CN1614032A - Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence - Google Patents
Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence Download PDFInfo
- Publication number
- CN1614032A CN1614032A CN 200310105037 CN200310105037A CN1614032A CN 1614032 A CN1614032 A CN 1614032A CN 200310105037 CN200310105037 CN 200310105037 CN 200310105037 A CN200310105037 A CN 200310105037A CN 1614032 A CN1614032 A CN 1614032A
- Authority
- CN
- China
- Prior art keywords
- dna
- sequence
- kelp
- authentication method
- kelp gametophyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention was involved in identification of kelp gametocyte (belonging to economic alga), namely the identification method of kelp gametocyte and part or the whole DNA sequence. The steps were following: a) The whole DNA was extracted. b) The whole DNA from the extract was amplified by PCR method basing on the DNA as template. c) PCT products were purified. d) The sequence of the purified production was assayed to determine rDNA ITS region including whole sequence of ITS1,5.8S and ITS2. e) The sequence alignment soft and manual work was used to analyse the difference between DNA sequence and the sequence in data brary. ADVANTAGE-The invention provided an effective method to extract high quality DNA of kelp gametocyte. Sequence of rDNA ITS region of China coastal kelp gametocyte was determined.
Description
Technical field
The present invention relates to the authentication method of economical alga kelp gametophyte, i.e. the authentication method of various trait kelp gametophyte and adoptable part or all of dna sequence dna.
Background technology
Sea-tangle is that individuality is bigger in the seashore plant, and matter is gentle delicious, a kind of important economical alga that nutritive value and economic worth are higher, and applicating history is long.Kelp nourishing is worth high, always is considered as the sea by people and hides treasure, and amount of iodine also contains rich in protein, fat, amino acid, VITAMIN and inorganic salt etc. in addition up to 0.2-0.4%, and many countries are described as it " protective foods ".Sea-tangle is important industrial raw material, and comprehensive utilization can be extracted multiple Industrial products such as algin, iodine, N.F,USP MANNITOL, sargassun essence and Repone K.Laver also has important pharmaceutical use.Put down in writing in " Bencao Jingshu " of Ancient Times in China and the Compendium of Material Medica: sea-tangle cures mainly " 12 kinds the coalescent gas of oedema goiter and tumor, hemorrhoid ", " diuresis " etc.Because sea-tangle contains abundant iodine element, but effectively preventing thyromegaly iodine deficiencys such as (being commonly called as roughneck or big neck).The effect that often edible sea-tangle brings high blood pressure down in addition.Sea-tangle has in " Chinese marine alga will " record: narrow leaf sea-tangle (Laminaria angustata Kjellm.), palmate sea-tangle (L.digitata Lamx.), sea-tangle (L.japonica Aresch), Changhai band (L.longissima Mijabe), wrinkle sea-tangle (L.religiosa) and sugared sea-tangle (L.saccharina (L.) Lam.) etc.
Since sea-tangle female and male gametophytes separation method was set up, along with the development of breed and crossbreeding technology, many sea-tangle new variety with good character had obtained widespread use aborning.But different main laminarias or the gametophyte with same main laminaria of different good characters are difficult to make a distinction from naked eyes, mainly are by microscopic examination at present.Along with molecular biological fast development, district (ISSR) etc. had been widely used in the evaluation of important economical alga between some molecular marking techniques such as random primer amplification polymorphism (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and simple sequence repeated.Because this several marking methods stability and repeatability is not high, take time and effort, limited their application in practice greatly.
