CN105648105A - Method for identifying persimmon germplasm by using cpDNA (chloroplast deoxyribonucleic acid) molecular marker - Google Patents
Method for identifying persimmon germplasm by using cpDNA (chloroplast deoxyribonucleic acid) molecular marker Download PDFInfo
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Abstract
The invention relates to a method for identifying persimmon germplasm by using a cpDNA (chloroplast deoxyribonucleic acid) molecular marker. The amplification primer of the persimmon cpDNA molecular marker is disclosed as SEQ ID NO:1 or SEQ ID NO:2. The method comprises the following steps: carrying out PCR (polymerase chain reaction) amplification on standard germplasm by using the molecular marker primers; carrying out PCR amplification on the target germplasm by using the molecular marker primers, wherein the target germplasm is a germplasm to be identified; comparing the amplification results obtained in the two steps above, and judging whether the comparison results are consistent to carry out germplasm identification, wherein the forward primer and reverse primer of the molecular marker are respectively disclosed as SEQ ID NO:1 and SEQ ID NO:2. The method is simple to operate, has the advantages of favorable reproducibility and high efficiency, and can effectively identify whether the unknown material is a persimmon species or other Diospyros plants.
Description
Technical field
A kind of method that the present invention relates to Fructus Kaki Idioplasm identification, particularly relates to a kind of method that the cpDNA of utilization molecular marker carries out Fructus Kaki Idioplasm identification.
Background technology
Global Diospyros (Diospyrosspp.) plant about totally 190 kinds, wherein has arbor, shrub, and what have fallen leaves also has evergreenness, and major part is distributed in Perenniporia martius. China's calamander aboundresources, economic use value is higher, is mainly distributed on southwest and South China.
China's calamander of the report such as Zuo great Xun (1984) has 58 kinds and mutation (type) (not including abroad introducing kind), wherein endemic species 27 kinds. Recording in Chinese edition " Chinese Plants will " (1987), there are 64 kinds and mutation (type) in China, wherein has 57 kinds, 6 mutation, 1 modification, 1 cultigen. Wang Renzi (1988) etc. report that China's calamander has 66 kinds, including from abroad introducing kind. English edition " Chinese Plants will " (1996) has 64 kinds and mutation (60 kinds, 4 mutation) though also recording China's calamander, but its content is not the same with Chinese edition " Chinese Plants will ". The report such as Zhang Hongling (1994), Luo Zhengrong (1996) and Yang Yong (2005) is all consistent with Chinese edition " Chinese Plants will ". Inventor amounts to 32 parts of data according to " Chinese Plants will " (Chinese and English version) and each place " flora " or " silva ", adds up known China calamander and has 78 kinds and mutation (type) (68 kinds, 8 mutation, 2 modification). Additionally, the Southern Mountain Areas of China of China's length and breadth of land especially southwest and mountain area, south China also exist substantial amounts of wild Diospyros resource, the classification position of these wild resources is indefinite, plants for Fructus Kaki (product) or other calamanders need further to differentiate.
As can be seen here, China's calamander resource category is recorded and is differed, local flora is not the same to the statistics of calamander with national flora, many calamander resource classification status are indefinite, the collection of China's calamander resource, preservation and utilization are caused certain impact by this, and then affect carrying out of calamander cross-breeding work.
It is presently mainly and carries out plant resources discriminating classification from morphology and molecular biology, traditional form differential method depends on phenotypic difference, and the various factors such as the morphological characters climate of phenotype, environment, nutritional status and biological phase, and form differential method workload is big, differentiate that cycle length, cost are high. In the discriminating sort research of xylophyta, apply RAPD, SSR, AFLP equimolecular labelling technique more, but these labellings are adopt the technological development without whole genome sequence information to obtain mostly, though having certain using value, but randomness is strong, poor stability.And owing to calamander resource ploidy is complicated, its Matrix attachment region structure and sequence are also complicated and changeable, and compared to karyogene, Chloroplast gene structure and sequence are very conservative, but do not find so far about utilizing cpDNA molecular marker to carry out the report of phyletic evolution and sibship research between calamander resource kind.
