CN103173562A - Construction method of date tree SSR (Simple Sequence repeat) marker molecular genetic map - Google Patents
Construction method of date tree SSR (Simple Sequence repeat) marker molecular genetic map Download PDFInfo
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Abstract
The invention discloses a construction method of a date tree SSR (Simple Sequence repeat) marker molecular genetic map. The construction method comprises the following steps of: (1) extracting genomic DNAs (deoxyribonucleic acids) of parents and offsprings of a date tree to be detected; (2) screening SSR primers by using parent materials; (3) carrying out PCR (Polymerase Chain Reaction) amplification by respectively using the SSR primers and respectively taking the genomic DNAs (deoxyribonucleic acids) of the parents and offsprings of the date tree to be detected as templates; and (4) analyzing PCR amplification results by using biology software, and constructing the date tree SSR marker molecular genetic map. According to the construction method, the high-efficiency low-cost date tree SSR marker technical system is constructed, and obtained SSR markers and a core primer group have the advantages of high efficiency, stability, co-dominance and the like. The set of markers can be used for researches such as the date tree genetic map construction, genetic diversity analysis, identification of variety, molecular mark assisted breeding and the like and promoting the development of date tree genetics and breeding technique.
Description
Technical field
The present invention relates to molecular biology, genetics and genomics field, refer to especially a kind of construction process of jujube tree SSR tagged molecule genetic map.
Background technology
Jujube tree belongs to the Rhamnaceae zizyphus, is the peculiar fruit tree that originates in China, and germ plasm resource is extremely abundant, and genetic background is complicated, has at least so far the cultivation history of 7000.In long-term natural evolvement and artificial selection process, formed abundant variation, have been found that so far and jujube cultivars and the good type put down in writing reach kind more than 880.But owing to lacking a large amount of polymorphic molecular markers, the research of its genetics and genomics aspect relatively lags behind.
Shen Lianying has built first jujube tree molecular genetic linkage map (Shen connects the QTL Position Research of English .2005. jujube (Ziziphus jujuba Mill) genetic linkage maps structure and proterties), comprise 333 AFLP (the amplified fragment length polymorphism Restriction fragment length polymorphism markers) mark that is distributed in 14 linkage groups, covering gene group length 1237.4cM, the mean chart distance is 3.9cM.Qi Jing etc. are (neat quiet, Dong Zhen, Mao Yongmin, Deng. the structure of jujube dense genetic map spectrum and the qtl analysis of trunk diameter. forest-science, 2009,45 (8): 44-49) this collection of illustrative plates is encrypted to 388 AFLP marks and 35 RAPD (random amplified polymorphic DNA is the randomly amplified polymorphic DNA mark) mark by 15 linkage groups covering gene group total length 1309.4cM, mean distance 3.1cM between mark.But this collection of illustrative plates lacks the codominant marker of versatility, relatively is difficult to integrate.Therefore, with codominance SSR mark construction framework genetic map, the development of jujube tree molecular breeding is had great importance.
RAPD, AFLP and SRAP (Sequence-related amplified polymorphism is SRAP) mark etc. is used to assortment and the Genetic relationship of jujube tree.But these marks add the appearance of new mark due to reasons such as dominant characteristics, and are gradually less for genetic map construction.SSR (Simple sequence repeat simple repeated sequence) mark, be again little satellite (Microsatellites) mark, have advantages of that quantity is abundant, that codominance, repeatability, experimental technique are easy etc. is outstanding, therefore more and more be applied to genetic map construction, especially the framework genetic map construction of numerous species as the grappling mark.In addition, SSR also is widely used in the structure of analysis of genetic diversity, finger printing, genetic map.The inventor utilizes magnesphere to develop 40 pairs of winter jujube SSR marking primer 2s, screening polymorphic primer wherein builds the molecular genetic linkage map of jujube tree, thereby carry out the QTL location of correlated character, can promote the Genetic Control research of jujube tree important character and the process of molecular mark.
Summary of the invention
The construction process that the purpose of this invention is to provide a kind of jujube tree SSR tagged molecule genetic map.
A kind of construction process of jujube tree SSR tagged molecule genetic map is characterized in that, comprises the steps:
1) extraction of DNA: the genomic dna that extracts jujube tree parent to be measured and filial generation;
2) screening of SSR labeled primer: with parent material screening SSR labeled primer;
3) pcr amplification: utilize respectively above-mentioned SSR labeled primer, take the genomic dna of jujube tree parent to be measured and filial generation as masterplate, carry out pcr amplification separately;
4) map construction: utilize biological software to analyze the pcr amplification result, build jujube tree SSR tagged molecule genetic map.