RDNA is the gene of coding ribosome-RNA(rRNA), and with mode from beginning to end repeated arrangement on karyomit(e), the copy number in each cell is between 1000 to 10000 in plant nucleolus.Coding region, non-coding region and nontranscribed domain that evolutionary rate does not wait have been comprised in the rDNA, ITS (Internal transcribed spacer) is between the non-encoding transcription transcribed spacer of (ITS2) between (ITS1) and 5.8S and the 26S between 18S and the 5.8S in the rDNA, studies show that at present, the base-pair sequence of rDNA genes such as 18S, 5.8S and 26S is very conservative, and the difference between different plants is very little; The base-pair sequence that is positioned at intergenic ITS1 of plant rDNA and ITS2 then differs greatly.Because ITS is present in the high multiple rDNA, fast and the fragment length of evolutionary rate is little, and (ITS1 is between 100-400bp in red algae, ITS2 is between 400-1400bp), add that coordinate to evolve (concerted evolution) makes this fragment very consistent between the genome different repeat units, thereby very be suitable for the research of botanical system growth and evolution and Idioplasm identification.Especially in the last few years, be based upon the foundation and the widespread use of direct sequence measurement on the PCR basis, between belonging in the ITS sequential analysis has become on the sequence level section and the plant of subordinate between planting named, classification, sibship is determined and the important means of phyletic evolution research.
Summary of the invention
The purpose of this invention is to provide a kind of authentication method and adoptable part or all of dna sequence dna of various trait kelp gametophyte, from molecular level, the various trait kelp gametophyte is carried out discriminatory analysis, make the result more accurate, more reliable.
For achieving the above object, the present invention has at first set up the preparation method of a cover kelp gametophyte high quality DNA, then the different gametophyte rDNA of sea-tangle ITS region sequence is checked order, and has obtained the rDNA ITS complete sequence of (comprising ITS1,5.8S and ITS2).
The present invention utilizes rDNA ITS district dna sequence dna to differentiate that the method for various trait kelp gametophyte comprises the steps: to carry out the collection of frond material, evaluation and preservation work;
(1) total DNA extraction: get the frond material of fresh and healthy, after handling with sterilized water,, extract total DNA at liquid nitrogen kind grind into powder;
(2) pcr amplification: the DNA that obtains with step (1) is a template, with the primer of oligonucleotide as polymerase chain reaction (PCR), and amplification ITS section, used primer picks up from the conservative section of frond 5.8S and 28S respectively,
Adopt forward primer P1:5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ',
Reverse primer P4:5 ' AATCCTGGTTAGTTTCTTTTCCTC3 ' utilizes this primer that total DNA is carried out pcr amplification reaction;
(3) PCR product purification;
(4) determined dna sequence: the DNA behind the purifying carries out sequencing (using direct sequencing technologies), establishes rDNA ITS district, comprises the complete sequence in ITS1,5.8S and ITS2 district;
(5) dna sequence data analysis:
The sequence one in rDNA ITS district to be analyzed is arranged software with contraposition and is carried out the contraposition arrangement, and be aided with artificial check and correction after measured, with the difference between each sequence in phylogenetic analysis each sample dna sequence dna of software analysis and the database.
Each sample repeats 3 times to order-checking at least from DNA extraction.
Described total DNA extraction can adopt the method for CTAB extracting and Glassmilk purifying; Or after fresh frond added the liquid nitrogen grind into powder, transfer to and extract in the damping fluid, use chloroform behind the 63-65 ℃ of water-bath 30-60min: the primary isoamyl alcohol extracting is once; Add the KAC of the 4M of 10%SDS and 1/3 volume in the supernatant liquor, use chloroform behind the ice bath 10-20min: primary isoamyl alcohol extracting is more once got supernatant liquor and is added 2/3 volume isopropanol precipitating DNA; DNA is dissolved in the TE damping fluid (pH 7.5 for 10mM Tris-HCl, 1mMEDTA), adds to use chloroform after Rnase removes RNA: the primary isoamyl alcohol extracting once adds 1/3 volume isopropanol precipitating DNA, and dried DNA is dissolved among the TE again; Purification part DNA; Extract damping fluid and consist of 10mmol L
-1Tris.HCl, pH 8.0,20mmol L
-1EDTA, 1.4M NaCl, 2%CTAB, 1.5%SDS; 20 μ L reaction solutions of pcr amplification contain 10mmol/LTris-HCl, and pH 8.3,50mmol/L KCl, 1.5mmol/L MgCl
2, Taq polymerase1U, 4 kinds of each 2mmol/L of dNTP, two each 4-6mmol/L of primer, dna profiling 20-60ng; The reaction cycle parameter of pcr amplification is 96 ℃ of pre-sex change 3 minutes; 94 ℃ 1 minute, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute; 2 circulations; 94 ℃ of 10 second, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute, 30-38 circulation; 72 ℃ were extended 5 minutes; The primer that the amplification back order-checking of ITS district is adopted is,
Forward primer P1:5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ',
Reverse primer P4:5 ' AATCCTGGTTAGTTTCTTTTCCTC3 '.
