genetic resources is constructed by using a chloroplast gene fragment rps16 intron; a trnL-F intergenic region; a nuclear gene internal transcribed spacer (ITS) fragment and a combination of the above three gene fragments.
Description
PHYLOGENETIC CLASSIFICATION METHOD OF ALLIUM VEGETABLE
GENETIC RESOURCES BASED ON DIFFERENT MOLECULAR MARKERS
AND USE THEREOF
[0001] The present disclosure belongs to the technical field of phylogenetic classification for resources, in particular to a phylogenetic classification method of
Allium vegetable genetic resources based on different molecular markers and use thereof.
[0002] At present, Allium vegetables are mostly classified based on single gene fragments at a molecular level. Due to a limited amount of data carrying less genetic information, there are often certain defects in the classification of species with minimal interspecific differences, restricting the development of classification and identification as well as resources utilization for the A/lium vegetables.
[0003] Through the above analysis, the existing problems and defects in the prior art are as follows: at present, the A/lium vegetables are mostly classified based on single gene fragments at the molecular level. Due to the limited amount of data carrying less genetic information, there are often certain defects in the classification of species with minimal interspecific differences, restricting the development of the classification and identification as well as the resources utilization for the Allium vegetables.
[0004] The significance of solving the above problems and defects 1s as follows: to analyze differences in classification results of A/lium vegetable genetic resources based on different sequence sets, to seek a method suitable for classification, identification and phylogeny of the Allium vegetable genetic resources, thereby providing new ideas for subsequent researches of the Allium vegetable genetic resources.
[0005] Aiming at the problems in the prior art, the present disclosure provides a phylogenetic classification method of Allium vegetable genetic resources based on different molecular markers and use thereof.
[0006] The present disclosure includes the following steps:
[0007] step 1, selection of test materials;
[0008] step 2, extraction and detection of a plant genomic DNA;
[0009] step 3, amplification and sequencing of a sequence;
[0010] step 4, assembly and feature analysis of the sequence; and
[0011] step 5, construction of a phylogenetic tree.
[0012] Further, in step 1, the test materials of the Allium vegetable genetic resources may be derived from the Mid-Term Bank of National Vegetable Germplasm Resources, the National Asexual Propagation and Perennial Vegetable Germplasm resources Garden and the Germplasm Bank of Wild Species.
[0013] Further, in step 2, the extraction and detection of a plant genomic DNA may include:
[0014] Extracting the plant genomic DNA, followed by detection with 1% agarose gel electrophoresis to determine whether the plant genomic DNA is successfully extracted and a mass of the plant genomic DNA.
[0015] Further, in step 3, the amplification and sequencing of a sequence may include: amplification of an internal transcribed spacer (ITS) sequence, an rps 16 intron sequence, and a tmL-F intergenic region sequence.
[0016] Further, in step 4, the sequence may be subjected to the assembly and feature analysis.
[0017] Further, in step 5, the phylogenetic tree may be constructed.
[0018] Combined with all the above technical solutions, the present disclosure has the advantages and positive effects as follows: the present disclosure provides a phylogenetic classification method of Allium vegetable genetic resources based on different molecular markers; through comparative analysis of a chloroplast genome of main A/ium plants, it is found that a rps16 intron and a trnL-F intergenic region in the chloroplast genome are highly variable regions due to obvious sequence differences; the present disclosure provides more locus information for classification and identification, genetic differences and phylogeny of species.
[0019] FIG. 1 shows a flow chart of a phylogenetic classification method of A/iwm vegetable genetic resources based on different molecular markers provided by examples of the present disclosure;
[0020] FIG. 2 (a) shows a schematic diagram of an amplification result of an 77S gene of a test plant sample provided by examples of the present disclosure;
[0021] FIG. 2 (b) shows a schematic diagram of an amplification result of an rps/6 gene of a test plant sample provided by examples of the present disclosure;
[0022] FIG. 2 (c) shows a schematic diagram of an amplification result of a frnl-F gene of a test plant sample provided by examples of the present disclosure;
[0023] FIG. 3 shows a schematic diagram of a phylogenetic tree of the A//ium vegetable genetic resources constructed based on an ITS fragment provided by examples of the present disclosure;
[0024] FIG. 4 shows a schematic diagram of a phylogenetic tree of the A//ium vegetable genetic resources constructed based on an rps16 fragment provided by examples of the present disclosure;
[0025] FIG. 5 shows a schematic diagram of a phylogenetic tree of the A//ium vegetable genetic resources constructed based on a trnL-F fragment provided by examples of the present disclosure;
[0026] FIG. 6 shows a schematic diagram of a phylogenetic tree of the A?!ium vegetable genetic resources constructed based on an ITS+rpsl6+trnL-F fragment provided by examples of the present disclosure; and
[0027] FIG. 7 shows a schematic diagram of classification and comparison of 78 Allium vegetable genetic resources based on different molecular markers provided by examples of the present disclosure.
