CN111733273A - DNA barcode sequence and method for identifying lycium species by using same - Google Patents
DNA barcode sequence and method for identifying lycium species by using same Download PDFInfo
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Abstract
The invention discloses a DNA barcode sequence and a method for identifying medlar species by using the same, belonging to the technical field of medlar species identification. The invention relates to a method for identifying variety of medlar and determining interspecies relationship of medlar by using a DNA bar code of psbH-petB of medlar, and simultaneously obtains a psbH-petB bar code database on the basis of the method.
Description
Technical Field
The invention belongs to the technical field of identification of lycium species, and particularly relates to a DNA (deoxyribonucleic acid) barcode sequence and a method for identifying the lycium species by using the DNA barcode sequence.
Background
Fructus Lycii (Lycium barbarum L.) is a famous and precious genuine medicinal material in China, and has effects of resisting oxidation, resisting tumor, softening blood vessel, reducing blood lipid, lowering blood sugar, promoting vital essence generation, protecting liver, improving eyesight, and enhancing immunity. Because genuine herbs and non-genuine herbs have the same basic origin or are closely related, they have high similarity in external form, habit, tissue structure and chemical components, so that the traditional identification method is difficult to identify accurately.
The DNA barcode is a new technology for identifying and identifying target species by using one or a few DNA fragments, has the characteristics of simple and convenient operation, high accuracy, quick identification and the like, and has become an attractive new direction and research hotspot in modern biology research. However, the existing barcode database of lycium is relatively deficient in species abundance and quantity, and still needs a lot of research. In recent years, researchers at home and abroad have conducted active research and study on DNA barcode gene sequences suitable for identifying plants.
The invention patent with publication number CN106282390A, Ningxia wolfberry fruit and wolfberry fruit SNP fast identification method, discloses a Ningxia wolfberry fruit and wolfberry fruit SNP fast identification method, which comprises the following steps: 1) amplifying a sequence containing a sequence shown in SEQ ID No.1 by using a sample DNA to be detected as a template; 2) sequencing the amplified product, analyzing the sequence shown by SEQ ID No.1, identifying the amplified product as the Lycium barbarum if the 97 th position and the 173 th position of the amplified product are C and the 200 th position is G from the 5 'end of the sequence shown by SEQ ID No.1, and identifying the amplified product as the Lycium barbarum if the 97 th position and the 173 th position of the amplified product are T and the 200 th position is A from the 5' end of the amplified product of SEQ ID No. 1. The invention can realize the rapid and accurate identification of the original plants, medicinal materials and crude decoction pieces of the Ningxia wolfberry and the wolfberry.
An article "identification of male sterile materials of Chinese wolfberry based on nrDNAITS sequence" discloses analysis of nrDNAITS (ribosomal DNA intragenic transcribed spacer) sequences in 7 parts of conventional Chinese wolfberry germplasm and 1 part of DNA of male sterile materials of Chinese wolfberry, and identification of male sterile materials of Chinese wolfberry from the molecular level. Extracting the DNA of the leaves of the Chinese wolfberry by adopting an improved CTAB method, amplifying and cloning nrdNAITS zones in the DNA by utilizing a synthesized specific primer, and sequencing and analyzing a target fragment. The result shows that the base sequences of the nrdNAITS regions of the male sterile material of the medlar and other 7 conventional medlar germplasms are obtained by first sequencing, the length of the whole ITS sequence is 559-633 bp, the average is 612bp, and 160 variation sites account for 25.3% in total; conserved site 473, accounting for 74.7%; 67 transition sites and 31 transversion sites. It is shown that the sequence analysis of the nrdNAITS zone can be used as a new identification method for identifying the male sterile material of the medlar.
However, in the prior art, the problems of delayed excavation and utilization of excellent resources of the medlar, unclear genetic background of medlar germplasm resources, unclear interspecific relations and the like still exist, and a unified rapid identification method for multiple varieties in different regions and different germplasm sources is still lacked.
Disclosure of Invention
The invention aims at the problems that the existing technology has lag in the excavation and utilization of excellent resources of medlar, unclear genetic background of medlar germplasm resources, unclear interspecific relationship, indistinguishable medlar species with similar appearance, habit and organizational structure and the like, particularly aiming at the problems that the barcode database of the lycium is relatively deficient in species abundance and quantity and the like, on the basis of the unique lycium barbarum germplasm resource garden in China and the breeding material obtained by carrying out new lycium barbarum variety breeding for a long time, meanwhile, the molecular marker rapid identification method of the lycium barbarum with representative germplasm in lycium barbarum, lycium barbarum and the like, local varieties of lycium barbarum, Sichuan, northern, Xinjiang, Yunnan, Hebei and the like, hybrid populations, space mutation populations, ploidy populations and the like is provided, and the molecular marker rapid identification method can be applied to identification of lycium barbarum species.
The invention aims to provide a method for identifying a Chinese wolfberry variety and determining the interspecies relationship of Chinese wolfberry, and simultaneously obtain a psbH-petB bar code database on the basis of the method, and by comparing a psbH-petB sequence of a sample to be identified with a sequence in the psbH-petB bar code database, the Chinese wolfberry variety can be effectively identified, the interspecies relationship of the Chinese wolfberry is determined, and an effective basis is provided for the Chinese wolfberry variety.
The technical scheme of the invention is as follows:
the first purpose of the invention is to provide a method for identifying the variety of the medlar by using a DNA bar code, wherein the DNA bar code is a psbH-petB bar code.