The applicant has obtained 3 kinds of sea-tangle (Laminaria japonica with various trait from the Institute of Oceanology of the Chinese Academy of Sciences, L.saccharina (L.) Lamouroux f., L.angustata Kjellman) 2 gametophytes of 17 gametophytes and a kind of bulk kelp (Macrocystis pyrifera (L.) Ag.), totally 19 samples, all samples all passes through the algae expert statement; The invention provides the specific DNA sequence of their different gametophytic generation ITS1,5.8S and ITS2 amplification, it is China's economic marine alga kelp gametophyte ITS district dna sequence dna, have any one group of base sequence in sequence table SEQ ID NO:1～19, it can be applied in the different kelp gametophyte authentication methods.
Analytical results shows, the ITS sequence length of several various trait kelp gametophytes is about 660bp, with the sequence similarity degree of the kelp gametophyte of having registered among the Genebank (ITS1,5.8S rDNA and ITS2) more than 90%, illustrate that the conservative property of kelp gametophyte ITS sequence is more intense.If do not consider the room, China's economic marine alga sea-tangle (Laminaria japonica) gametophyte entire I TS section length is 658bp, narrow leaf sea-tangle (Laminaria angustata kjellman) is 660bp, and sugared sea-tangle (Laminaria saccharina Lamouroux f.) is 664bp; If consider the room then all be 673bp.And measured kelp gametophyte ITS district nucleotide diversity rate if consider point mutation and some disappearance, is 0.61-0.91% in planting; Between planting, do not consider a disappearance, be 0.59-3.41%, if the consideration point lacks then is 2.23-6.24%.
The present invention has following advantage:
1. the present invention has at first set up the preparation method of a cover kelp gametophyte high quality DNA.
2. the method that the invention provides the dna sequence dna that utilizes the economical alga kelp gametophyte and utilize this sequence that the various trait kelp gametophyte is identified is also promptly utilized all DNA sequence of the ITS1 of economical alga kelp gametophyte and 5.8S rDNA and ITS2 and is utilized the gametophytic method of its various trait of this Sequence Identification.
3. the present invention checks order to the different gametophytic rDNA ITS region sequences of Chinese economical alga sea-tangle, has obtained the rDNA ITS complete sequence of (comprising ITS1,5.8S and ITS2).
Description of drawings
Fig. 1 for utilization homology primer P1 and P4 to the template DNA electrophoresis result synoptic diagram that increases.
Embodiment
Describe described method below in detail:
Embodiment 1
(1) total DNA extraction
Fresh frond adds the liquid nitrogen grind into powder, transfers to and extracts damping fluid [10mmol L
-1Tris.HCl, pH 8.0,20mmol L
-1Ethylenediaminetetra-acetic acid (EDTA), 1.4MNaCl, 2%cetyltrimethyl ammonium bromide (CTAB), 1.5%sodium dodecylsulfate (SDS)] in, use chloroform behind 65 ℃ of water-bath 1h: primary isoamyl alcohol (24: 1) extracting is once.Add the KAC of the 4M of 10%SDS and 1/3 volume in the supernatant liquor, use chloroform behind the ice bath 10min: primary isoamyl alcohol (24: 1) extracting is more once got supernatant liquor and is added 2/3 volume isopropanol precipitating DNA.DNA is dissolved among the TE, adds to use chloroform after Rnase removes RNA: primary isoamyl alcohol (24: 1) extracting once adds 1/3 volume isopropanol precipitating DNA, and dried DNA is dissolved among the TE again.The test kit that part DNA provides with Beijing Biological Development science and technology company limited (article number MK 001-3) carries out purifying.The agarose gel electrophoresis poststaining shows that the genomic dna size of extracting is more than 23Kb.