[0028] Aiming at the problems in the prior art, the present disclosure provides a phylogenetic classification method of Allium vegetable genetic resources based on different molecular markers and use thereof. The present disclosure will be described in detail below in conjunction with the accompanying drawings.
[0029] As shown in FIG. 1, the phylogenetic classification method of Allium vegetable genetic resources based on different molecular markers includes the following steps:
[0030] S101, selection of test materials;
[0031] S102, extraction and detection of a plant genomic DNA;
[0032] S103, amplification and sequencing of a sequence;
[0033] S104, assembly and feature analysis of the sequence; and
[0034] S105, construction of a phylogenetic tree.
[0035] The present disclosure is further described below with reference to specific examples.
[0036] 1. Overview of the present disclosure
[0037] In the present disclosure, through comparative analysis of a chloroplast genome of main A/lium plants, it is found that a rps 16 intron and a trnL-F intergenic region in the chloroplast genome are highly variable regions due to obvious sequence differences, the present disclosure provides more locus information for classification and identification, genetic differences and phylogeny of species.
[0038] 2. Materials and methods
[0039] 2.1. Test materials
[0040] 78 test materials of the Allium vegetable genetic resources are derived from the
Mid-Term Bank of National Vegetable Germplasm Resources, the National Asexual
Propagation and Perennial Vegetable Germplasm resources Garden and the Germplasm
Bank of Wild Species, including a total of 28 species of common A//ium vegetables, such as Allium tuberosum, Allium fistulosum L. var. gigantum Makino, Allium fistulosum L. var. caespitosum Makino and Allium cepa L., covering 7 groups classified under the genus Al/ium in China (Table 1).
[0041] Table 1 Test materials
[0042] 2.2. Extraction and detection of a plant genomic DNA
[0043] The plant genomic DNA is extracted, followed by detection with 1% agarose gel electrophoresis to determine whether the plant genomic DNA is successfully extracted and a mass of the plant genomic DNA.
[0044] 2.3. Amplification and sequencing of a sequence
[0045] In the present disclosure, amplification primers of an ITS sequence are designed by WHITE and the like, including universal primers, forward primers and reverse primers. The amplification primers of the rpsl6 intron sequence use the forward primer and reverse primer designed by OXELMAN, and the amplification primers of the trnL-
F gene spacer sequence use the forward primer and reverse primer designed by
TABERLET et al. the primers are synthesized by BGI Group (Beijing). An amplification reaction system is set to 50 pL.
[0046] 3. Results and analysis
[0047] 3.1. Amplification results of the sequence
The ITS sequence is amplified by primers ITS1 and ITS4 using the genomic DNA of 78
Allium vegetable genetic resources samples as templates. FIG. 2(a) shows a partial amplification result: at a standard molecular weight of about 700 bp, each sample has a clear band, which can be considered as an ITS sequence of a target band. FIG. 2(b) shows a partial amplification result of the rps16 intron sequence at a standard molecular weight of about 800 bp. Similarly, PCR products of the tmL-F intergenic region of the 78 Allium vegetable genetic resources are as shown in FIG. 2(c), exhibiting a partial amplification result of the materials.
[0048] 3.2. Feature analysis of the sequence 5 [0049] A sequence length and a guanine-cytosine (GC) content are counted for the ITS, rps16 intron and trnL-F intergenic region of the 78 different Allium vegetable genetic resources (Table 2). The lengths of ITS sequences of all the test materials are between 625-687 bp, where Allium wallichii Kunth in a Sect. Bromatorrhiza group has the longest sequence, and Allium fistulosum L. var. viviparum Makino in a Sect. Schoenoprasum group has the shortest sequence, and a difference between the above two is only 62 bp.
[0050] Table 2 Basic characteristics of ITS, rps16, trnL-F sequences of different species
[0051] 3.3. Phylogenetic analysis
[0052] 3.3.1. Classification results of A/lium vegetable genetic resources based on ITS sequence
[0053] A phylogenetic tree of 78 Allium vegetable genetic resources is constructed by a nuclear gene ITS sequence (FIG. 3). The results show that the 4//ium vegetable genetic resources in each group can be basically distinguished.