Wherein, described step 1), jujube tree parent to be measured is as maternal, take azalea as male parent take the winter jujube; Described filial generation is 149 strain F
1The filial generation in generation.
Wherein, described step 1) the CTAB method after improvement is used in the extraction of DNA described in, and concrete steps are as follows:
(1) getting the addition of C TAB extracting solution, to put into 65 ℃ of water-bath preheatings stand-by;
(2) get Chinese Jujube 0.5g, put into mortar, add a small amount of PVP-40, be ground into powder rapidly in liquid nitrogen, be transferred in the centrifuge tube of CTAB extracting solution that 1ml65 ℃ of preheating is housed (with before adding 2% beta-mercaptoethanol mixing) (with before doing sterilising treatment);
(3) with described centrifuge tube vortex thermal agitation, fully mixing, put into 65 ℃ of water-baths, and water-bath 50min stirs up and down centrifuge tube every 10min and helps abundant mixing;
(4) centrifugal 10min under 12000rpm normal temperature;
(5) get supernatant liquor and (do not touch separation surface when noting drawing in new centrifuge tube, in order to avoid bring impurity into), add equal-volume water-saturated phenol/chloroform/primary isoamyl alcohol mixed solution (25: 24: 1) (matching while using), put upside down gently mixing, room temperature is placed 10min;
(6) centrifugal 10min under 12000rpm normal temperature;
(7) get step (6) gained supernatant liquor in new centrifuge tube, add equal-volume chloroform/primary isoamyl alcohol (24: 1) extracting again;
(8) get step (7) gained supernatant liquor in new centrifuge tube, add the equal-volume Virahol, put upside down gently mixing, be positioned in-20 ℃ of refrigerators and precipitate 20min;
(9) choose the DNA flocks in the 2ml centrifuge tube, with 70% ethanol washing and precipitating 2 times, each 30min;
(10) dry up precipitation with sterile wind in Bechtop, add 100 μ l TE damping fluid dissolving DNAs, stand-by in-20 ℃ of preservations.
Wherein, described step 2) described in, the screening of SSR primer is based on magnesphere, exploitation winter jujube SSR primer utilizes maternal winter jujube and male parent azalea to carry out the SSR primer screening, selects that in maternal winter jujube and male parent, at least one party shows as the primer of heterozygosis as core primers.
Wherein, 240 pairs of primers that SSR primer described step 2) is as shown in table 3.
Wherein, 170 pairs of core primers that SSR primer described step 2) is as shown in table 3.
Wherein, described step 2) also comprise the quality of the DNA of Detection and Extraction.
What wherein, described step 3) pcr amplification adopted is the three-primer method, namely adds M13 tail sequence, special reverse primer by 5 ' end at forward primer and with fluorescently-labeled universal M13 primer.
Wherein, described as shown in table 1 with fluorescently-labeled universal M13 primer.
Wherein, described step 3) also comprise after pcr amplification the result after pcr amplification is detected, adopt capillary electrophoresis to detect.
Wherein, described step 4) described in, biological software is Genemarker V1.75 software, and the data after reading are used for subsequent analysis after correcting through the FlexiBinv2 program.
Wherein, described step 4) respectively the pcr amplification result is carried out χ2-test,chi-square test in, analyze it and whether meet Mendel's law of segregation, find inclined to one side separation marking and remove under 5% conspicuous level, (1: 1,1: 2: 1,1: 1: 1: labeling pattern 1) was converted into unified form, selected full sibs population genetic map construction software FslinkageMAP to carry out the structure of molecular genetic linkage map with three kinds of unpack formats respectively.
Beneficial effect of the present invention is as follows:
1,170 pairs of core primers of jujube tree are provided, have set up efficient jujube tree SSR labeled analysis technical system.
2, adopt the FslinkageMAP mapping software, can utilize the unavailable mark of Mapmaker and Joinmap, improve the utilising efficiency of mark.
3, adopt capillary electrophoresis SSR genotyping technology, improve efficient and the accuracy of experiment.
4, adopt the pcr amplification system of 8 μ l in SSR labeled analysis process, reduce experimental cost.
The present invention utilizes magnesphere to develop 40 pairs of winter jujube SSR primer 2s, screening SSR primer wherein builds the genetic map of jujube tree, thereby carry out the QTL location of correlated character, can promote the Genetic Control research of jujube tree important character and the process of molecular mark.
