CN103667487B - Based on the method for EST-SSR Marker Identification Loquat Cultivars - Google Patents
Based on the method for EST-SSR Marker Identification Loquat Cultivars Download PDFInfo
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Abstract
The invention discloses a kind of method based on EST-SSR Marker Identification Loquat Cultivars, belong to molecular biology molecule marker field.The present invention is directed to the est sequence design SSR primer of loquat, after screening, obtain 5 to the primer for cultivar identification, after extracting the DNA of Loquat Cultivars, utilize the DNA of primer amplification Loquat Cultivars one by one, the variety plot with unique specificity bands of a spectrum obtained is branched away, sets up tree-shaped discriminating figure.Primer of the present invention is through and strictly screens acquisition, just 18 Loquat Cultivars can be made a distinction by means of only 5 pairs of EST-SSR primers, easy to operate, quick and precisely; Method of the present invention can realize 18 Loquat Cultivars under any circumstance to distinguish fast and accurately, and tree-shaped discriminating figure has more intuitive than clustering tree, just can find out the primer distinguishing any two kinds according to tree-shaped qualification figure.
Description
Technical field
The present invention relates to molecular biology molecule marker field, relate in particular to a kind of method based on EST-SSR Marker Identification Loquat Cultivars.
Background technology
Loquat (Eriobotrya japonica Lindl, 2n=2x=34) be the Rosaceae (Rosaceae) Maloideae (Maloideae) Eriobotrya, have in Subtropic of China area and Asia other countries and plant widely.Loquat originates from the southeast (Lin and Hu, 2000) of China, has the cultivation history of more than 2,000 year in China, and germ plasm resource is enriched.Loquat belongs to subtropical evergreen fruit trees, and according to the feature of fruit, loquat can be divided into red sand and white sand kind (Fu et al., 2012); How spherical in shape ripe loquat is or oval, and soft and succulency, is rich in L-glutamic acid, aldehydes matter (especially flavonoid compound), the nutritive ingredient such as VITAMIN and carotenoid, is important health fruit (Cao etal., 2009; Fu et al., 2011).Loquat except eating raw, or processed can, jam, fruit cream, jelly and fruit wine good raw material.
Due to development and the growth of loquat industry in recent years, the popularization of the new quality product kind of Loquat Cultivars and deeply, and the destruction of resource environment, Loquat Cultivars has the trend towards the development of unicity aspect, the situation that in resource conservation, recurrent resource is obscured, the importance of research of fruit germplasm resource also causes the attention of more and more people.Therefore, set up the fast and reliable authenticate technology system of Loquat Cultivars effective utilization of Loquat Variety Germplasm and the sustainable development of protection and loquat industry are had great importance.
The morphology cultivar identification method that current tradition is single cannot meet the requirement effectively distinguishing or differentiate numerous kinds.The generation of molecule marker breaches the bottleneck of genetics research, overcomes traditional morphological markers, cytological marker and biochemical marker and is subject to extraneous factor, have incomparable advantage.In conventional RAPD, RFLP, SSR, AFLP equimolecular labeling technique, SSR marker because of having simplicity, polymorphism highly, codominant, efficiency advantages of higher is widely used in plant variety qualification, genetic linkage maps, the research such as analysis of genetic diversity and comparative genomics.It, with quick, easy and do not need to predict the characteristic and advantage of genome sequence, has been widely used in the aspects such as the sort research of kind, the Study on Genetic Basis of germ plasm resource, the structure of genetic map.Along with the rapid growth of EST quantity, EST develops into the general data source finding the polymorphic molecular markers such as SSR gradually.Compared with genome SSR, EST-SSR derives from the coding region of genetic transcription, directly can reflect the diversity of genes involved, and be easy to mark function gene, can serve the researchs such as fruit tree marker-assisted breeding, Molecular Phylogeny and cultivar identification better.Existing EST-SSR is in the Idioplasm identification of fruit tree crop, and the computer software that adopts draws digitized battlefield environment more, and carries out cluster analysis in conjunction with statistics software, but the result of cluster analysis often cannot be used for Variety identification.And such research often adopts 0-1 fingerprint and constructs clustering tree.When Quality Identification, operational ton is very large, does not possess ageing, judges by clustering tree is bad the primer distinguishing kind, and directly perceived not.