[0054] 3.3.2. Classification results of Allium vegetable genetic resources based on rps 16 intron sequence
[0055] A phylogenetic tree of 78 Allium vegetable genetic resources is constructed by a chloroplast gene rps16 intron sequence (FIG. 4). Materials of a Sect. Porrum group are gathered into one, including four kinds: Allium sativum, Allium ampeloprasum L., Allium longicuspis, and Allium porrum.
[0056] 3.3.3. Classification results of Allium vegetable genetic resources based on trnL-
F intergenic region sequence
[0057] A phylogenetic tree of 78 Allium vegetable genetic resources is constructed by a chloroplast gene trnL-F intergenic region sequence (FIG. 5). The Allium sativum,
Allium ampeloprasum L., Allium longicuspis, and Allium porrum in the materials are grouped into a same branch, forming the Sect. Porrum group, which are more closely related; while one Allium porrum is divided into the Sect. Schoenoprasum group.
[0058] 3.3.4. Classification results of Allium vegetable genetic resources based on
ITS-+rps16+trnL-F sequence
[0059] A phylogenetic tree of 78 Allium vegetable genetic resources is constructed by an ITS+rpsl6+tmL-F sequence (FIG. 6)Most of the Allium sativum, Allium ampeloprasum L., Allium longicuspis, and Allium porrum in the materials are grouped into a same branch, forming the Sect. Porrum group, which are more closely related; while there are also a small amount of materials that have been aggregated into other groups. Table 3 shows classification and comparison of 78 Allium vegetable genetic resources based on different molecular markers.
[0060] Table 1 Test materials
[0061]
Species
No. Accessions Species No. Accessions Species
Allium Allium T14
A003 tuberosum tuberosum 1 Allium sativum Allium sativum
Allium Allium N08
A056 fuberosum tuberosum 96 Allium longicuspis Allium longicuspis
Allium Allium N08
Al87 tuberosum tuberosum 97 Allium longicuspis Allium longicuspis
Allium Allium N09
A197 wberosum tuberosum 00 Allium longicuspis Allium longicuspis
Allium Allium N09
A256 tuberosum tuberosum 05 Allium longicuspis Allium longicuspis
Allium Allium N09 1060 tuberosum tuberosum 08 Allium longicuspis Allium longicuspis
Allium Allium 103 Allium Allium
BO10 fistulosum Jistulosum 9 schoenoprasum schoenoprasum
Allium Allium 103 Allium Allium
B038 fistulosum fistulosum 9-1 schoenoprasum schoenoprasum
Allium Allium 103
B043 fistulosum Jistulosum 6 Allium ramosum Allium ramosum
Allium Allium 103
B045 fistulosum fistulosum 8 Allium ramosum Allium ramosum
Allium Allium 104
B224 fistulosum fistulosum 2 Allium ramosum Allium ramosum
Allium Allium GS
C001 ascalonicum ascalonicum YJ Allium ramosum Allium ramosum
Allium Allium 106
C021 ascalonicum ascalonicum 1 Allium ramosum Allium ramosum
Allium Allium 102
C032 ascalonicum ascalonicum 6 Allium mongolicum Allium mongolicum
Allium Allium 104
C040 ascalonicum ascalonicum 3 Allium mongolicum Allium mongolicum
Allium Allium 103 1049 ascalonicum ascalonicum 5 Allium mongolicum Allium mongolicum
Allium Allium 105 1050 ascalonicum ascalonicum 6 Allium mongolicum Allium mongolicum
Allium Allium 105 1051 ascalonicum ascalonicum 7 Allium mongolicum Allium mongolicum
Allium Allium 1053 ascalonicum ascalonicum 168 Allium wallichii Allium wallichii 100