The identifiable species of Lycium barbarum include Lycium barbarum No.1 (l. barbararum lin), Lycium barbarum No.3, Lycium barbarum No. 4, Lycium barbarum No. 6, Lycium barbarum No. 9, Lycium barbarum (l. bararum Linn. var. aurantianum k.f. china barngar. nov.nov.), Lycium planum barbarum (Lycium barbarum Bianguo), Lycium ruthenicum (Lycium ruthenicum murr.), Lycium ninghamii No. 5 (l.bararum Linn), Lycium northern Lycium barbarum (Lycium chinense. var. potaninii (Pojark.) a. lu.), Lycium barbarum, 9001, Lycium ninghamatum, Lycium barbarum (Lycium chinense) q. syzeng), Lycium cannabifolia (l. bararum), Lycium barbarum (baion. yunna. i), Lycium barbarum (lyrum).
Preferably, the method for identifying the variety of the Chinese wolfberry based on the DNA bar code comprises the following steps:
1) extracting genome DNA from a medlar sample;
2) amplifying a sequence fragment of the psbH-petB barcode by using the genomic DNA as a template and primers shown as nucleotide sequences SEQ ID NO.25 and SEQ ID NO.26 to obtain a PCR product;
3) sequencing the PCR amplification product;
4) and (5) constructing a phylogenetic tree and identifying the medlar.
The invention obtains psbH-petB sequences by screening, carries out alignment arrangement by homology comparison, analyzes and calculates the base composition of a target sequence, the base variation frequency among sequences, the conversion and inversion frequency among sequences and the ratio thereof, compares the distribution of sequence intraspecific and interspecific differences, constructs a phylogenetic tree, establishes an identification technical system of psbH-petB DNA bar codes and is used for identifying Chinese wolfberry varieties.
Preferably, in the step 1), the sample genomic DNA is extracted by using a kit.
More preferably, in the step 1), the Kit is a DNA secure Plant Kit.
More preferably, the step of DNA extraction using the kit is as follows:
(1) extraction of DNA
Collecting fresh and tender leaves of a Chinese wolfberry Plant as a Chinese wolfberry sample, washing, freezing and storing at-80 ℃, and extracting the total DNA of the Chinese wolfberry sample by adopting a novel Plant genome DNA extraction Kit (DNA secure Plant Kit);
(2) DNA concentration and purity measurement
And (2) respectively carrying out agarose gel electrophoresis detection and ultraviolet spectrophotometer detection on the DNA extracted in the step (1).
Preferably, in the step (1), the kit extraction method comprises the following steps:
firstly, 100g of sample is ground in a multifunctional high-efficiency biological sample preparation instrument for 22 times/s for 2 min. 400ul of buffer LP1 and 6ul of RNase A (10mg/ml) were immediately added thereto, vortexed for 1min, and allowed to stand at room temperature for 10 min.
② adding 130ul buffer solution LP2, mixing well, and vortex shaking for 1 min.
③ centrifuging at 12000rpm for 5min, and transferring the supernatant to a new centrifuge tube.
Adding 1.5 times of buffer LP3 (testing whether absolute ethyl alcohol is added before use), immediately and fully shaking and mixing for 15sec, wherein flocculent precipitates may appear.
Fifthly, adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000rpm for 30s, pouring the waste liquid, and placing an adsorption column CB3 into the collecting pipe.
Sixthly, 600ul of rinsing liquid PW (whether absolute ethyl alcohol is added in the adsorption column CB3 is checked before use) is added into the adsorption column CB3, the adsorption column CB3 is placed into a collecting pipe after the adsorption column CB3 is centrifuged at 12000rpm for 30 s. (Note: if the adsorption column membrane is green, 500ul of absolute ethanol was added to adsorption column CB3, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and adsorption column CB3 was put into the collection tube)
And seventhly, repeating the operation step 6.
And eighthly, putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, and pouring the waste liquid. The adsorption column CB3 was left at room temperature for 15min to thoroughly dry the residual rinse solution in the adsorption material.
Ninthly, transferring the adsorption column CB3 into a clean centrifugal tube, hanging and dripping 100ul of elution buffer TE into the middle part of the adsorption film, placing the adsorption film at room temperature for 2min, centrifuging the adsorption film at 12000rpm for 2min, and collecting the solution into the centrifugal tube.
Preferably, in the steps (2) -r, the operation is as follows:
1.2% agarose gel was prepared with 1.2g agarose and 100ml 1 × TAE buffer, and 4ul ddH was added to the PCR tube2The detection system of O +1ul DNA sample (undiluted) +1ul 6 loading buffer was subjected to agarose gel electrophoresis. The results were observed under a UV gel imaging system as shown in FIG. 1.
Preferably, in the steps (2) - ②, the operation is as follows:
preheating the ultraviolet spectrophotometer in advance, adding 99ul ddH into the PCR tube2O +1ul DNA samples (undiluted) were tested. The detection result shows the concentration and OD of the sample260/OD280Ratio, OD260/OD280The ratio should be 1.7-1.9, if elution is performed without using elution buffer, ddH is used2O, ratio is low because the pH and the presence of ions affect the light absorption but do not indicate low purity.
Preferably, in the step 2), the reaction system for PCR amplification is: firstly, pre-denaturation is carried out for 2min at 94 ℃; ② denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s (the annealing temperature can be adjusted between 58 ℃ and 60 ℃), extension at 72 ℃ for 2min, and 35 cycles; ③ keeping the temperature at 72 ℃ for 10 min; and fourthly, storing at 4 ℃. The PCR product was subjected to 1.0% agarose gel electrophoresis. After the gel electrophoresis detection, the amplification result is observed under an ultraviolet gel imaging system as shown in figure 2.
Preferably, in the step 3), the sequencing step is as follows:
(a) cloning of PCR products:
and (3) recovering the target band by using an AxyPrep DNA gel recovery kit, recovering and detecting by using 1.2% agarose gel electrophoresis, and taking the purified target DNA as a sequencing template. The recovered product was ligated to a T vector (pGEM-T) using pLB zero background rapid cloning kit, and then transformed into E.coli DH 5. alpha. for culture. Positive colonies were screened for AMP resistance and colony PCR was performed. Wherein, the amplification result observed under the ultraviolet gel imaging system is shown in figure 3.