Summary of the invention
The object of this invention is to provide a kind of method based on EST-SSR Marker Identification Loquat Cultivars, for distinguishing fast, identifying Loquat Cultivars.
For achieving the above object, technical solution of the present invention is as follows:
Based on a method for EST-SSR Marker Identification Loquat Cultivars, its be used for identifying that the primer of Loquat Cultivars is that SSR primer by designing for loquat est sequence filters out through 3 times the primer that band is clear, polymorphism is good, specifically comprise:
18 important Loquat Cultivars that the present invention mainly collects for this seminar are distinguished, and these 18 Loquat Cultivars comprise: 1. ' large five-pointed star ', 2. ' large room ', 3. ' Ninghai is white ', 4. ' on small stream white sand ', 5. ' Luoyang blue or green ', 6. ' water chestnut kind ', 7. ' hat is beautiful ', 8. ' egg albumen ', 9. ' blue or green plant ', 10. ' white jade ', 11. ' sweet kinds ', 12. ' red late ', 13. ' six points of kinds ', 14. ' Bai Maowu ', 15. ' large rose-red robes ', 16. ' Chinese white poplar piers ', 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae)s ', 18. ' mounds '.
For above-mentioned kind, utilize above-mentioned primer Rapid identification to distinguish the method for Loquat Cultivars, specifically comprise the steps:
(1) the loquat DNA needing to carry out identifying, distinguishing is extracted, diluted for use;
(2) primer EJ-38 is utilized to carry out pcr amplification to loquat DNA and carry out PAGE gel detection, all characteristic spectrum belt is there is at 223bp, 227bp, and 245bp atypism bands of a spectrum, 1. ' large five-pointed star ', 13. ' six points of kinds ', 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ' three Loquat Cultivars is divided into A group; All there is characteristic spectrum belt at 227bp, 245bp, at 223bp atypism bands of a spectrum, 3. ' Ninghai is white ', 4. ' on small stream white sand ', 6. ' water chestnut kind ', 7. ' hat beautiful ', 10. ' white jade ', 14. ' Bai Maowu ' six Loquat Cultivars are divided into B group; At 223bp, 227bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 245bp, be numbered 5. ' Luoyang is blue or green ', 9. ' blue or green kind ', 11. ' sweet kinds ', 15. ' large rose-red robes ', 18. ' mound ' five Loquat Cultivars and be divided into C group; At 223bp, 245bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 227bp, be numbered 8. ' egg albumen ', 16. ' Chinese white poplar pier ' two Loquat Cultivars is divided into D group; At 223bp, 227bp, 245bp all atypism bands of a spectrum, be numbered 2. ' large rooms ', 12. ' red late ' two Loquat Cultivars is divided into E group, and enters step (3);
(3) utilize primer EJ-25 to carry out pcr amplification further and carry out PAGE gel detection, distinguishing the Loquat Cultivars in A, B, C, D, E five groups according to the characteristic spectrum belt occurred at 160bp, 210bp, 216bp and 220bp:
In A group, at 160bp place without characteristic strip to occur at 210bp place characteristic spectrum belt then kind 1. ' large five-pointed star ' distinguished; If all there is characteristic spectrum belt at 160bp, 220bp, kind 13. ' six points of kinds ' distinguished; 160bp, 210bp all occur characteristic spectrum belt then kind 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ' distinguished;
In B group, all characteristic spectrum belt is there is at 216bp, then kind 7. ' hat is beautiful ' is distinguished, and 3. ' Ninghai is white ', 4. ' on small stream white sand ', 6. ' water chestnut kind ', 10. ' white jade ', 14. ' Bai Maowu ' five kinds at 216bp atypism bands of a spectrum, enter step (4);
In C group, have characteristic spectrum belt at 220bp, at 160bp place without characteristic strip, kind 11. ' sweet kind ' is identified out by differentiation; And have characteristic spectrum belt at 210bp, at 160bp place without characteristic strip, kind 18. ' mound ' identified go out, its excess-three kind 5. ' Luoyang is blue or green ', 9. ' blue or green plant ', 15. ' large rose-red robes ' then enter step (4);
In D group, at 160bp place without characteristic strip, kind 8. ' egg albumen ' is identified out by differentiation; And characteristic spectrum belt is had at 160bp place, kind 16. ' Chinese white poplar pier ' then distinguished.