Summary of the invention
The present invention is to solve that the problems referred to above provide a kind of method that the cpDNA of utilization molecular marker carries out Fructus Kaki Idioplasm identification, the cpRNA molecular marker distinguishing ability that the method utilizes is strong and favorable reproducibility, can effectively differentiate that unknown resources is Fructus Kaki kind or is other calamanders, there is provided important technology support for the discriminating of China's calamander resource, phylogenetic analysis and Genetic relationship simultaneously, China's calamander cross-breeding and exploitation are significant.
The invention provides the amplimer of a kind of Fructus Kaki cpDNA molecular marker, as shown in SEQIDNO:1 or SEQIDNO:2.
Present invention also offers the screening technique of the amplimer of a kind of described Fructus Kaki cpDNA molecular marker, comprise the steps: that the cpDNA to standard species matter checks order; The cpDNA of target species matter is checked order, and target species matter is kind matter to be identified; The sequencing result that two above step obtains is compared, it is thus achieved that primer is also designed in diff area; After pcr amplification, purification and sequence of resurveying, screen the amplimer that can effectively differentiate Fructus Kaki kind and other calamanders.
Further, described diff area is the fragment more than 20bp.
Further, the quantity of described standard species matter is 3 or more than 3.
Present invention also offers a kind of method that the cpDNA of utilization molecular marker carries out Fructus Kaki Idioplasm identification, comprise the steps: to utilize molecular marker primer PCR amplification standard species matter; Utilizing molecular marker primer PCR amplification target species matter, target species matter is kind matter to be identified; Being compared by the amplification that two above step obtains, according to whether comparison result unanimously carries out Idioplasm identification, the forward primer of molecular marker used and reverse primer are respectively as shown in SEQIDNO:1 and SEQIDNO:2.
Described utilize molecular marker primer PCR amplification step before also include extracting the chloroplast complete genome DNA of standard species matter and target species matter.
Further, the chloroplast complete genome DNA of described extraction standard species matter and target species matter comprises the steps:
Step 1, adding 800 �� lCTABDNA Extraction buffers in 1.5ml centrifuge tube, described Extraction buffer includes pH8.0100mMTris-HCl, 1.4MNaCl and pH8.020mMEDTA, adds 30 �� l beta-mercaptoethanols, and mixing is placed on 65 DEG C of water-baths preheatings;
Step 2, take 0.3g blade, add liquid nitrogen and be fully ground to Powdered, be transferred quickly in the centrifuge tube in step 1, crack 2h in 65 DEG C of water-baths, period vibration mixing 3-5 time;
After step 3, water-bath terminate, take out centrifuge tube cooling, in the centrifugal 12min of room temperature 12000rpm;
Step 4, centrifugal after with liquid-transfering gun, 700 �� l supernatant are proceeded in another centrifuge tube of 1.5ml, add the mixed solution of 700 �� l chloroforms and isoamyl alcohol, wherein chloroform is 24:1 with the volume ratio of isoamyl alcohol, gently reverse mixing 10min, the centrifugal 10min of 12000rpm at 4 DEG C;
Step 5,2-3 step 4 of repetition, till without white precipitate layer;
Step 6, being proceeded to by supernatant in the another centrifuge tube of 1.5ml, add the isopropanol of 700 �� l pre-coolings, mix gently to occurring that transparent cotton-shaped DNA precipitates, at 4 DEG C, the centrifugal 6min of 10000rpm, abandons supernatant;
Adding the alcoholic solution that percent by volume is 75% of 1000 �� l pre-coolings, washing precipitation 2 times in step 7, centrifuge tube after step 6, each 10000rpm is centrifuged 6min, abandons ethanol, air-dry under room temperature volatilizees completely to ethanol;
Step 8, centrifuge tube after step 7 add 500 �� 11MNaCl, after abundant dissolving DNA, adds 2 �� l10mg/mlRNase, in 37 DEG C of water-bath 1h;
Step 9, centrifuge tube adds after step 8 1ml dehydrated alcohol ,-20 DEG C of precipitation DNA15h after mixing;
Step 10, at 4 DEG C the centrifugal 10min of 8000rpm, abandon supernatant, percent by volume be 75% alcoholic solution wash DNA2 time, each 8000rpm is centrifuged 6min, abandons ethanol, and room temperature is air-dry volatilizees completely to ethanol;
Step 11, adding 200 �� lTE buffer components dissolving DNAs, DNA sample is placed in-20 DEG C and saves backup.