(b) Sequencing and analysis of the sequence:
the colonies of positive clones were subjected to DNA sequencing and to homology alignment with the sequences published in NCBI and the sequences were analyzed.
Preferably, said step (b) operates as follows:
after PCR detection is carried out on positive colonies, the colonies containing target fragments are cultured by an LB liquid culture medium, 3 colonies are selected from each material and sent to a company Limited in Biotechnology engineering (Shanghai) to be sequenced by a Sanger method, and a psbH-petB sequence is obtained.
And carrying out homology comparison on the obtained DNA barcode gene sequence and published sequences in an NCBI database by using DNAMAN, and respectively carrying out alignment arrangement on the DNA barcode gene sequence of the medlar by using a Clustal X program. The phylogenetic analysis software MEGA7.0 is used to calculate the base composition of the target sequence, the base variation frequency among sequences, the conversion and inversion frequency among sequences and the ratio thereof, and the phylogenetic tree is constructed by comparing the distribution of the intraspecific and interspecific differences of the sequences. And establishing a psbH-petB bar code database for identifying the variety of the medlar.
The second purpose of the invention is to provide a wolfberry sample psbH-petB barcode database constructed based on the method, which comprises 24 psbH-petB barcodes, and the nucleotide sequence of the psbH-petB barcodes is shown in SEQ ID NO. 1-24.
The third purpose of the invention is to provide the application of identifying the variety of the medlar based on the psbH-petB bar code database of the medlar sample.
Preferably, the application of identifying the variety of the medlar based on the psbH-petB bar code database of the medlar sample comprises the following steps:
and comparing the psbH-petB bar code sequence of the sample to be identified with the sequences in the psbH-petB bar code database to identify the variety of the medlar and determine the interspecies relationship of the medlar.
By comparing the psbH-petB sequence of the sample to be identified with the sequence in the psbH-petB bar code database of the medlar sample, the species of medlar can be effectively identified, the interspecific relationship of medlar can be determined, and an effective basis is provided for classification and identification of the variety of medlar by determining the interspecific relationship of medlar to be detected and medlar in the bar code database.
Preferably, the method for obtaining the psbH-petB barcode sequence of the sample to be identified also comprises the steps of extracting genomic DNA, carrying out PCR amplification and sequencing PCR amplification products to obtain a corresponding sequence, and the operation steps are the same as the steps 1), 2) and 3) in the method for identifying the variety of the Chinese wolfberry by using the DNA barcode.
The invention has the beneficial effects that:
(1) the identification method for the Chinese wolfberry variety is established by utilizing the psbH-petB barcode gene for the first time. The method realizes effective expansion of the barcode database of the lycium in species abundance and quantity, and can effectively and quickly identify multiple species of different species sources, namely flat fruit lycium barbarum, black fruit lycium barbarum, yellow fruit lycium barbarum, round fruit lycium barbarum, red branch lycium barbarum, hybrid population, aerospace mutagenesis population, ploidy population and the like in different areas, namely local varieties of Ningxia lycium barbarum, Sichuan, northern, Xinjiang, Yunnan, Hebei and the like.
(2) Genetic diversity and genetic relationship of Lycium are revealed based on the psbH-petB gene. Meanwhile, an effective basis is provided for variety identification, classification and phylogenetic research of the medlar.
(3) Accurately identifying the variety of the Chinese wolfberry based on the psbH-petB gene.
(4) The invention establishes a psbH-petB bar code database of the medlar sample based on the identification method, wherein the psbH-petB bar code database comprises local varieties of Ningxia medlar, Sichuan, North, Xinjiang, Yunnan, Hebei, Sichuan and the like, and simultaneously comprises varieties of Lycium ruthenicum Murr, Lycium xanthocarpum, Lycium circulans, Lycium barbarum and the like, and different medlar sources of hybrid groups, space mutation groups, ploidy groups and the like, and are the medlar with representative germplasm in the whole country, so that effective basis can be provided for classification and identification of medlar varieties.
By comparing the psbH-petB bar code of the sample to be identified with the sequence in the psbH-petB bar code database of the medlar sample, the medlar species can be effectively identified, the interspecific relationship of medlar can be determined, and an effective basis is provided for classification and identification of medlar species.
Drawings
Fig. 1 is a DNA extraction detection result of a lycium barbarum sample according to embodiment 1 of the present invention, where a lane M: marker (DL2000DNA molecular marker); lanes 1-24 are the results of DNA extraction assays on samples of Lycium barbarum numbered 1-24 of example 1 of the present invention;
FIG. 2: the results of PCR amplification of the psbH-petB sequence of a portion of the Lycium barbarum sample of example 1 of the invention are shown in lane M: marker (DL2000DNA molecular marker), lanes 1-2: PCR products for both numbers 15; lanes 3-4: PCR products for both numbers 16; lanes 5-6: PCR products for both numbers 17; lanes 7-8: PCR products for all numbers 18; lanes 9-10: PCR products for both accession No. 19; lanes 11-12: the PCR products are all numbered 20, wherein lanes 1-2, 3-4, 5-6, 7-8, 9-10, 11-12 are PCR verifications performed twice for different samples.
FIG. 3: the results of cloning of the psbH-petB sequence for a portion of the samples of example 1 of the invention, in which lane M: marker (DL2000DNA molecular marker), lanes 1-6: cloning result of rpoB sequence of the medlar sample with number 11; lanes 7-12: the clone results of rpoB sequence of the Lycium barbarum sample No. 12 are positive clones in lanes 1-8 and 10, and negative clones in lanes 9 and 11-12 (results of multiple replicates).
FIG. 4: an NJ phylogenetic tree constructed for the psbH-petB barcodes in the wolfberry sample psbH-petB barcode database in example 1 of the present invention.