In E group, at 210bp atypism bands of a spectrum, kind 2. ' large room ' is identified out by differentiation; And characteristic spectrum belt is had at 210bp place, kind 12. ' red late ' is out identified.
(4) utilize primer EJ-20 to carry out pcr amplification, and carry out PAGE gel detection,
In B group, occur characteristic spectrum belt at 200bp place, kind 3. ' Ninghai is white ', 6. ' water chestnut kind ', 14. ' Bai Maowu ' three kinds are divided into one group; Kind 4. ' on small stream white sand ' and 10. ' white jades ', at 200bp place atypism bands of a spectrum, are divided into another group, continue to enter step (5);
In C group, have characteristic spectrum belt at 200bp, kind 15. ' large rose-red robe ' is out identified, and kind 5. ' Luoyang is blue or green ' and 9. ' blue or green kind ' then enters step (5);
(5) utilize primer EJ-16 and EJ-40 to carry out pcr amplification respectively, and carry out PAGE gel detection,
In B group, utilize primer EJ-40 increase to kind 3. ' Ninghai is white ', 6. ' water chestnut kind ', 14. ' Bai Maowu ' three kinds and detect, all occur characteristic spectrum belt at 236bp, 238bp place, kind 3. ' Ninghai is white ' is out identified; Only there is characteristic spectrum belt at 236bp place, and be kind 14. ' Bai Maowu ' at 238bp place without specific spectruming belt; Only there is characteristic spectrum belt at 238bp place, and be kind 6. ' water chestnut kind ' at 236bp place without specific spectruming belt;
Utilize primer EJ-16 to carry out amplification PAGE to kind 4. ' on small stream white sand ' and 10. ' white jades ' to detect, occurring characteristic spectrum belt at 172bp place, is kind 4. ' on small stream white sand '; Without this specific band is then kind 10. ' white jade ', and so far all Loquat Cultivars of B group are all out identified.
In C group, utilize primer EJ-16 increase to kind 5. ' Luoyang is blue or green ' and 9. ' blue or green plant ' and carry out PAGE detection, occurring characteristic spectrum belt at 172bp place, is kind 9. ' green grass or young crops kind '; Without this specific band is then kind 5. ' Luoyang is blue or green ', and so far all Loquat Cultivars of C group are also all out identified.
In aforesaid method, EST-SSR amplification is the PCR reaction system of 20 μ l: 10 μ l 2 × Taqmix, each 0.8 μ L of primer, 3 μ L DNA 30-50ng, appropriate ddH
2o to cumulative volume 20 μ L.Response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, renaturation 53 ~ 60 DEG C of 40s, 72 DEG C extend 1min, 35 circulations; Last 72 DEG C of condition downward-extension 10min.Get 5 μ L products after pcr amplification and carry out agarose 1.5% and PAGE electrophoresis detection, draw Loquat Cultivars qualification collection of illustrative plates according to the polymorphic bands of amplification.
In above-mentioned EST-SSR amplified reaction, the annealing temperature of primer used is as following table:
The present invention just can distinguish the loquat of 18 different varietiess by 5 primers, in order to clear, intactly represent the primer that qualification adopts and the kind that identifies, conveniently other people distinguish Loquat Cultivars, identify simultaneously, and the present invention also establishes " tree-shaped differentiates figure ".Described tree-shaped discriminating figure is PCR-based amplification, the result that bands of a spectrum form after PAGE gel detection counts, tree-shaped differentiates that figure is using primer as node, it is the differentiation result of this primer pair different varieties after node, comprise the classification distinguished or the kind directly distinguished, branch after node also marked the foundation that Different Results is distinguished mutually, the difference namely between bands of a spectrum.Such as: 223bp (+) represents there is characteristic spectrum belt at 223bp; 223bp (-) represents at 223bp atypism bands of a spectrum.
Utilize method of the present invention, and " tree-shaped differentiates figure ", greatly can facilitate qualification and the differentiation of above-mentioned 18 Loquat Cultivars, and, when known need to distinguish kind, the primer distinguishing these kinds can be found in " tree-shaped differentiate figure ", convenient and swift.