Description of drawings
Fig. 1 is the jujube tree SSR tagged molecule genetic map of the embodiment of the present invention.
Embodiment
For making the technical problem to be solved in the present invention, technical scheme and advantage clearer, the below will be described in detail at specific embodiment.If do not specialize, the conventional means that in embodiment, technique means used is known for those of ordinary skills, the raw materials used commercial goods that is.
The construction process of the jujube tree SSR tagged molecule genetic map that the embodiment of the present invention provides comprises:
Step 1 is extracted DNA
The genome DNA extracting method of described jujube tree adopts the CTAB method after improveing, and concrete steps are as follows:
Step 11 is got the addition of C TAB extracting solution, and to put into 65 ℃ of water-bath preheatings stand-by;
Step 12 is got Chinese Jujube 0.5g, put into mortar, add a small amount of PVP-40, be ground into powder rapidly in liquid nitrogen, be transferred in the centrifuge tube of CTAB extracting solution that 1ml65 ℃ of preheating is housed (with before adding 2% beta-mercaptoethanol mixing) (with before doing sterilising treatment);
Step 13 vortex thermal agitation, fully mixing, put into 65 ℃ of water-baths, and water-bath 50min stirs up and down centrifuge tube every 10min and helps abundant mixing;
Centrifugal 10min under step 1412000rpm normal temperature;
Step 15 is got supernatant liquor and (do not touch separation surface when noting drawing in new centrifuge tube, in order to avoid bring impurity into), add equal-volume water-saturated phenol/chloroform/primary isoamyl alcohol mixed solution (25: 24: 1) (matching while using), put upside down gently mixing, room temperature is placed 10min;
Centrifugal 10min under step 1612000rpm normal temperature;
Step 17 is got step 16 gained supernatant liquor in new centrifuge tube, adds equal-volume chloroform/primary isoamyl alcohol (24: 1) extracting again;
Step 18 is got step 17 gained supernatant liquor in new centrifuge tube, adds the equal-volume-20 ℃ cold isopropanol precipitating DNA of chance, puts upside down gently mixing, is positioned in-20 ℃ of refrigerators and precipitates 20min;
Step 19 is chosen the DNA flocks in the 2ml centrifuge tube, with 70% ethanol washing and precipitating 2 times, and each 30min;
Step 20 dries up precipitation with sterile wind in Bechtop, add 100 μ l TE damping fluid dissolving DNAs, and is stand-by in-20 ℃ of preservations.
Extract take jujube cultivars ' winter jujube ' as maternal DNA, the DNA take ' azalea ' as male parent, and 149 strain F with this method
1The DNA in generation obtains respectively DNA sample liquid separately.
Step 2 detects DNA
Get respectively the DNA sample liquid of 3 μ l step 1 gained, measure the value of its DNA content (μ g/ml), A260nm/A280nm and in the absorption peak at 260nm place, the purity of detection DNA on the ultramicron ultraviolet spectrophotometer.
Getting respectively 5 μ l step 1 gained DNA sample mix 1 μ l6 * Loading Buffer, is to detect the integrity of DNA in 1% agarose gel electrophoresis in concentration.
Step 3 screening, amplification and detection
select the inventor to utilize 240 couple ' winter jujube ' SSR labeled primer of magnesphere exploitation, utilize 2 parent materials ' winter jujube ' and ' azalea ' to carry out the SSR primer screening, SSR labeled primer (seeing Table 3:240 to SSR primer information table) is synthetic by Jin Weizhi (Beijing) bio tech ltd, select the primer that in ' winter jujube ' and ' azalea ' at least one party shows as heterozygosis, and the random choose daughter DNA carries out augmentation detection, pick out expanding effect stable, without assorted band, 170 pairs of SSR core primers (being shown in Table 3) that signal is stronger are used for the structure of molecular genetic linkage map.
The present invention utilizes three-primer method PCR (namely to add M13 tail sequence, special reverse primer by 5 ' end at forward primer and with fluorescently-labeled universal M13 primer, fluorescently-labeled universal M13 primer sees Table 1 (M13 fluorescent primer table), utilizes capillary electrophoresis technique to detect simultaneously a plurality of SSR site of different fluorochrome labels) the SSR site of increasing.