(2) pcr amplification
The special primer of kelp gametophyte ITS district amplification is P1 and P4, and sequence is as follows:
P1 (forward) is 5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ', is positioned on the 18S,
P4 (oppositely) is 5 ' AATCCTGGTTAGTTTCTTTTCCTC 3 ', is positioned on the 26S.
PCR reaction amplified reaction is finished in the system of 20 μ L, and reaction solution contains 10mmol/L Tris-HCl, and pH 8.3,50mmol/L KCl, 1.5mmol/L MgCl
2, Taq polymerase 1U, 4 kinds of each 2mmol/L of dNTP, two each 5mmol/L of primer, the about 50ng of dna profiling.The reaction cycle parameter is 96 ℃ of pre-sex change 3 minutes; 94 ℃ 1 minute, 55 ℃ of 10 second, 72 ℃ 1 minute, 2 circulations; 94 ℃ of 10 second, 55 ℃ of 10 second, 72 ℃ 1 minute, 35 circulations; 72 ℃ were extended 5 minutes.With ddH
2O replaces template DNA to do blank.
(3) PCR product purification
PCR product Watson test kit purifying, concrete operations are undertaken by the test kit instruction manual.
(4) determined dna sequence
Use BigDye
TMSequencing kit carries out sequencing reaction, and the sequencing reaction parameter is: Mix 1 μ L, the about 30-50ng of the DNA behind the purifying, Primer 1.5pmol, buffer 1.2 μ L, ddH
2O is an amount of.The sequencing reaction condition is: 95 ℃ of 1min, 95 ℃ of 30s, 50 ℃ of 30s, 60 ℃ of 4min, 35 each circulation altogether.With carrying out sequencing on the full-automatic sequenator of ABI PRISM 377-96.
(5) dna sequence data analysis
The start-stop scope of ITS1 and ITS2 is with reference to part kelp gametophyte ITS1,5.8S rDNA and the ITS2 sequence registered among the Genbank, with the arrangement of comparing of Clustal W software, and is aided with artificial check and correction behind institute's calling sequence input computer.Analyze difference between each sample dna sequence dna respectively with MEGA 2.1 and PHYLIP 3.6 analysis software package, and make up sibship.
Embodiment 2
Be that primer and reactive component are system with P1:5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ' and P4:5 ' AATCCTGGTTAGTTTCTTTTCCTC 3 ' in the present embodiment, adopt two cover pcr amplification programs to increase.
Program a:95 ℃ 5 minutes; 90 ℃ 1 minute, 50 ℃ 2 minutes, 5 circulations; 72 ℃ 1 minute; 90 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.
Program b:96 ℃ 3 minutes; 94 ℃ 1 minute, 55 ℃ of 10 second, 72 ℃ 1 minute, 2 circulations; 94 ℃ of 10 second, 55 ℃ of 10 second, 72 ℃ 1 minute, 35 circulations; 72 ℃ 5 minutes.It is band about 700bp that electrophoresis result display routine a and program b can both amplify a size.But the banding pattern instability of program a amplification, measure also bigger, with its order-checking demonstrate signal a little less than, sample segment need be resurveyed.And the product of program b amplification is used to the result that checks order and can directly draw.The primer P1 of design and the better homology of P4 are described, reliability is higher, and response procedures is had dependency.
Embodiment 3
Utilize the primer P1 and P4 amplification sea-tangle " 901 " the gametophyte different generations of better homology
(1) plant total DNA extraction to be detected
Sample to be checked is clean with aseptic water washing, grinding powder in liquid nitrogen.The CTAB extracting that provides according to this patent and the method for Glassmilk purifying are extracted genome DNA.
(2) quality examination of template DNA
Utilization primer P1 and P4 (P1, P4 sequence are seen embodiment 1) treat the inspection sample total DNA and increase, and detect the fragment that whether can amplify about about 700bp and the brightness of amplified fragments, with the quality of validation template DNA, the concentration of adjustment template DNA.
Pcr amplification reaction is finished in the system of 20 μ L, and reaction solution contains 10mmol/L Tris-HCl, and pH 8.3,50mmol/L KCl, 1.5mmol/L MgCl
2, Taq polymerase 1U, 4 kinds of each 2mmol/L of dNTP, two each 5mmol/L of primer, the about 50ng of dna profiling.The reaction cycle parameter is 96 ℃ of pre-sex change 3 minutes; 94 ℃ 1 minute, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute; 2 circulations; 94 ℃ of 10 second, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute, 30-38 circulation; 72 ℃ were extended 5 minutes.With ddH
2O replaces template DNA to do blank.