Preferably, described pcr amplification condition is as follows:
Described pcr amplification reaction system is as follows:
Described pcr amplification program is: (1) 94 DEG C of denaturation 3min; (2) 94 DEG C of degeneration 45s; (3) 52 DEG C of annealing 1min; (4) 72 DEG C extend 50s; (5) return step (2), repeat 35 circulations; (6) 4 DEG C of preservations.
Present invention also offers a kind of Fructus Kaki genetic resources method for analyzing diversity, for molecular marker Amplification Analysis method, utilize the amplimer of described Fructus Kaki cpDNA molecular marker to carry out Amplification Analysis.
Present invention also offers the detection kit of a kind of amplimer comprising described Fructus Kaki cpDNA molecular marker.
The present invention is by comparison after the chloroplast genome sequencing of 5 parts of calamander materials, determine diff area and design primer, through pcr amplification, purification and sequence of resurveying, screening 1 pair of specific primer that can be used for differentiating Fructus Kaki kind and other calamanders, described specific primer can effectively differentiate that unknown material is Fructus Kaki kind or is other calamanders. The method has simple to operate, favorable reproducibility, efficiency high, important technological means is provided to the discriminating of phylogeny and Fructus Kaki novel species between research Fructus Kaki kind, also provide important technology support for the discriminating of China's calamander resource, phylogenetic analysis and Genetic relationship, China's calamander cross-breeding and exploitation are significant.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes cpDNA molecular marker to carry out differentiating the phylogeny tree graph that open country, Yunnan hair Fructus Kaki is other calamanders in the method for Fructus Kaki Idioplasm identification.
Detailed description of the invention
Further illustrating the present invention below by specific embodiment utilizes cpDNA molecular marker to carry out the technical scheme of method of Fructus Kaki Idioplasm identification.
First embodiment, calamander material acquisition
15 parts of calamander materials are acquired from economic forest research and development centre of China Forestry Science Research Institute Fructus Kaki Germplasm Resources, 1 portion of calamander material to be identified-open country, Yunnan hair Fructus Kaki (formal name used at school D.spp.) is gathered from botanical garden, Chinese Academy of Sciences Xishuangbanna, record from current document and traditional form taxonomy cannot differentiate that open country, Yunnan hair Fructus Kaki is Fructus Kaki kind or is other calamanders, as shown in table 1 from 15 parts of sample specifying informations of forest-science academy of China.