FIG. 5: the NJ phylogenetic tree of a part of psbH-petB bar codes in the wolfberry sample and the database to be identified in the experimental example 1 of the invention.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The present invention is further illustrated by the following specific examples.
Example 1 identification of Lycium barbarum samples and construction of a PsbH-petB Bar code database for Lycium barbarum samples
The invention will be further illustrated by the following examples.
1. Samples of the Lycium barbarum sample psbH-petB Bar code database
To construct a psbH-petB barcode database of Lycium barbarum samples, 24 total Lycium barbarum samples from different regions with similar morphology were collected and detailed in Table 1:
TABLE 1 Lycium plant samples (Lycium samples psbH-petB Bar code database)
(Note: variety numbers 11, 22-23 are from open literature 1: "Wang, Wan Asia Jun, An Wei, et al." identifying 21 parts of the public variety in Lycium plants [ J ]. Jiangsu agricultural science, 2019, 47(01):64-67. ", based on psbA-trnH sequence bar code," "variety number 13 is from open literature 2:" Shigang, 18 parts of Ningxia Lycium resources genetic diversity based on nrDNA ITS sequence [ J ]. Anhui agricultural science (24): 10379: "10379 and 10380." Table 1 "", variety number 22 is from literature 4: Shiqiu, 18 parts of Ningxia Lycium resources genetic diversity based on nrDNA ITS sequence [ J ]. Anhui agricultural science (24):10379 and 10380. Table 1; variety number 12 is from literature 3: "jump, Anhua, Zhao Hua, etc.;" SSR Piezhijiang agriculture, molecular marker development, J. university of the Hist History J. molecular marker, 2019,36(02): 215-; number 14 resource number: 1111C0003121000044, classification code: 11132115107)
2. Identification method of wolfberry sample and construction method of psbH-petB barcode database of wolfberry sample
1) Extracting genome DNA from a medlar sample;
collecting the fresh and tender leaves of the 24 parts of Chinese wolfberry plants in a 5ml freezing tube at the base of the Chinese wolfberry engineering and technology research institute of Ningxia agriculture and forestry academy of sciences, immediately placing the leaves into liquid nitrogen after marking, and preserving the leaves at minus 80 ℃. Sampling time: 6 months 2018, sampling site: the germplasm resource library of national forest trees of medlar in Ningxia Yichuan city is shown in table 1 in detail.
The total DNA is extracted by adopting a novel Plant genome DNA extraction Kit (DNA sure Plant Kit), and the extraction method comprises the following steps:
(1) extraction of DNA:
collecting fresh and tender leaves of a Chinese wolfberry Plant as a sample, washing, freezing and storing at-80 ℃, and extracting the total DNA of the Chinese wolfberry by adopting a novel Plant genome DNA extraction Kit (DNAsecure Plant Kit); the extraction method of the kit comprises the following steps:
firstly, 100g of sample is ground in a multifunctional high-efficiency biological sample preparation instrument for 22 times/s for 2 min. 400ul of buffer LP1 and 6ul of RNase A (10mg/ml) were immediately added thereto, vortexed for 1min, and allowed to stand at room temperature for 10 min.
② adding 130ul buffer solution LP2, mixing well, and vortex shaking for 1 min.
③ centrifuging at 12000rpm for 5min, and transferring the supernatant to a new centrifuge tube.
Adding 1.5 times of buffer LP3 (testing whether absolute ethyl alcohol is added before use), immediately and fully shaking and mixing for 15sec, wherein flocculent precipitates may appear.
Fifthly, adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000rpm for 30s, pouring the waste liquid, and placing an adsorption column CB3 into the collecting pipe.
Sixthly, 600ul of rinsing liquid PW (whether absolute ethyl alcohol is added in the adsorption column CB3 is checked before use) is added into the adsorption column CB3, the adsorption column CB3 is placed into a collecting pipe after the adsorption column CB3 is centrifuged at 12000rpm for 30 s. (Note: if the adsorption column membrane is green, 500ul of absolute ethanol was added to adsorption column CB3, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and adsorption column CB3 was put into the collection tube)
And seventhly, repeating the operation step 6.
And eighthly, putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, and pouring the waste liquid. The adsorption column CB3 was left at room temperature for 15min to thoroughly dry the residual rinse solution in the adsorption material.
Ninthly, transferring the adsorption column CB3 into a clean centrifugal tube, hanging and dripping 100ul of elution buffer TE into the middle part of the adsorption film, placing the adsorption film at room temperature for 2min, centrifuging the adsorption film at 12000rpm for 2min, and collecting the solution into the centrifugal tube.
(2) And (3) detecting the DNA concentration and purity:
and (2) respectively carrying out agarose gel electrophoresis detection and ultraviolet spectrophotometer detection on the DNA extracted in the step (1).
In the steps (2) -phi, the operation is as follows:
1.2% agarose gel was prepared with 1.2g agarose and 100ml 1 × TAE buffer, and 4ul ddH was added to the PCR tube2The detection system of O +1ul DNA sample (undiluted) +1ul 6 loading buffer was subjected to agarose gel electrophoresis. The results were observed under a uv gel imaging system (figure 1).
In the step (2) - ②, the operation is as follows:
preheating the ultraviolet spectrophotometer in advance, adding 99ul ddH into the PCR tube2O +1ul DNA samples (undiluted) were tested. The detection result shows the concentration and OD of the sample260/OD280Ratio, OD260/OD280The ratio should be 1.7-1.9, if elution is performed without using elution buffer, ddH is used2O, the ratio is low because the pH and the presence of ions affect the light absorption, but does not indicate a low purity。
2) Amplifying a sequence fragment of the psbH-petB barcode by using the genomic DNA as a template and primers shown as nucleotide sequences SEQ ID NO.37 and SEQ ID NO.38 to obtain a PCR product;
adding reagents required by amplification such as primers and the like into the DNA obtained in the step 1) as a template to perform PCR amplification. Specific primers and amplification systems are shown in tables 2 and 3.