D006 Allium cepa Allium cepa 4 Allium caeruleum Allium caeruleum 100
D027 Allium cepa Allium cepa 9 Allium strictum Allium strictum
D048 Allium cepa Allium cepa 1 Allium plurifoliatum Allium plurifoliotum 101
D055 Allium cepa Allium cepa 2 Allium wallichii Allium wallichii 101 Allium cyathophorum Allium cyathophorum
D067 Allium cepa Allium cepa 8 var. farreri var. farreri
Allium 102 Allium Allium
E003 porrum Allium porrum 1 przewalskianum przewalskianum
Allium 102
E004 porrum Allium porrum 4 Allium sikkimense Allium sikkimense
Allium 102
E006 porrum Allium porrum 5 Allium sikkimense Allium sikkimense
Allium 102
E007 DOFTUM Allium porrum 7 Allium macrostemon Allium macrostemon
Allium 102
E008 porrum Allium porrum 8 Allium taishanense Allium taishanense
Allium ampeloprasu Allium 103
H002 m ampaloprasum 0 Allium polyrhizum Allium pohrhizum
Allium
NOS ampeloprasu Allium 103 7 m ampaloprasum 3 Allium tenuissimum Allium tenuissimum
Allium
NO35 ampeloprasu Allium 103 3 m ampaloprasum 4 Allium anisopodium Allium anisopodium
Allium
N054 ampeloprasu Allium 104 0 m ampaloprasum 0 Unknown 2 unknow2
Allium
N101 ampeloprasu Allium 104 3 m ampaloprasum 1 Allium chrysonthum Allium chrysanthum
Allium
N107 ampeloprasu Allium 104 6 m ampaloprasum 4 Allium victorialis Allium victorialis
N022 Allium Allium 104 Allium Allium 0 sativum sativum 6 schoenoprosum schoenoprosum
N060 Allium Allium 104 Allium fistulosum. Allium fisulosum 8 sativum sativum 7 ViViparun1 var. viviparum
N073 Allium Allium 104 3 salivim sativim 8 ZZ1489
N081 Allium Allium 105 7 sativum sativum 8 Allium hookeri Allium hookeri
[0062] Table 2 Basic characteristics of ITS, rps16, trnL-F sequences of different species 00631
No. Accessions ITS rps16 tmnL-F
GC GC GC
Length content Length content Length content (bp) (%0) (bp) (%) (bp) (%)
A003 Allium tuberosum 681 45.08 776 29.90 662 36.25
A056 Allium tuberosum 671 45.45 795 29.31 662 36.25
A187 Allium tuberosum 681 45.08 794 29.35 662 36.25
A197 Alkum tuberosum 678 46.90 836 29.07 662 36.10
A256 Allium tuberosum 681 45.23 795 29.31 662 36.25 1060 Allium tuberosum 681 45.23 794 29.35 662 36.25
BO10 Allium fistulosum 679 47.28 616 31.33 674 36.20
B038 Allium fistulosum 679 47.28 613 31.32 674 36.20
B043 Alim fistulosum 679 47.28 614 31.43 674 36.20
B045 Allium fistulosum 679 47.28 613 31.32 674 36.35
B224 Allium fistulosum 678 46.76 829 28.35 662 36.10
C001 Allium ascalonicum 979 47.28 616 31.33 674 36.20
C021 Allium ascalonicum 979 47.28 612 31.37 674 36.20
C032 Allium ascalonicum 679 47.28 612 31.37 674 36.35
C040 Allium ascalonicum 679 47.28 614 31.43 674 36.20 1049 Allium ascalonicum 077 47.12 839 29.32 698 35.96
I050 Allium ascalonicum 977 47.12 839 29.32 698 35.96 1051 Allium ascalonicum 677 47.12 782 28.39 677 35.45 1053 Allium ascalonicum 977 47.12 838 29.24 698 35.96
D006 Allium cepa 677 47.12 839 29.32 698 35.96
D027 Allium cepa 677 47.12 839 29.32 698 35.96
D048 Allium cepa 677 47.12 839 29.32 698 35.96
D055 Allium cepa 677 47.12 839 29.32 698 35.96
D067 Allium cepa 677 47.12 839 29.32 698 35.96
E003 Allium porrum 678 46.76 858 29.02 662 36.10
E004 Allium porrum 678 46.76 858 29.02 662 36.10
E006 Allium porrum 678 46.76 858 28.90 662 36.10