(3) utilization homology primer P1 and P4 increase to template DNA.
Electrophoresis result demonstration amplified production is very desirable (to be illustrated in fig. 1 shown below, M:100bp Ladder3,4,5 is followed successively by sea-tangle " 901 " gametophyte 6,7,8 generation ITS districts amplification collection of illustrative plates among the figure), so perhaps being used for the gametophytic generation of other marine algas of Laminariales, homology primer P1 and P4 identify.
The sequence subordinate list
(complete sequence in China's economic marine alga kelp gametophyte rDNAITS district (comprising ITS1,5.8S and ITS2))
1. blade is longer, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria.japonica Aresh) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
2. blade is longer, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonica Aresh) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGA
ACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAAT
CATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAG
CATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
3. iodine content height, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonica Aresh) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGA
ACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAAT
CATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAG
CATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
4. iodine content height, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonica Aresh) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCTCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGA
ACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAAT
CATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAG
CATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
5. blade broad, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonica Aresh) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGA
ACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAAT
CATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAG
CATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTCTGGGTCAGCGTCGGAAACCGTACCACTTTCGAT
6. blade broad, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonica Aresh) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGA
ACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAAT
CATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAG
CATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTCTGGGTCAGCGTCGGAAACCGTACCACTTTCGTT
7. blade is thin, wide, long, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonicaAresh) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTCGGAAACCGTACCACTTTCGTT
8. blade is thin, wide, long, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonicaAresh) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTCGGAAACCGTACCACTTTCGTT
9. blade is thick, wide, crisp, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonicaAresh) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
10. blade is thick, wide, crisp, come from Chinese Academy of Sciences ocean the ITS district complete sequence of sea-tangle (Laminaria japonicaAresh) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
11. come from Chinese Academy of Sciences ocean the ITS2 district complete sequence of bulk kelp (Macrocystis pyrifera) microgametophyte
CTCTCTCTCTCTCTCTCTCTCGAGCGAGCGAGCGAACGAGGGGCCGG
CGAGAGGGGGCGCGGACTCTGAGTGTTCCGGAGTCTCCCACGCTCCG
AGTGCACCTAATCGCGTGAACGAAGCCTCTCGCGCCCTGCCGCACAG
AGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGACTCACGAAAA
CGTGCGCAGGACATGCGGGCTGCTTCCTTCCGGCGCTCCAGCATATC
TCTGCTGGAATCCGTACACCACTTTCGTT
12. come from Chinese Academy of Sciences ocean the ITS1 and the 5.8S rDNA district complete sequence of bulk kelp (Macrocystis pyrifera) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGAGA
CTCTGCGCCCGACTCACGGAGGGCGCGTTTCTACACCCCGAGAAAGG
ATTCGTTATGCGAAGTTGGGCGAGGGGCGCCTCGCCGAGAGCTCAAT
CAAGCGCTCTCGAACCAAAGCGCACCCCACATTTCAACCCCATTAAA
CTCTGAATCTGAACTAAAAGGGGTGCCGCGCTCGCCGCGGCTCCCCC
AACCTTTTAACGTTGTCTTTCAGCGACGGATGTCTTGGCTCCCACAAC
GATGAAGAACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATC
CAGTGAATCATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTC
CTGGGAGCATGCTTGTCGGAGTGTCTGTT
13. come from the ITS district complete sequence of sugared sea-tangle (Lamimaria saccharina (L.) Lamouroux f.) microgametophyte of the U.S.