Designing a primer as follows:
TABLE 2 Universal primers for the DNA barcode gene psbH-petB
(ii) PCR amplification System:
the genomic DNA of the test material was subjected to PCR amplification using the above primers, and the amplification system is shown in Table 3:
TABLE 3 DNA Bar code reaction System
In the step 2), the reaction system of PCR amplification is as follows: firstly, pre-denaturation is carried out for 2min at 94 ℃; ② denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s (the annealing temperature can be adjusted between 58 ℃ and 60 ℃), extension at 72 ℃ for 2min, and 35 cycles; ③ keeping the temperature at 72 ℃ for 10 min; and fourthly, storing at 4 ℃. The PCR product was electrophoresed on 1.0% agarose gel and electrophoresed. After the gel electrophoresis detection, the amplification result is observed under an ultraviolet gel imaging system, taking the sample number of 15-20 as an example, and referring to the attached figure 2, the amplification result shows that the length of the psbH-petB sequence is about 1800.
3) Sequencing the PCR amplification product, wherein the sequencing steps are as follows:
(a) cloning of PCR products:
and (3) recovering the target band by using an AxyPrep DNA gel recovery kit, recovering and detecting by using 1.2% agarose gel electrophoresis, and taking the purified target DNA as a sequencing template. The recovered product was ligated to a T vector (pGEM-T) using pLB zero background rapid cloning kit, and then transformed into E.coli DH 5. alpha. for culture. Wherein, the amplification result is observed under an ultraviolet gel imaging system, the medlar with the numbers of 11 and 12 is taken as an example, and the psbH-petB gene has good amplification result, clear bands and obvious cloning result according to the analysis shown in figure 3 and referring to figure 3.
(b) Sequencing and analysis of the sequence:
DNA sequencing of colonies of positive clones and homology alignment with sequences published in NCBI, sequence analysis, step (b) operating as follows:
after PCR detection is carried out on positive colonies, the colonies containing target fragments are cultured by an LB liquid culture medium, 3 colonies are selected from each material and sent to a company Limited in Biotechnology engineering (Shanghai) to be sequenced by a Sanger method, and a psbH-petB sequence is obtained.
4) And (5) constructing a phylogenetic tree and identifying the medlar.
And carrying out homology comparison on the obtained DNA barcode gene sequence and published sequences in an NCBI database by using DNAMAN, and respectively carrying out alignment arrangement on the DNA barcode gene sequence of the medlar by using a Clustal X program. The phylogenetic analysis software MEGA7.0 is used to calculate the base composition of the target sequence, the base variation frequency among sequences, the conversion and inversion frequency among sequences and the ratio thereof, and the phylogenetic tree is constructed by comparing the distribution of the intraspecific and interspecific differences of the sequences. And establishing a psbH-petB bar code database for identifying the variety of the medlar.
The total length of the psbH-petB barcode sequence is 1878bp, the conserved sites are 1865 and account for 99.3%, the mutation sites are 5 and account for 0.3% by using phylogenetic analysis software MEGA7.0, wherein the information sites are 3, the ancestral sites are 2, the base transition value is 0.6, and the average value of GC content is 36.4%.
After sequence comparison analysis, 4 parts of Huangguo change, Lycium ruthenicum Murr, Ningnong wolfberry No. 5 and Changji wolfberry are transformed from T to G at 359bp, from A to G at 447bp and from A to C at 1051 bp. A base C is inserted into 1248bp of Ningnong Qiqi No. 5; 7bp base, namely TATTTTA, is inserted into the Yunnan medlar at 658 bp; the Chinese wolfberry is converted from C to A at 1070bp and from C to T at 1395 bp.
Phylogenetic trees were constructed by comparing the distribution of sequence intraspecific and interspecific differences (FIG. 4). The cluster map of the psbH-petB barcode sequence is divided into two major branches, wherein the Huangguoyuan, the Lycium ruthenicum Murr, the Ningnong Lycium Chinense No. 5 and the Changji Lycium Chinense are gathered into one major branch, and the parent relationship of the major branch with other varieties is far. The other 20 germplasm were gathered together with closest relationship. 4 varieties of space mutation, yellow fruit change, black fruit medlar and Changji medlar of the hybrid variety of the red fruit medlar and the black fruit can be identified according to the psbH-petB sequence, and the self-expanding support rate of the branch of the cluster map is higher and is 94%.
Therefore, the DNA bar code provided by the invention can be applied to the construction of the medlar phylogenetic tree and further used for researching the phylogeny of medlar intra-species and interspecies. Further proves the effectiveness and feasibility of the DNA bar code provided by the invention in the variety identification, classification and phylogenetic research of the medlar.
Meanwhile, the embodiment of the invention constructs a psbH-petB bar code database based on a bar code psbH-petB sequence, and the database comprises Ningqi No.1, Ningqi No.3, Ningqi No. 4, Ningqi No. 6, Ningnongqi No. 9, Huangguoqi changed medlar, Flat medlar, Black medlar, Ningnongqi No. 5, northern medlar, round medlar, 9001, Ningxia yellow medlar, Sichuan medlar, hemp leaf medlar, white medlar, Chinese medlar, Yunnan medlar, trailing medlar, purple handle medlar, Jiangbu medlar, Heqin medlar, Changji medlar, Xin medlar and other different medlar sources, which are the medlar with representative germplasm in the whole country, thereby providing an effective basis for classification and identification of medlar.
By comparing the psbH-petB sequence of the sample to be identified with the sequence in the psbH-petB bar code database of the medlar sample, the species of medlar can be effectively identified, the interspecific relationship of medlar can be determined, and an effective basis is provided for classification and identification of the variety of medlar by determining the interspecific relationship of medlar to be detected and medlar in the bar code database.