E007 Allium porrum 679 47.28 856 28.97 674 36.20
E008 Allium porrum 658 46.05 858 29.02 662 36.10
Allium 676 46.89 857 28.94 662 36.10
H002 ampeloprasum
N015 Allium 678 46.76 857 28.94 662 36.10 7 ampeloprasum
N035 Allium 634 47.63 790 29.11 662 35.20 3 ampeloprasum
N054 Allium 678 47.49 785 29.30 668 35.48 0 ampeloprasum
NIO1 Allium 678 46.76 848 29.13 662 36.10 3 ampeloprasum
N107 Allium 678 47.79 785 29.30 668 35.48 6 ampeloprasum
N022 678 47.49 790 28.86 662 35.05 0 Allium sativum
N060 678 47.64 786 29.13 662 35.20 8 Allium sativum
N073 678 47.49 790 28.86 662 35.05 3 Allium sativum
N081 678 47.49 790 28.86 662 35.05 7 Allium sativum
Tl141 Allium sativum 678 47.35 790 28.86 662 35.05
N089 678 47.49 789 29.02 662 35.20 6 Allium longicuspis
N089 678 47.49 769 28.87 662 35.20 7 Allium longicuspis
N090 678 47.49 813 28.91 662 35.05 0 Allium longicuspis
N090 637 47.41 790 20.11 662 35.20 3 Allium longicuspis
N090 678 47.49 790 29.11 662 35.20 8 Allium longicuspis
Allium 679 47.28 612 31.37 672 36.01 1039 schoenoprasum
Allium 679 47.28 613 31.32 647 35.86 1039 schoenoprasum
I03G Allium ramosum 682 45.16 793 29.89 662 36.25 1038 Allium ramosum 637 46.31 793 29.26 662 36.25 1042 Allium ramosum 679 45.07 777 29.34 646 35.29
GSY 681 45.23 794 29.35 662 36.25
J Allium ramosum 1061 Allium ramosum 640 45.78 793 29.26 662 36.25 1043 Allium mongolicum 682 43.99 789 30.29 678 35.99 1056 Allium mongolicum 682 46.48 791 29.33 677 36.19 1057 Allium mongolicum 675 46.67 805 29.32 677 36.19 1004 Allium caeruleum 669 50.52 554 32.67 673 35.36
Allium 677 47.71 789 29.40 673 36.26 011 plurifoliatum
Allium 650 48.31 780 30.77 653 35.68 cyathophorum var. 1018 farreri
Allium 636 46.70 616 31.33 679 35.79 1021 przewalskianum 1024 Allium sikkimense 669 46.94 667 29.54 653 35.68 1025 Allium sikkimense 669 46.94 616 31.33 592 35.81
Allium 641 52.57 697 30.42 674 35.16 1027 macrostemon 1028 Allium taishanense 681 42.58 788 29.95 678 36.14 1033 Allium tenuissimum 682 45.16 768 30.21 662 36.25
Allium 679 45.21 796 29.65 677 35.75 1034 anisopodium 1040 Unknown 682 45.16 793 29.89 662 36.25
Allium 637 46.31 793 29.26 662 36.25 1041 chrysanthum 1044 Allium victorialis 682 48.39 784 29.97 662 36.25
Allium 668 47.01 616 31.33 673 35.66 1046 schoenoprosum
Allium fistulosum. 625 46.72 780 29.87 673 35.96 1047 viviparum 1048 Unknoun 679 45.36 799 29.66 677 35.75 1058 Allium hookeri 652 47.09 832 29.45 649 35.75
[0064] Table 3 Classification and comparison of 78 Allium vegetable genetic resources based on different molecular markers
[0065]
ITS rpsl6 trnL-F ITS+rpsl6+tmL-F
A197. B224. E003. E004, E006, E008. H002. N0157, N0220, N0340.
N0608, N0733. N0817, N0896, N0897, N0900, N0908, N1076, T141
Sect. Porrum ee
NI1013 NI1013.N03353. N1013,N0353, N0353. N0905
N0905, E007 N0903 q B010, B038, B043. B045, C001, C021. C032, C040. 1039, 1039-1
Sehcenaprasiom E007 1046, 1030, E007 1046, E007, 1044, 1021, 1025 1012 7 D006, D027, D048, D035, D067, 1049. 1050, 1051, 1033
Sect. Cepa 1046 1041 1047 1046, 1047 1011
A003, A056, A187, A256, GSYJ. 1009, 1018, 1026, 1028. 1033, 1034, 1036, 1038. 1042, 1043, 1048, 1055, 1036. 1057, 1060, 1061 1011, 1021, 1024. 1011, 1040, 1011, 1021, 1021, 1024, 1025.