The specifying information of 1,15 calamander materials of table
Second embodiment, calamander material genomic DNA extraction
The CTAB method adopting improvement extracts chloroplast complete genome DNA (cpDNA) from the blade of above-mentioned 16 parts of calamander material samples, and concrete grammar is as follows:
Step 201, in 1.5ml centrifuge tube, add 800 �� lCTABDNA Extraction buffers, described Extraction buffer includes 100mMTris-HCl (pH8.0), 1.4MNaCl and 20mMEDTA (pH8.0), adding 30 �� l beta-mercaptoethanols, mixing is placed on 65 DEG C of water-bath preheatings;
Step 202, take 0.3g blade, add liquid nitrogen and be fully ground to Powdered, be transferred quickly in the centrifuge tube in step 201, crack 2h in 65 DEG C of water-baths, period vibration mixing 3-5 time;
After step 203, water-bath terminate, take out centrifuge tube cooling, in the centrifugal 12min of room temperature 12000rpm;
Step 204, centrifugal after with liquid-transfering gun, 700 �� l supernatant are proceeded in another centrifuge tube of 1.5ml, add the mixed solution (chloroform and isoamyl alcohol volume ratio 24:1) of 700 �� l chloroforms and isoamyl alcohol, reverse mixing 10min gently, the centrifugal 10min of 12000rpm at 4 DEG C;
Step 205,2-3 step 204 of repetition, till without white precipitate layer;
Step 206, being proceeded to by supernatant in the another centrifuge tube of 1.5ml, add the isopropanol of 700 �� l pre-coolings, mix gently to occurring that transparent cotton-shaped DNA precipitates, at 4 DEG C, the centrifugal 6min of 10000rpm, abandons supernatant;
Adding the alcoholic solution that percent by volume is 75% of 1000 �� l pre-coolings, washing precipitation 2 times in step 207, centrifuge tube after step 206, each 10000rpm is centrifuged 6min, abandons ethanol, air-dry under room temperature volatilizees completely to ethanol;
Step 208, centrifuge tube after step 207 add 500 �� 11MNaCl, after abundant dissolving DNA, adds 2 �� lRNase (10mg/ml), in 37 DEG C of water-bath 1h;
Step 209, centrifuge tube after step 208 add 1ml dehydrated alcohol ,-20 DEG C of precipitation DNA15h after mixing;
Step 210, at 4 DEG C the centrifugal 10min of 8000rpm, abandon supernatant, percent by volume be 75% alcoholic solution wash DNA2 time, each 8000rpm is centrifuged 6min, abandons ethanol, and room temperature is air-dry volatilizees completely to ethanol;
Step 211, adding 200 �� lTE buffer (10mMTris-HCl, 1mMEDTA, pH=8.0) dissolving DNAs, DNA sample is placed in-20 DEG C and saves backup.
3rd embodiment, primer screening
1, genes of interest fragment is determined
Choose representative Fructus Kaki kind (Mopan Persimmon) and the chloroplast STb gene sample of other calamanders (gold Fructus Jujubae Fructus Kaki, Fructus Dispyroris Loti, Zhejiang Fructus Kaki and wild kaki persimmon) material;
The cpDNA of above-mentioned 5 parts of calamander materials is checked order, passes through Multiple Sequence Alignment, it is determined that an insertion/deletion genes of interest fragment more than 20bp;
2, screening primer
Design primer according to above-mentioned purpose genetic fragment, carry out pcr amplification according to following system (as shown in table 2) and condition:
Table 2, PCR reaction system
PCR reaction condition is:
The PCR primer that said method obtains is detected by the agarose gel electrophoresis that the Gelred concentration dyeed is 2.0%. Bio-red company gel imaging system is adopted to observe and record bands of a spectrum. Utilizing UniQ column type to reclaim test kit (Shanghai biological engineering company limited) and reclaim purification amplified production, products therefrom carries out two-way order-checking on automatic sequencer.
By above-mentioned pcr amplification reaction and sequence of resurveying, finishing screen selects the specific primer that can be used for differentiating Fructus Kaki kind and other calamanders a pair. Primer information is as follows:
Forward primer: 5 '-TCAAAGAGGGAGGTTTCT-3 ', as shown in SEQIDNO:1;
Reverse primer: 5 '-CCGTTATTCGGAGGTTCG-3 ' are as shown in SEQIDNO:2.
4th embodiment, phylogenetic tree structure
16 parts of calamander materials of the primer pair of method and screening described in the 3rd embodiment is utilized to carry out pcr amplification reaction and sequence of resurveying.