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
AGCCGCCGGTTTCGGCGGCCTCTGTACCCCGAGAAAGAACTCGTTAT
GCGAAGTTGGGCGAGGGGCGCCTCGCCGAGAGTTTTTGTAAGAAGA
GCTCTCGAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGA
ATCTGAACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTT
TAACGTTGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAA
GAACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGA
ATCATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGG
AGCATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTC
CTGTCTCACGACGAGGGGGTCGCGGCGGCGGACTTTGAGTGTTCCGG
AGTTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCG
CGCCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACT
CTCGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTC
CGACGCCGGCCCGGTAAAAAAGGTCGGCGTTGGAAACCGTACCACTT
TCGTT
14. come from the ITS district complete sequence of sugared sea-tangle (Lamimaria saccharina (L.) Lamouroux f.) megagametophyte of the U.S.
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
AGCCGCCGGTTTCGGCGGCCTCTGTACCCCGAGAAAGAACTCGTTAT
GCGAAGTTGGGCGAGGGGCGCCTCGCCGAGAGTTTTTGTAAGAAGA
GCTCTCGAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGA
ATCTGAACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTT
TAACGTTGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAA
GAACGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGA
ATCATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGG
AGCATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTC
CTGTCTCACGACGAGGGGGTCGCGGCGGCGGACTTTGAGTGTTCCGG
AGTTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCG
CGCCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACT
CTCGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTC
CGACGCCGGCCCGGTAAAAAAGGTCGGCGTTGGAAACCGTACCACTT
TCGTT
15. come from the sea-tangle (Laminaria japonica Aresh) of Japan, the ITS district complete sequence of its filial generation microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTG
TGAGGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTAT
GCGAAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCC
CTCGAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATC
TGAACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAA
CGTTGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAA
CGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATC
ATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGC
ATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTG
TCTCACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGT
TCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGC
CCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTC
GACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGA
CGCCGACCCTTCTGGGTCAGCGTCGGAAACCGTACCACTTTCGTT
16. come from Chinese Academy of Sciences ocean the ITS district complete sequence of narrow leaf sea-tangle (Laminaria angustata Kjellman) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTTGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGTCTGTAAAATGGCTCTCG
AATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGAA
CTCAAAGGGGCGCTGCGCTAGTCGCGGCTCCCCCAACCTTTAACGTT
GTAAAACTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAA
CGCAGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATC
ATCAAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGC
ATGCTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTG
TCTCACGACGAGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGT
TCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGC
CCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTC
GACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGA
CGCCGACCCTTTTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
17. come from Chinese Academy of Sciences ocean the ITS district complete sequence of narrow leaf sea-tangle (Laminaria angustata Kjellman) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTTGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGTCTGTAAAATGGCTCTCG
AATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGAA
CTCAAAGGGGCGCTGCGCTAGTCGCGGCTCCCCCAACCTTTAACGTT
GTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGCA
GCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATCA
AAACTTTGAAACGCATCTTGCGCTTTCCGGGATACTCCTGGGAACAT
GCTTTGTCGGGAATGGCTTGTTGACACCACTCGCCCCTCTTCTCTCCT
GTCTCACGACGAGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAG
TTCCCATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCG
CCCTGCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCT
CGACTCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCG
ACGCCGACCCTTTTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
18. come from Chinese Academy of Sciences ocean the ITS district complete sequence of parthenogenesis sea-tangle (Laminaria japonica) microgametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
19. come from Chinese Academy of Sciences ocean the ITS district complete sequence of parthenogenesis sea-tangle (Laminaria japonica) megagametophyte
CCGAAAGCGGGTTCGTTCAATCCCCCCCGCTCTATAAATTGTCTGTGA
GGCCGCTTCGTGCGGCCTCTTTACCCCGAGAAAGAATTCGTTATGCG
AAGTTGGGCGAGGGGCGCCTCGCCGAGAGCCTGTGAAAAGGCCCTC
GAATCAAAGCGCACCCCACATTTCAACCCATTAAACTCTGAATCTGA
ACTCAAAGGGGCGCTGCGCTAGCCGCGGCTCCCCCAACCTTTAACGT
TGTCTTTCAGCGACGGATGTCTTGGCTCCCACAACGATGAAGAACGC
AGCGAAATGCGATACGTCTTGCGACTTGCAGAATCCAGTGAATCATC
AAAACTTTGAACGCATCTTGCGCTTCCGGGATACTCCTGGGAGCATG
CTTGTCGGAGTGTCTGTTGACACCACTCGCCCCTCTTCTCTCCTGTCT
CACGACGGGGGAGTCGCGGCGGCGGACTTTGAGTGTTCCGGAGTTCC
CATGCTCCGAGTGCACCTAATCTCGTGAACGAAGCCTCTCGCGCCCT
GCCGCACAGAGTTGTTGACGGCGCTCGCTTCGGCGGCGACTCTCGAC
TCACCAAACGTGCGCAGGATGCCTGCCTCATTCCGGCGCTCCGACGC
CGACCCTTCTGGGTCAGCGTTGGAAACCGTACCACTTTCGTT
Claims (10)
1. various trait kelp gametophyte authentication method is characterized in that step is as follows:
(1) total DNA extraction: get the frond material of fresh and healthy, after handling with sterilized water,, extract total DNA at liquid nitrogen kind grind into powder;
(2) pcr amplification:
Adopt forward primer P1:5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ',
Reverse primer P4:5 ' AATCCTGGTTAGTTTCTTTTCCTC3 ' utilizes this primer that total DNA is carried out pcr amplification reaction;
(3) PCR product purification;
(4) determined dna sequence: the DNA behind the purifying carries out sequencing, establishes rDNA ITS district, comprises the complete sequence in ITS1,5.8S and ITS2 district;
(5) dna sequence data analysis:
The sequence one in rDNA ITS district to be analyzed is arranged software with contraposition and is carried out the contraposition arrangement, and be aided with artificial check and correction after measured, with the difference between each sequence in phylogenetic analysis each sample dna sequence dna of software analysis and the database.
2. the authentication method of various trait kelp gametophyte according to claim 1 is characterized in that: described total DNA extraction is the method that adopts CTAB extracting and Glassmilk purifying.
3. the authentication method of various trait kelp gametophyte according to claim 1, it is characterized in that: described total DNA extraction is after fresh frond adds the liquid nitrogen grind into powder, transfer to and extract in the damping fluid, use chloroform behind the 63-65 ℃ of water-bath 30-60min: the primary isoamyl alcohol extracting once; Add the KAC of the 4M of 10%SDS and 1/3 volume in the supernatant liquor, use chloroform behind the ice bath 10-20min: primary isoamyl alcohol extracting is more once got supernatant liquor and is added 2/3 volume isopropanol precipitating DNA; DNA is dissolved in the TE damping fluid, adds to use chloroform after Rnase removes RNA: the primary isoamyl alcohol extracting once adds 1/3 volume isopropanol precipitating DNA, and dried DNA is dissolved among the TE again; Purification part DNA.
4. the authentication method of various trait kelp gametophyte according to claim 3 is characterized in that: described extraction damping fluid consists of 10mmol L
-1Tris.HCl, pH8.0,20mmol L
-1EDTA, 1.4M NaCl, 2%CTAB, 1.5%SDS.
5. the authentication method of various trait kelp gametophyte according to claim 1 is characterized in that: 20 μ L reaction solutions of pcr amplification contain 10mmol/L Tris-HCl, pH8.3,50mmol/LKCl, 1.5mmol/L MgCl
2, Taq polymerase 1U, 4 kinds of each 2mmol/L of dNTP, two each 4-6mmol/L of primer, dna profiling 20-60ng.
6. the authentication method of various trait kelp gametophyte according to claim 1 is characterized in that: the reaction cycle parameter of pcr amplification is 96 ℃ of pre-sex change 3 minutes; 94 ℃ 1 minute, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute; 2 circulations; 94 ℃ of 10 second, 53-57 ℃ of 10 seconds, 72 ℃ 1 minute, 30-38 circulation; 72 ℃ were extended 5 minutes.
7. the authentication method of various trait kelp gametophyte according to claim 1 is characterized in that: the primer that the amplification back order-checking of described ITS district is adopted is,
Forward primer P1:5 ' AGAAGTCGTAACAAGGTTTCCGTAGG3 ',
Reverse primer P4:5 ' AATCCTGGTTAGTTTCTTTTCCTC3 '.