Sect. Rhizirdinan 10251030, 1040, 1004, 1027 1024, 1025. 1030. 1040, 168, 168. 1004, 1027. 1030, 168, 1058, 1038, 1012. 1004, 1047, N0333. 1012, 1044, 1041 1027, N1013
N0905
Sect. 1012, 1058 1012, 1038. 168
Bromatorrhiza
Sect. 1004, 1027. 1040
Haplostemon
Sect. Anguium 1044, 1041 1044
<110> Institute of Vegetables and Flowers, Chinese Academy of
Agricultural Sciences <120> PHYLOGENETIC CLASSIFICATION METHOD OF ALLIUM
VEGETABLE RESOURCES
BASED ON DIFFERENT MOLECULAR MARKERS AND USE THEREOF
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<210> 2 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for amplification of rps16 intron
<400> 2 gtggtagaaa gcaacgtgcg actttcggga tcgaacatca attgcaac 48 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for amplification of trnL-F intergenic region <400> 3 cgaaatcggt agacgctacg atttgaactg gtgacacgag 40 i xml versicn=Nl, ON encoding=TUTFE-8" 7 2 <!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing 1.3//EN" "ST26Sequencelisting V1 3.dtd"> 3 <3T26%squencelisting dudVersion=svi_an T11aName="HRIPIOI11TI0LL, main soïtwaceNems=*WIPO Sagueance? soitwareVersion="2.1.0% productions ie=vR023-07-11">
A <ApplicantFilieReference>HKIJP2021111011</ApplicantFileRefaerence> > <FariiestPricritvAppiicaticonidentification> 6 <IPOfficetoderCN</IPOfficeloder 7 <ApplicetionNumerTezt>202110499035.4</ArplicationNumberText> 8 <FilingDate>»2021-05-08«</FilingDatex 3 </EarliestPriorityapplicationidentification»> <ApplicantiName languagelode="en“>Institute of Vegetables and Flowers, Chinese
Academy of Agricultural Sciences</ApplicantName> iA <InventlonTitle languagelode="en">PHYLOGENETIC CLASSIFICATION METHOD OF ALLIUM
VEGETABLE GENETIC RESOURCES BASED ON DIFFERENT MOLECULAR MARKERS AND USE
THEREOF / InventicnTitle>
Le <JequanceTotalQuantity>6</SaquenceTotaluantity> 13 “<SequenceData seguanoelhNumbag="1%> 14 <INSDSeq>
Ls <IN3DSeq length»19</INSDSeq length> ia ZINSDSegq molitypes>DNAC/INSDSeq moltyper> i <IN3DSeq divisior>PAT</INSDIeqg division» 13 <INSDSeq feature-table>
Le <INSDFeature> <INSDFeaturs Keyrmisc feature</INSDFzature key> zl <INSDFeature location>l..19</IN3DFeature location» 22 <INSDhFeature quels» 22 <INSDQualifier id="gir> 24 <IN3DQualifier name>note</INSDQualifier name> <INSDQualifier valuerforward primer sequence for amplification of ITS /INSDQualifier value» zó «</INSDOQualifier»> wi </THSDFeaturs guals> 28 </INSDFeatures 23 <INSDPFSsarure:» 20 <IN3DFeature key>source</IiN3DFeature key» 31 <IN3DFeature lowation>l..19</INSDFeaturs location» 32 <INSDFeature guals> 33 <INSDOualifier> 34 <INSDQualifier name>PCR primers“/INSDQualifier name> <INSDQualifler valuve>fwd name: ITS1, fwd seq: tccgtaggtgaacctgcgg, rev name: ITS4, rev seq: tcctcegettattgatatge-/INSDOualifier value» 24 </INSDQualifier> u 37 <INSDOQualifieg> 385 <INSDQualifier name>mol type“/INSDQuali fier name> 38 <INSDQualifier valuerother DNA</INSDQualifier value» </INSDOualifier> 41 <INSDQualifler id="gd™> 47 <INSDQualifier name>organism</INSDQualifier name> 43 <IN3DQualifier value>synthetic construct</INSDQualifier value» 44 </INSDOQuali fier 4% </INSDFesature duals» 46 </INSDFeature> 47 </INSDSeg feature-tabhle> 43 <INSDSeq sequsnce>teegtaggtgaacctgegg</INEDS ag sequences 4% </INSDSeg> </Seguencedata> bi <SequenceData sequence lDNumben="2%> 52 <INSDSeq> 53 <IN3DSeqy Leng:th>20</IN5DSeq length 54 <INSDSeq moltype>DNA</INSDSeg moltype»
Sh <INSDSeq division>PAT</INSDSeg division» <INSDSeq feature-table> 57 <INSDFeature> 5a <INSDFeature keyrmisc feature</INSDFeature key> 53 <INSDFearure location>l..20</INSDFeature location» ai <INSDFeature guals> oi <INSDQualifler ìd='qg3">