To sequence ClustalX software (Thompson measured by 15 parts of calamander materials and calamander to be identified material-open country, Yunnan hair Fructus Kaki, 1997) para-position arrangement is carried out, then MEGA software is utilized, use contiguous method (TheNeighbor-Jioning, NJ) initial tree is set up, adopt method of maximum likelihood (MaximumCompositeLikelihood again, ML) find most likelihood tree, and utilize bootstrap (repeating for 1000 times) to check the confidence level of each branch.
The result of Fig. 1 shows: identifying through said method, open country, Yunnan hair Fructus Kaki is other calamanders, namely just can be differentiated primer to other calamanders with 1.
Claims (10)
1. the amplimer of a Fructus Kaki cpDNA molecular marker, it is characterised in that as shown in SEQIDNO:1 or SEQIDNO:2.
2. the screening technique of the amplimer of Fructus Kaki cpDNA molecular marker described in a claim 1, it is characterised in that comprise the steps: that the cpDNA to standard species matter checks order; The cpDNA of target species matter is checked order, and target species matter is kind matter to be identified; The sequencing result that two above step obtains is compared, it is thus achieved that primer is also designed in diff area; After pcr amplification, purification and sequence of resurveying, screen the amplimer that can effectively differentiate Fructus Kaki kind and other calamanders.
3. method according to claim 2, it is characterised in that described diff area is the fragment more than 20bp.
4. according to the method in claim 2 or 3, it is characterised in that the quantity of described standard species matter is 3 or more than 3.
5. one kind utilizes the method that cpDNA molecular marker carries out Fructus Kaki Idioplasm identification, it is characterised in that comprise the steps: to utilize molecular marker primer PCR amplification standard species matter; Utilizing molecular marker primer PCR amplification target species matter, target species matter is kind matter to be identified; Being compared by the amplification that two above step obtains, according to whether comparison result unanimously carries out Idioplasm identification, the forward primer of molecular marker used and reverse primer are respectively as shown in SEQIDNO:1 and SEQIDNO:2.
6. method according to claim 5, it is characterised in that described utilize molecular marker primer PCR amplification step before also include extracting the chloroplast complete genome DNA of standard species matter and target species matter.
7. method according to claim 6, it is characterised in that the chloroplast complete genome DNA of described extraction standard species matter and target species matter comprises the steps:
Step 1, adding 800 �� lCTABDNA Extraction buffers in 1.5ml centrifuge tube, described Extraction buffer includes pH8.0100mMTris-HCl, 1.4MNaCl and pH8.020mMEDTA, adds 30 �� l beta-mercaptoethanols, and mixing is placed on 65 DEG C of water-baths preheatings;
Step 2, take 0.3g blade, add liquid nitrogen and be fully ground to Powdered, be transferred quickly in the centrifuge tube in step 1, crack 2h in 65 DEG C of water-baths, period vibration mixing 3-5 time;
After step 3, water-bath terminate, take out centrifuge tube cooling, in the centrifugal 12min of room temperature 12000rpm;
Step 4, centrifugal after with liquid-transfering gun, 700 �� l supernatant are proceeded in another centrifuge tube of 1.5ml, add the mixed solution of 700 �� l chloroforms and isoamyl alcohol, wherein chloroform is 24:1 with the volume ratio of isoamyl alcohol, gently reverse mixing 10min, the centrifugal 10min of 12000rpm at 4 DEG C;
Step 5,2-3 step 4 of repetition, till without white precipitate layer;
Step 6, being proceeded to by supernatant in the another centrifuge tube of 1.5ml, add the isopropanol of 700 �� l pre-coolings, mix gently to occurring that transparent cotton-shaped DNA precipitates, at 4 DEG C, the centrifugal 6min of 10000rpm, abandons supernatant;
Adding the alcoholic solution that percent by volume is 75% of 1000 �� l pre-coolings, washing precipitation 2 times in step 7, centrifuge tube after step 6, each 10000rpm is centrifuged 6min, abandons ethanol, air-dry under room temperature volatilizees completely to ethanol;
Step 8, centrifuge tube after step 7 add 500 �� 11MNaCl, after abundant dissolving DNA, adds 2 �� l10mg/mlRNase, in 37 DEG C of water-bath 1h;
Step 9, centrifuge tube after step 8 add 1ml dehydrated alcohol ,-20 DEG C of precipitation DNA15h after mixing;
Step 10, at 4 DEG C the centrifugal 10min of 8000rpm, abandon supernatant, percent by volume be 75% alcoholic solution wash DNA2 time, each 8000rpm is centrifuged 6min, abandons ethanol, and room temperature is air-dry volatilizees completely to ethanol;
Step 11, adding 200 �� lTE buffer components dissolving DNAs, DNA sample is placed in-20 DEG C and saves backup.