The PCR reaction system comprises: 2xTaq PCR premixed solution (Beijing rich step Deco skill Development Co., Ltd) 4 μ l, forward primer (1 μ M) 0.32 μ l, reverse primer (1 μ M) 1.28 μ l, M13 primer (1 μ M) 1.28 μ l, masterplate DNA0.80 μ l, ddH
2O0.32 μ l, cumulative volume 8.00 μ l.Pcr amplification program: step 1:94 ℃ 5 minutes; Step 2:94 ℃ 30 seconds, 55 ℃ 40 seconds, 72 ℃ 45 seconds, totally 30 the circulation; Step 3:94 ℃ 30 seconds, 53 ℃ 40 seconds, 72 ℃ 45 seconds, totally 8 the circulation; Step 4:72 ℃ 10 minutes; Step 5:10 ℃ of preservation.
Amplification send Jin Weizhi (Beijing) bio tech ltd to carry out capillary electrophoresis and detects.
Step 4 result treatment
Utilize Genemarker V1.75 (Soft Genetics LLC, USA) software to read the capillary electrophoresis raw data, the data after reading are used for subsequent analysis after correcting through the FlexiBinv2 program.In the screening process of 240 pairs of SSR primers, pick out altogether 170 pairs of amplifications and stablize, substantially build for follow-up molecular linkage map without assorted band and the stronger SSR core primers of signal.
Step 5 separation marking partially detects
Respectively the filial generation separation case of 170 amplifications is carried out χ2-test,chi-square test, analyze it and whether meet Mendel's law of segregation, find inclined to one side separation marking under 5% conspicuous level, there are 57 pairs to be marked under 5% conspicuous level and to be defined as inclined to one side separation marking, segregation ratio is 33.5% partially, when building the Genetic Linkage Map spectrum, inclined to one side separation marking is removed.
The structure of step 6 molecular genetic linkage map
Respectively with the type of 113 unpack formats that amplify (1: 1,1: 2: 1,1: 1: 1: 1) be converted into unified form, the jujube tree molecular genetic linkage map that builds with full sibs population genetic map construction software FslinkageMAP.
Fig. 1 is the jujube tree SSR tagged molecule genetic map of the embodiment of the present invention, and left data represents two relative distances between mark, and the right represents marker number used.According to the collection of illustrative plates result, 113 marks of mapping, there are 106 marks to be positioned on collection of illustrative plates, the mark utilization ratio is 93.8%, constructs one and comprises 16 linkage groups, and overall length is the genetic linkage maps of 579.86cM, the mean chart distance is 5.47cM, the longest linkage group comprises 15 marks, and overall length is 91.66cM, and mean chart is apart from being 6.11cM (2:SSR is marked at distribution on collection of illustrative plates referring to table); The shortest linkage group comprises 4 marks, and overall length is 3.90cM, and the mean chart distance is 0.98cM.What table 2 showed is detailed figure spectrum information.
Table 1
Table 2
Table 3
Claims (10)
1. the construction process of a jujube tree SSR tagged molecule genetic map, is characterized in that, comprises the steps:
1) extraction of DNA: the genomic dna that extracts jujube tree parent to be measured and filial generation;
2) screening of SSR labeled primer: with parent material screening SSR labeled primer;
3) pcr amplification: utilize respectively above-mentioned SSR labeled primer, take the genomic dna of jujube tree parent to be measured and filial generation as masterplate, carry out pcr amplification separately;
4) map construction: utilize biological software to analyze the pcr amplification result, find inclined to one side separation marking and remove under under 5% conspicuous level, build jujube tree SSR tagged molecule genetic map.
2. the construction process of jujube tree SSR tagged molecule genetic map according to claim 1, is characterized in that, jujube tree parent to be measured described in described step 1) is as maternal, take azalea as male parent take the winter jujube; Described filial generation is 149 strain F
1The filial generation in generation.
3. the construction process of jujube tree SSR tagged molecule genetic map according to claim 1, is characterized in that, the CTAB method after improvement is used in the extraction of DNA described in described step 1), and concrete steps are as follows:
(1) getting the addition of C TAB extracting solution, to put into 65 ℃ of water-bath preheatings stand-by;
(2) get Chinese Jujube 0.5g, put into mortar, add a small amount of PVP-40, be ground into powder rapidly in liquid nitrogen, be transferred in the centrifuge tube of the CTAB extracting solution that 1ml65 ℃ of preheating is housed;
(3) vortex thermal agitation, fully mixing, put into 65 ℃ of water-baths, and water-bath 50min stirs centrifuge tube up and down every 10min, fully mixing;
(4) centrifugal 10min under 12000rpm normal temperature;
(5) get supernatant liquor in new centrifuge tube, add equal-volume water-saturated phenol/chloroform/primary isoamyl alcohol mixed solution, its proportioning is 25:24:1, puts upside down gently mixing, and room temperature is placed 10min;
(6) centrifugal 10min under 12000rpm normal temperature;
(7) get step (6) gained supernatant liquor in new centrifuge tube, add isopyknic chloroform/primary isoamyl alcohol mixed solution, its proportioning is 24:1, extracting again;
(8) get step (7) gained supernatant liquor in new centrifuge tube, add the equal-volume Virahol, put upside down gently mixing, be positioned in-20 ℃ of refrigerators and precipitate 20min;
(9) choose the DNA flocks in the 2ml centrifuge tube, with 70% ethanol washing and precipitating 2 times, each 30min;
(10) dry up precipitation with sterile wind in Bechtop, add 100 μ l TE damping fluid dissolving DNAs, stand-by in-20 ℃ of preservations.