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TO <INSDFeature quals> u
JL <IN3DQualifiers
GE <INSDQualifier name>PCR primers</INSDQualifilsr named 72 <INSDQualifier value>fwd name: ITS1, fwd seq: tccgtaggtgaacctgcgg, rev name: ITS4, rev seq: tecteegettattgatatge-/INSDQuali fier value» a </INSDQuali fier» in <INSDQualifier> 7E <INSDQualifier name>mol type</INSDQualifier name> 77 <INSDQualifler valverother DNA</INSDQualifier value» 78 </INSDOualifier> 73 <INSDOualifier id="qd4">
Sn <IN3DQualifier namevorganism“/INSDQualifier name> 21 <INSDQualifier valuersynthetic construct</INSDQuallifier value»
BZ <{INSDQualifier» 5% </INSDFeature quals>
G4 </INSDFeature> u “/INSDSeqg fesature-table> a <“INSDSeg sequencer»tectceegcttattgatatge</INSD5eq sequence» 27 </INSDSeq> u a8 </SeguenceDatas 59 <SequenceData zaquencalDNumben=n3n>
GO <INSDSeqd»>
Gl <INSDSeq length>24</INSD5eq length»
Gi <INSDSeq moltype>DNA</INSDSeg moltype>
EN <INSDSeq divislion»PAT</INSDSeqg division» 34 <INSDSeg feature~tablex 35 <INSDPsaturer
Ga <IN3DFeature keysmisc feature /INSDFeature key» a <IN3DFeature location>l..24</INSDFeaturs locations os <INSDFsature qualsg>
GG <INSDQualifier id="g5">
LOG CINSDQualifisr name>note</INSDQvali fier name>
Ol <INSDQualifier valus>forward primer sequence for amplification of rpsl16 intron</INSDOualifier valus> 103 </TNSDQualifier> u 103 </INSDFeature guals> 104 </INSDFeature>
LOS <INSDFearure>
LOG <INSDFeature key>source</INSDFeature key>
LT <INSDFeature location>l..24</INSDFeature locations 108 <INSDFealurse guals> 103 <INSDOQualifier» iijn <IN3DQualifier name>PCR primers</iN3DQualifier name> iid <INSDQualifier valiue>fwd name: rpsF, fwd seq: gtggtagaaagcaacgtgcgactt, rev name: rpsR, rev seq: tegggatcgaacatcaattgcaac:/INSDQualifier value»
Az </INSDOualifier>
LLS <IN3DQualifiers iid <INSDQualifier name>mol type</INSDQualifier name> ils <INSDOualifier valuesother DNA</INS3SDO0uelifier value» iis </INSDQualifier> u
Li <INSDQualiflisr id='gô">
Lin <IN3DQualifier namerorganism“/INSDQuali fier name>
Lis <INSDQualifier valuersynthetic construct</INSDQualifier values»
LED </INSDOualiLfier»> zl </INSDFeaturs duals? 127 </INSDFeaturer» 123 </INBDSeq feature-table> 124 <IN3DSeq seguencergtggtagaaagcaacgtgegactt</INSDSeq sequence 12% </INSDSeg> i2ë </SegusnceData»>
La? <Sequencebata zsegvenceliNumber="4*>
LEH <IN3D3eq> 140 <INSDSeq length>24</IN3DSeq length» ijd <INSDSeq moltype>DNA</INSDSeq moltype> 131 <IN3DSeq division»PAT</INSD3eq division» i122 <INSDSeq feature-table> 1373 <INSDFeabture> 134 <INSDFeature key>misc feature</INZDFeature key> 13 <INSDFeature location>l..24</INSDFeature location> 136 <INSDFeaturs quals> u 137 <INSDQuelifler in="g7">
Lig <INSDQualifier name>note</INSDQualifier name> 129 <IN3DQualifier value>reverse primer sequence for amplification of rpsl16 intron“/INSDQualifier value» 140 </INSDQuali fier»
Lal </INSDFeature qguals»
TAZ </IN3DFeature> 143 <INSDFeature> idd <INSDFeaturs keyrsource</INSDFeaturs key 145 <INSDFeature location>l..24</INSlFeature location» 14a <INSDFeature guals> 147 <INSDOQualifier> 148 <IN3DQualifier name>PCR primers</INSDQualifier name> ido <INSDQualifier valuerfwd name: rpsF, fwd seq: gtggtagaaagcaacgtgcgactt, rev name: rpsR, rev seq: tegggatcgaacatcaattgcaac“/INSDQvali fier value>
LEG </INSDOualifier> 151 <INSDOQualifier» 152 <IN3DQualifier namedmol type</INSDQualifisr name> 153 <INSDUvaelifier valuerother DNA</INSDGualifier value> 154 </INSDOuali fier» u 15% <INSDOQualifier id="g8*>