Experimental example 1 identification of variety of Lycium barbarum by barcode database
1. Sampling
And comparing and identifying 18 parts of to-be-detected medlar samples with barcodes of psbH-petB barcode databases of the part of medlar samples in the embodiment 1 by selecting 18 parts of to-be-detected medlar samples with the serial numbers of Ningqi No.2, Ningqi No. 5, Ningqi No. 7, Mongolian medlar No.1, Ningqi No.1, AN-YN-02, strain 6, rhubarb fruit, jute leaf, 332, HZ-13-01, ZH-13-08, W-12-27, W-11-15, ZH-13-08-02, W-13-30, W-13-26 and W-12-26, wherein the 18 parts of to-be-detected medlar cannot be simply distinguished by shape structure and color and cannot be identified by using a morphological method. The experimental example was identified using the DNA barcode technique.
TABLE 4 number of sample and production area information of fructus Lycii to be tested
DNA extraction and concentration detection, PCR amplification, PCR product cloning, sequence sequencing and analysis were the same as in example 1.
3. Analysis of sequence results
The total length of the psbH-petB barcode sequence is 1878bp, the conserved sites are 1874 and account for 99.7%, the mutation sites are 3 and account for 0.2% by MEGA7.0 analysis, wherein the information sites are 3, the ancestral sites are 0, the base transition value is 0.6, and the average value of GC content is 36.4%.
After sequence comparison and analysis, 6 parts of black-and-hetero aerospace mutant HZ-13-01, ZH-13-08, W-12-27, W-11-15, W-13-26, W-12-26, yellow fruit mutation (a bar code database, number 6) and Lycium ruthenicum Murr (a bar code database, number 8) to be detected are converted from T to G at 359bp, from A to G at 447bp and from A to C at 1052 bp; inserting 7bp base, namely TATTTTA, into 658bp positions of a sample AN-YN-02, a strain 6 and Yunnan medlar (a bar code database, the number is 18) to be detected; 1 base A is inserted into 775bp of a sample ZH-13-08-02 to be detected. And secondly, calculating genetic distances by adopting a K2P model (Kimura 2-parameter model) shown in a table 5, wherein the minimum genetic distance between the lycium ruthenicum varieties and the black hybrid aerospace induced varieties is 0.00000, and the maximum genetic distance between the lycium ruthenicum varieties and the lycium ruthenicum varieties is 0.001606.
As can be seen by locus analysis and genetic distance analysis, 359bp, 447bp and 1052bp are characteristic loci for identifying the variety of the Lycium ruthenicum Murr and the variety of the Lycium ruthenicum Murr.
Phylogenetic trees were constructed by comparing the distribution of sequence intraspecific and interspecific differences (FIG. 5). Wherein 6 parts of the black hybrid space-induced variety to be tested HZ-13-01, ZH-13-08, W-12-27, W-11-15, W-13-26 and W-12-26 are gathered into one branch with yellow fruit change and black fruit medlar, the genetic relationship is recent, the self-expansion rate is 94%, and the reliability is high. The rest 12 germplasms to be tested are gathered together with Ningqi No.1 (barcode database, number 1), hemp leaf (barcode database, number 15) and Yunnan medlar, and the genetic relationship is nearest. The hemp leaves and hemp leaves of the sample to be tested are the current breed of the Ningxia wolfberry cultivar, the Ningqi No.2 sample to be tested is obtained by selection from a production park of the hemp leaves, and the Ningqi dish No.1 sample to be tested is obtained by hybridization and breeding of the Ningqi No.1 sample and wild wolfberry. The result shows that the clustering analysis is consistent with the pedigree source, and the reliability is high.
TABLE 5 genetic distance analysis of psbH-petB identification of variety of Lycium barbarum
SEQUENCE LISTING
<110> research institute of Lycium barbarum engineering technology of Ningxia academy of sciences and forestry
<120> DNA barcode sequence and method for identifying lycium species thereof
<130>1
<160>26
<170>PatentIn version 3.5
<210>1
<211>1870
<212>DNA
<213>Ningqi No.1 (L. barbarum Linn)
<400>1
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>2
<211>1870
<212>DNA
<213>Ningqi No.3 (L. barbarum Linn)
<400>2
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>3
<211>1870
<212>DNA
<213>Ningqi No.4 (L. barbarum Linn)
<400>3
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>4
<211>1870
<212>DNA
<213>Ningqi No.6 (L. barbarum Linn)
<400>4
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>5
<211>1870
<212>DNA
<213>L. barbarum Linn
<400>5
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>6
<211>1870
<212>DNA
<213>L. barbarum Linn. var. auranticarpum K.F.Ching var. nov.
<400>6
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcga 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattaata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aatcaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>7
<211>1870
<212>DNA
<213>Lycium barbarum Bianguo
<400>7
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>8
<211>1870
<212>DNA
<213>Lycium ruthenicum Murr.
<400>8
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcga 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattaata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aatcaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>9
<211>1871
<212>DNA
<213>L. barbarum Linn
<400>9
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcga 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattaata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgtgtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aatcaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc ccacgtcaat atattttatt 1260
gtttaggggg aattacgctt acttgttttt tagtacaagt agctactggg tttgctatga 1320
ctttttacta tcgtccgacc gttactgagg cttttgcttc tgttcaatac ataatgactg 1380
aagccaactt tggttggtta atccgatcag ttcatcgatg gtcggcaagt atgatggtcc 1440
taatgatgat cctgcatgta tttcgtgtgt atctcaccgg cggatttaaa aaacctcgcg 1500
aattgacttg ggttacaggc gtggttctag ctgtattaac cgcatctttt ggcgtaactg 1560
gttattcctt acctcgggac caaattggtt attgggcagt gaaaatagta acaggtgtcc 1620
ctgacgctat tcctgtaata ggatcacctt tggtcgaatt attgcgcgga agcgctagtg 1680
tgggacaatc cactttgact cgtttttata gtttacacac ttttgtattg ccgcttctta 1740
ctgccgtatt tatgttaatg cactttccaa tgatacgtaa acaaggtatt tctgggcctt 1800
tatagagcaa agaaaaatat atcctaaata tttgtaatca atcatttatc acttggtgga 1860
ggaatatata g 1871
<210>10
<211>1870
<212>DNA
<213>Lycium chinense MilL. var. potaninii (Pojark.) A. M. Lu
<400>10
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>11
<211>1870
<212>DNA
<213>lycium
<400>11
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>12
<211>1870
<212>DNA
<213>lycium
<400>12
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>13
<211>1870
<212>DNA
<213>lycium
<400>13
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>14
<211>1870
<212>DNA
<213>Lycium chinense
<400>14
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>15
<211>1870
<212>DNA
<213>Damaye (L. barbarum Linn)
<400>15
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>16
<211>1870
<212>DNA
<213>Baihua(L. barbarum)
<400>16
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>17
<211>1870
<212>DNA
<213>L. chinense Mill. var.