4. the construction process of jujube tree SSR tagged molecule genetic map according to claim 2, is characterized in that described step 2) described in the SSR labeled primer be 240 pairs of primers shown in table 3; Described step 2) described in, the screening of SSR labeled primer is based on magnesphere, exploitation winter jujube SSR primer, utilize maternal winter jujube and male parent azalea to carry out the screening of SSR labeled primer, select that in maternal winter jujube and male parent, at least one party shows as the primer of heterozygosis as core primers.
5. the construction process of jujube tree SSR tagged molecule genetic map according to claim 4, is characterized in that, described core primers is 170 pairs of core primers shown in table 3.
6. the construction process of according to claim 4 or 5 described jujube tree SSR tagged molecule genetic maps, is characterized in that described step 2) comprise the quality of the DNA of Detection and Extraction.
7. the construction process of jujube tree SSR tagged molecule genetic map according to claim 6, it is characterized in that, what described step 3) pcr amplification adopted is the three-primer method, namely adds M13 tail sequence, special reverse primer by 5 ' end at forward primer and with fluorescently-labeled universal M13 primer.
8. the construction process of jujube tree SSR tagged molecule genetic map according to claim 7, is characterized in that, also comprises after described step 3) pcr amplification the result after pcr amplification is detected, and adopts capillary electrophoresis to detect.
9. the construction process of jujube tree SSR tagged molecule genetic map according to claim 8, it is characterized in that, biological software described in described step 4) is Genemarker V1.75 software, and the data after reading are used for subsequent analysis after correcting through the FlexiBinv2 program.
10. the construction process of jujube tree SSR tagged molecule genetic map according to claim 1, it is characterized in that, respectively the pcr amplification result is carried out χ2-test,chi-square test in described step 4), analyze it and whether meet Mendel's law of segregation, find inclined to one side separation marking and remove under 5% conspicuous level, labeling pattern with three kinds of unpack format: 1:1,1:2:1,1:1:1:1 is converted into unified form respectively, selects full sibs population genetic map construction software FslinkageMAP to carry out the structure of molecular genetic linkage map.
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| CN104726598A (en) * | 2015-03-27 | 2015-06-24 | 北京农学院 | Primer for identifying 11 Beijing local jujube varieties and application of primer |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
| CN107151696A (en) * | 2017-02-27 | 2017-09-12 | 浙江理工大学 | A kind of germplasm identification method of fringe chinaroot greenbrier |
| CN107944227A (en) * | 2017-12-08 | 2018-04-20 | 江汉大学 | A kind of mark bearing calibration of genetic map and device |
| CN108192994A (en) * | 2018-03-15 | 2018-06-22 | 山东省林业科学研究院 | Chinese scholar tree DNA fingerprinting library and its construction method based on SSR marker |
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| KR101860232B1 (en) | 2016-12-05 | 2018-05-23 | 충북대학교 산학협력단 | Method for discrimination of jujube varieties using SSR marker |
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| CN104726598A (en) * | 2015-03-27 | 2015-06-24 | 北京农学院 | Primer for identifying 11 Beijing local jujube varieties and application of primer |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
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| CN107151696B (en) * | 2017-02-27 | 2020-06-05 | 浙江理工大学 | Method for identifying germplasm resource of smilax china |
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| CN107944227B (en) * | 2017-12-08 | 2021-06-29 | 江汉大学 | Genetic map marking correction method and device |
| CN108192994A (en) * | 2018-03-15 | 2018-06-22 | 山东省林业科学研究院 | Chinese scholar tree DNA fingerprinting library and its construction method based on SSR marker |
| CN108192994B (en) * | 2018-03-15 | 2020-07-21 | 山东省林业科学研究院 | Chinese scholar tree DNA fingerprint spectrum library based on SSR markers and construction method thereof |
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