8. the partial dna sequence that can adopt in the authentication method of the described various trait kelp gametophyte of claim 1, it is characterized in that: it is China's economic marine alga various trait kelp gametophyte ITS district dna sequence dna, has any one group of base sequence in sequence table SEQ ID NO:1～19.
9. the partial dna sequence that can adopt in the various trait kelp gametophyte authentication method according to claim 8, it is characterized in that: if do not consider the room, China's economic marine alga sea-tangle entire I TS section length is 658bp, and narrow leaf sea-tangle is 660bp, and sugared sea-tangle is 664bp; If consider the room then all be 673bp.
10. the partial dna sequence that can adopt in the various trait kelp gametophyte authentication method according to claim 8, it is characterized in that: measured kelp gametophyte ITS district nucleotide diversity rate, if consider point mutation and some disappearance, in planting, be 0.61-0.91%; Between planting, do not consider a disappearance, be 0.59-3.41%, if the consideration point lacks then is 2.23-6.24%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101050372A CN100406574C (en) | 2003-11-07 | 2003-11-07 | Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101050372A CN100406574C (en) | 2003-11-07 | 2003-11-07 | Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1614032A true CN1614032A (en) | 2005-05-11 |
| CN100406574C CN100406574C (en) | 2008-07-30 |
Family
ID=34757165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101050372A Expired - Fee Related CN100406574C (en) | 2003-11-07 | 2003-11-07 | Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100406574C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
-
2003
- 2003-11-07 CN CNB2003101050372A patent/CN100406574C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
| CN102807978B (en) * | 2012-04-17 | 2014-02-26 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100406574C (en) | 2008-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1156587C (en) | Chinese dendrobe plant and its medicinal materials DNA molecular diagnosis method | |
| CN1283780C (en) | Method for constructing genetic engineering fungus of monascus with no citrinin | |
| CN101074435A (en) | Alpha-galactosidase gene, its coding protein, production and use | |
| CN1487997A (en) | Gene HD3A inducing flowering of plant and utilization thereof | |
| CN1718742A (en) | Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia | |
| CN1294255C (en) | Toxicide enzyme with aflatoxin activity conversion and gene for coding the enzyme | |
| CN1582335A (en) | Rice transposon genes | |
| CN1854154A (en) | Rice blast resistant related protein, its coding gene and use | |
| CN1844377A (en) | 9-cis epoxy carotinoid dioxygenase in Stylosanthes guianensis and genes encoding same and use thereof | |
| CN1814787A (en) | Ramie micro-satellite DNA Label | |
| CN1861791A (en) | Recessive gene xa13 of rice bacterial blight resistance and its allelic dominant gene xa13 | |
| CN101045933A (en) | Cryophilous proteinase gene mcp01 and its prepn process | |
| CN1886521A (en) | Molecular marker associated with CMV resistance and use thereof | |
| CN1160459C (en) | Cytochrome BD type quinol oxidase gene of brevibacterium lactofermentum | |
| CN1289523C (en) | Paddy rice potassium, sodium ion transport gene and its application | |
| CN1693473A (en) | Method of prepairing peltoglucose syrup and high temperature resistant glucose isomerase mutant | |
| CN1614032A (en) | Identification of kelp gametocyte with different behaviors and acceptable partial DNA sequence | |
| CN1163604C (en) | Attenuated virus oka strain gene 62 and method for identifying virus strain for attenuated live vaccine by using same | |
| CN1262654C (en) | Corn height related gene and coding protein and uses | |
| CN1614031A (en) | Method for economic alginate laver idioplasm resource identification and partial DNA sequence | |
| CN1239704C (en) | Gene associated with plant salt resistance and drought resistance, encoded protein and application thereof | |
| CN1641044A (en) | Asparagus assortative breading strain and wild type and correlated species identifying method | |
| CN1643151A (en) | Gene participating in acetic acid tolerance, acetic acid bacterium bred using the gene, and process for producing vinegar with the use of the acetic acid bacterium | |
| CN1796574A (en) | Method for authenticating gametocyte of wakame of economical alga from different breeding sea zones | |
| CN1566146A (en) | Paddy rice stalk extension gene, coded protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080730 Termination date: 20111107 |