<400>17
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttacatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaatttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>18
<211>1876
<212>DNA
<213>Lycium yunnanenseKuang et A.M.Lu
<400>18
cgtttattta caacggaatg gtatacaaag tcaacagatc tcaatgaata caataggatt 60
tatggctaca caaactgttg agaacagttc tagatctggt ccaagacgaa ctgcggtagg 120
agatttatta aaaccattga attcggaata tggtaaagta gctcctgggt ggggaactac 180
tcctttgatg ggtgtcgcaa tggccttatt tgcggtattt ctatctatta ttttggagat 240
ttataactct tccgttttat tggatggaat ttcaatgaat taggtctata agaaccccaa 300
agtccttgct tttgagtcca aaatgaatca tttagagctc cgatttctag tccattctat 360
tttcttttgg tagttcgatc gtggaatttc tttgtttctg tatttccgga gtatgagtgt 420
gtgacttgtt ataattgatc ctattgatag tacagagaat gggtctgtca tcttgataga 480
gatggttcta cttcgtcaga tatttattcg aatatttgga acacgaaata gattaagaaa 540
tatttgaact atgattcata cttaatattc agacctcgtg tccgggttcc aaaaaattgt 600
caaacaaaga attctaattt ctaaatcgaa agattctttt ctttcaaccc ctatttatat 660
tttatatttt gaccaaaagc aaaacctttc tttgaatttt tagtcattct atttattcag 720
ggaataagtg atgatccgag gattcttact cagggaatcc ttgatttgat ttaggttagg 780
tttttttatt gaatcatcgt ggttctagta tgaatctgag gttttaatcg attcataggg 840
tcttaacaag agaattccta tcaataataa agaaaacaaa taataaaagc catattccac 900
aaaaacaaat tatagggaaa aagagaattc aagaggccca taagtatcaa aataaagata 960
aagacgactg cgccaacttg atattttggt attatcgcca caaagaagag ctttcggatt 1020
ttccagagaa gatgggatca gaacttaata aatttaaaac tttctattcc atatccgttg 1080
caactagtat ttgggtgttt ttgcttgagc tgtacgagat gaaagtctca tatacggttc 1140
tcagaggggg agttccgcct atctcaataa agtatatgat tggttcgaag aacgtctcga 1200
gattcaagca attgcggatg atataactag taaatacgtt cctccccacg tcaatatatt 1260
ttattgttta gggggaatta cgcttacttg ttttttagta caagtagcta ctgggtttgc 1320
tatgactttt tactatcgtc cgaccgttac tgaggctttt gcttctgttc aatacataat 1380
gactgaagcc aactttggtt ggttaatccg atcagttcat cgatggtcgg caagtatgat 1440
ggtcctaatg atgatcctgc atgtatttcg tgtgtatctc accggcggat ttaaaaaacc 1500
tcgcgaattg acttgggtta caggcgtggt tctagctgta ttaaccgcat cttttggcgt 1560
aactggttat tccttacctc gggaccaaat tggttattgg gcagtgaaaa tagtaacagg 1620
tgtccctgac gctattcctg taataggatc acctttggtc gaattattgc gcggaagcgc 1680
tagtgtggga caatccactt tgactcgttt ttatagttta cacacttttg tattgccgct 1740
tcttactgcc gtatttatgt taatgcactt tccaatgata cgtaaacaag gtatttctgg 1800
gcctttatag agcaaagaaa aatatatcct aaatatttgt aatcaatcat ttatcacttg 1860
gtggaggaat atatag 1876
<210>19
<211>1870
<212>DNA
<213>Manshenggouqi (L.bararum)
<400>19
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>20
<211>1870
<212>DNA
<213>Ziguogouqi (L.barbarum)
<400>20
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>21
<211>1870
<212>DNA
<213>Lycium dasystemum
<400>21
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>22
<211>1870
<212>DNA
<213>lycium
<400>22
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>23
<211>1870
<212>DNA
<213>lycium
<400>23
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcga 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattaata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aatcaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>24
<211>1870
<212>DNA
<213>Lycium dasystemumPojark
<400>24
tcgtttattt acaacggaat ggtatacaaa gtcaacagat ctcaatgaat acaataggat 60
ttatggctac acaaactgtt gagaacagtt ctagatctgg tccaagacga actgcggtag 120
gagatttatt aaaaccattg aattcggaat atggtaaagt agctcctggg tggggaacta 180
ctcctttgat gggtgtcgca atggccttat ttgcggtatt tctatctatt attttggaga 240
tttataactc ttccgtttta ttggatggaa tttcaatgaa ttaggtctat aagaacccca 300
aagtccttgc ttttgagtcc aaaatgaatc atttagagct ccgatttcta gtccattcta 360
ttttcttttg gtagttcgat cgtggaattt ctttgtttct gtatttccgg agtatgagtg 420
tgtgacttgt tataattgat cctattgata gtacagagaa tgggtctgtc atcttgatag 480
agatggttct acttcgtcag atatttattc gaatatttgg aacacgaaat agattaagaa 540
atatttgaac tatgattcat acttaatatt cagacctcgt gtccgggttc caaaaaattg 600
tcaaacaaag aattctaatt tctaaatcga aagattcttt tctttcaacc cctatttata 660
ttttgaccaa aagcaaaacc tttctttgaa tttttagtca ttctatttat tcagggaata 720
agtgatgatc cgaggattct tactcaggga atccttgatt tgatttaggt taggtttttt 780
tattgaatca tcgtggttct agtatgaatc tgaggtttta atcgattcat agggtcttaa 840
caagagaatt cctatcaata ataaagaaaa caaataataa aagccatatt ccacaaaaac 900
aaattatagg gaaaaagaga attcaagagg cccataagta tcaaaataaa gataaagacg 960
actgcgccaa cttgatattt tggtattatc gccacaaaga agagctttcg gattttccag 1020