Lae <INSDOQualifier namerorganism</INSDQualifier name>
Ln? <INSDgQualifier value>synthetic construct“/INSDGualifier value» 158 </INSDOualifier> 153 </IN3DFeature guala>
Tan </INSDFeaturs> u
Lel </INSDSegy featurs-table>
Le: <INSDSeq seguence>tegggatcgaacatcaattgcaac</INSD3eq sequence»
LES </INSDSeg> 184 </SequenceData> is “<SequenceData segusnceliNumec=NS"> 18a <INSDSeqg> ia: <IN3DSeq length»20</INSDSeq length> 168 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 18% <IN3DSeq divisior>PAT</INSDIeqg division»
LFO <INSDSeq feature-table>
LUL <INS3DFesature>
LEE <INSDFeaturs Keyrmisc feature</INSDFzature key> 173 <INSDFeature location>l..20</IN3DFeature location» ijd <INSDhFeature quels» 175 <INSDQualifier id="g8%> ia <IN3DQualifier name>note</INSDQualifier name>
Li <INSDQualifier valuerforward primer sequence for amplification oftrnL-F intergenic region</INSDQualifier value» 175 </INSDOualiLfier»> ijn </INSDFeaturs duals? 18h </TNSDFeaturer
LEL <INSDFeature> i182 <IN3DFeature key>source</IN3DFeature key> 1873 <IN3DFeature location»l..20</INSDFeaturs locations 14 <INSDFsature qualsg>
TES <INSDVualifier>
LEE CINSDQualifisr name>PCR primers</INSDQualifisr name> iad <INSDQualifier value>fwd name: trnL F F, fwd seq: cgaaatcggtagacgctacg, rev name: trnL F R, rev seq: atttgaactggtgacacgag-/INSDQualifier value» iss </INSDOQuali fier iss <INSDOualifien>
LSD <INSDQualifier name>mol type“/INSDQualifier name>
LSL <INSDQualifier valuerother DNA</INSDQualifier value»
LGE </INSDOualiLfier»> 193 <INSDQualiifler id="g19'> ind <INSDQualifier name>organism</INSDQualifier name> ijb <IiNSDgualifier value>synthetic construct</INSDQualifier valued» 198 </INSDQuali fier»
LT </INSDFearure quals>
Le </INSDFeature>
Lah </INSDSeg feature-table> 200 <INSDSeq sequence>cgaaatcggtagacgctacg</INSDSeq sequence» 201 </INSDSeg> 202 </SeguenceData> 203 <SeguenceData semuenceIDNumber="6n> 204 <INSDSedg> 20% <INSDSeq length>20</INSD3eq lengths 208 <INSDSeq molitype>DNA</IN3DSeq moltype> wi’ <INSDSeq division>PAT</INSDSeq division» 208 <INSDSeq [eatureriabie> 203 <INSDPFSsarure:» zin <IN3DFeature keyrmisc feature</INSDFeature key> 21d <IN3DFeature lowation>l..20</INSDFeaturs location» 212 <INSDFeature guals> 243 <INSDQuaiifier id="gil’x» 24 <INSDOQualifier namernote</INSDQualifiesr name> ais <INSDQualifier value>reverse primer sequence for amplification oftrnL-F intergenic region</INSDQuslifier wvalue> 218 </INSDOualifier> u 2L7 </INSDFeature guals> 218 </INSDFealure> 21% <INSDFeature> 220 <INSDFeature key>source</INSDFeature key> met <INSDFeature location»l..20</INSDFeature locations
LE <INSDFealurse guals>
PP <INSDOQualifier» zld <IN3DQualifier name>PCR primers</iN3DQualifier name> 225 <INSDQualifier vaiue>fwd name: trnL F F, fwd seq: cgaaatcggtagacgctacg, rev name: trnL F R, rev seq: atttgaactggtgacacgag</INs5DQualifier value» zee «</INSDOQualifier»> wai <IN3DQualifiers
HER <INSDQualifier name>mol type</INSDQualifier name>
HES <INSDOualifier wvalue>other DNA</INSDCualifier value» 220 </INSDQualifier> u 221 <INSDOualifier id="gien> 232 <IN3DQualifier namerorganism“/INSDQuali fier name> 233 <INSDQualifier valuersynthetic construct</INSDQualifier valued acy </INSDOualifiers 235 </INSDFeaturs guals> 238 </TNSDFeaturer 227 </INBDSeq feature-table> 238 ZINSDSeq sequencsratttgaactggtgacacgag“/INSDSey seduenze> 238 </INSDSeg> 240 </SegusnceData»>
ZA </STi6SeguenceListing>
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