8. the method according to any one of claim 5-7, it is characterised in that described pcr amplification condition is as follows:
Described pcr amplification reaction system is as follows:
Described pcr amplification program is: (1) 94 DEG C of denaturation 3min; (2) 94 DEG C of degeneration 45s; (3) 52 DEG C of annealing 1min; (4) 72 DEG C extend 50s; (5) return step (2), repeat 35 circulations; (6) 4 DEG C of preservations.
9. a Fructus Kaki genetic resources method for analyzing diversity, for molecular marker Amplification Analysis method, it is characterised in that utilize the amplimer of Fructus Kaki cpDNA molecular marker described in claim 1 to carry out Amplification Analysis.
10. one kind comprises the detection kit of the amplimer of Fructus Kaki cpDNA molecular marker described in claim 1.
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| CN107142330A (en) * | 2017-07-11 | 2017-09-08 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using core ITS sequence |
| CN107312848A (en) * | 2017-07-13 | 2017-11-03 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using cpDNA sequences |
| CN108130379A (en) * | 2017-07-05 | 2018-06-08 | 华北水利水电大学 | Affiliation carries out mirror method for distinguishing between fortune paulownia and congener |
| CN111763761A (en) * | 2020-07-29 | 2020-10-13 | 三门峡职业技术学院 | Method for analyzing genetic structure of wild salvia miltiorrhiza population by virtue of cPDNA sequence YCF 1-RPS 15 |
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| CN112195271A (en) * | 2020-11-18 | 2021-01-08 | 国家林业和草原局泡桐研究开发中心 | Primer and method for early identifying natural deastringency character of Chinese sweet persimmon |
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| CN108130379A (en) * | 2017-07-05 | 2018-06-08 | 华北水利水电大学 | Affiliation carries out mirror method for distinguishing between fortune paulownia and congener |
| CN107142330A (en) * | 2017-07-11 | 2017-09-08 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using core ITS sequence |
| CN107142330B (en) * | 2017-07-11 | 2020-06-02 | 中国林业科学研究院林业研究所 | Method for carrying out hazel germplasm identification by using nuclear ITS sequence |
| CN107312848A (en) * | 2017-07-13 | 2017-11-03 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using cpDNA sequences |
| CN111763761A (en) * | 2020-07-29 | 2020-10-13 | 三门峡职业技术学院 | Method for analyzing genetic structure of wild salvia miltiorrhiza population by virtue of cPDNA sequence YCF 1-RPS 15 |
| CN111763761B (en) * | 2020-07-29 | 2022-11-25 | 三门峡职业技术学院 | Method for analyzing genetic structure of wild salvia miltiorrhiza population by using cpDNA sequence YCF1-RPS15 |
| CN112029893A (en) * | 2020-09-23 | 2020-12-04 | 国家林业和草原局泡桐研究开发中心 | Molecular marker primer for identifying sex of persimmon tree and identification method |
| CN112195271A (en) * | 2020-11-18 | 2021-01-08 | 国家林业和草原局泡桐研究开发中心 | Primer and method for early identifying natural deastringency character of Chinese sweet persimmon |
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