agaagatggg atcagaactt aataaattta aaactttcta ttccatatcc gttgcaacta 1080
gtatttgggt gtttttgctt gagctgtacg agatgaaagt ctcatatacg gttctcagag 1140
ggggagttcc gcctatctca ataaagtata tgattggttc gaagaacgtc tcgagattca 1200
agcaattgcg gatgatataa ctagtaaata cgttcctccc cacgtcaata tattttattg 1260
tttaggggga attacgctta cttgtttttt agtacaagta gctactgggt ttgctatgac 1320
tttttactat cgtccgaccg ttactgaggc ttttgcttct gttcaataca taatgactga 1380
agccaacttt ggttggttaa tccgatcagt tcatcgatgg tcggcaagta tgatggtcct 1440
aatgatgatc ctgcatgtat ttcgtgtgta tctcaccggc ggatttaaaa aacctcgcga 1500
attgacttgg gttacaggcg tggttctagc tgtattaacc gcatcttttg gcgtaactgg 1560
ttattcctta cctcgggacc aaattggtta ttgggcagtg aaaatagtaa caggtgtccc 1620
tgacgctatt cctgtaatag gatcaccttt ggtcgaatta ttgcgcggaa gcgctagtgt 1680
gggacaatcc actttgactc gtttttatag tttacacact tttgtattgc cgcttcttac 1740
tgccgtattt atgttaatgc actttccaat gatacgtaaa caaggtattt ctgggccttt 1800
atagagcaaa gaaaaatata tcctaaatat ttgtaatcaa tcatttatca cttggtggag 1860
gaatatatag 1870
<210>25
<211>22
<212>DNA
<213> Artificial sequence
<400>25
tcgtttattt acaacggaat gg 22
<210>26
<211>22
<212>DNA
<213> Artificial sequence
<400>26
ctatatattc ctccaccaag tg 22
Claims (8)
1. A method for identifying Chinese wolfberry varieties by using DNA bar codes is characterized in that: the DNA barcode is a psbH-petB barcode.
2. The method for identifying the variety of Lycium barbarum by using DNA barcode as claimed in claim 1, wherein the method comprises the steps of: the method comprises the following steps:
1) extracting genome DNA from a medlar sample;
2) amplifying a sequence fragment of the psbH-petB barcode by using the genomic DNA as a template and primers shown as nucleotide sequences SEQ ID NO.25 and SEQ ID NO.26 to obtain a PCR product;
3) sequencing the PCR amplification product;
4) and (5) constructing a phylogenetic tree and identifying the medlar.
3. The method for identifying the variety of Lycium chinense Miller by using DNA barcode as claimed in claim 2, wherein the method comprises the following steps: in the step 1), the sample genome DNA is extracted by using a kit.
4. The method for identifying the variety of Lycium chinense Miller by using DNA barcode as claimed in claim 3, wherein the method comprises the following steps: in the step 1), the extraction of DNA is as follows:
(1) extraction of DNA
Washing the medlar sample, freezing and storing at-80 ℃, and extracting the total DNA of the medlar sample by using a Kit (DNA secure Plant Kit);
(2) DNA concentration and purity measurement
And (2) respectively carrying out agarose gel electrophoresis detection and ultraviolet spectrophotometer detection on the total DNA extracted in the step (1).
5. The method for identifying the variety of Lycium chinense Miller by using DNA barcode as claimed in claim 2, wherein the method comprises the following steps: in the step 2), the reaction system of PCR amplification is as follows: firstly, pre-denaturation is carried out for 2min at 94 ℃; ② denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s (the annealing temperature can be adjusted between 58 ℃ and 60 ℃), extension at 72 ℃ for 2min, and 35 cycles; ③ keeping the temperature at 72 ℃ for 10 min; and fourthly, storing at 4 ℃.
6. A psbH-petB barcode database of a Lycium barbarum sample constructed by the method of any one of claims 1-5, comprising the psbH-petB barcode 24, having the nucleotide sequence set forth in SEQ ID nos. 1-24.
7. Use of the psbH-petB barcode database of a Lycium barbarum sample of claim 6 for identifying varieties of Lycium barbarum.
8. Use of the psbH-petB barcode database of a Lycium barbarum sample of claim 6 for identifying a variety of Lycium barbarum, wherein: the method comprises the following steps: and comparing the psbH-petB bar code sequence of the sample to be identified with the sequences in the psbH-petB bar code database to identify the variety of the medlar and determine the interspecies relationship of the medlar.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110229927A (en) * | 2019-06-20 | 2019-09-13 | 上海诺德生物实业有限公司 | A kind of method and its application based on DNA bar code identification black fruit fructus lycii |
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| CN110229927A (en) * | 2019-06-20 | 2019-09-13 | 上海诺德生物实业有限公司 | A kind of method and its application based on DNA bar code identification black fruit fructus lycii |
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