Effective effect of the present invention is: the EST-SSR primer used of quick differentiation Loquat Cultivars provided by the invention is through and strictly screens acquisition, there is higher stability and repeatability, by means of only 5 pairs of primers, 5 times 18 Loquat Cultivars just can easily make a distinction by PCR, easy to operate, quick and precisely; Method of the present invention can realize 18 Loquat Cultivars under any circumstance to distinguish fast and accurately, the tree-shaped discriminating figure of gained has more intuitive than clustering tree, the primer distinguishing any two kinds just can be found out according to tree-shaped qualification figure, the method can realize the Forepart identification of loquat nursery stock, and other species also have versatility widely.
Accompanying drawing explanation
Fig. 1 is the tree-shaped discriminating figure utilizing 5 pairs of primer pairs, 18 Loquat Cultivars to distinguish.
Fig. 2 utilizes the amplification electrophorogram of primer EJ-38 on 18 Loquat Cultivars.
Fig. 3 utilizes the DNA of primer EJ-25 to the 3rd group of kind to carry out EST-SSR to increase the amplification figure (line is specific spectruming belt, and the numeral of file is corresponding with table 1, and M represents DNA marker) obtained.
Fig. 4 utilizes primer EJ-20 (line is specific spectruming belt, and numeral is corresponding with table 1 to the amplification figure of 5,9,15 3 kinds in the 3rd group; M represents DNA marker).
Fig. 5 utilizes the amplification qualification of EJ-16 to kind in the 3rd group 5. ' Luoyang is blue or green ' and 9. ' blue or green plant ' to scheme (line is specific spectruming belt, and M represents DNA marker).
Fig. 6 is to kind 1. ' large five-pointed star ' and 8. ' egg albumen ', 3. ' Ninghai is white ' and 13. ' six points of kinds in embodiment ', (line is specific spectruming belt, and file numeral is corresponding with table 1 for the AFLP system of 14. ' Bai Maowu ' and 16. ' Chinese white poplar pier ', 2. ' large room ' and 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ', 4. ' on small stream white sand ' and 10. ' white jade '; M:DNA marker).
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described, better to understand the present invention.
Embodiment
Based on a method for EST-SSR Marker Identification Loquat Cultivars, concrete steps are as follows:
(1) DNA is extracted:
Loquat sample in the embodiment of the present invention is all common important Loquat Cultivars, mainly see " fruit tree will longan loquat volume ", (Qiu Wuling and Zhang Huizhi edits these 18 Loquat Cultivars, China Forestry Publishing House publishes), other part kinds are the loquat kind matter that Institute of Horticulture, Zhejiang Academy of Agricultural Sciences is newly collected in recent years.
In this step, with loquat spire for material, traditional CT AB method is utilized to extract sample DNA.By the loquat spire genomic dna extracted, detect through 0.8% agarose gel electrophoresis, the Bio-Photometer detection of nucleic acids instrument utilizing Eppendorf company to produce detects concentration and the purity of DNA, and final concentration is diluted to 30-50ng/ μ L, for pcr amplification.Each loquat numbering and variety name are in table 1.
Table a kind information used
(2) PCR reaction system and condition:
PCR reaction system is as follows: 10 μ l 2 × Taq mix, and each 0.8 μ L of primer, 3 μ L DNA, appropriate ddH2O are to cumulative volume 20 μ L.Response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, renaturation 53 ~ 60 DEG C of 40s, 72 DEG C extend 1min, 35 circulations; Last 72 DEG C of condition downward-extension 10min.Get 5 μ L products after pcr amplification and carry out agarose 1.5% electrophoresis detection, have the primer of desirable amplified production then to carry out the polymorphism of PAGE gel detection PCR primer.PCR reaction product utilizes 5% non-denaturing polyacrylamide gel to carry out in DYY-II type vertical slab electrophoresis instrument and DYC-30 type electrophoresis chamber (Liuyi Instruments Plant, Beijing's production).After loading under 170V and 150mA condition electrophoresis 120min, electrophoresis terminates the rear Silver Nitrate of 2 ‰ that utilizes and carries out silver dye, and utilize sodium hydroxide to develop the color, it is for subsequent use that white light preserves picture after taking pictures.
(3) Loquat Cultivars is distinguished fast:
The have no-trump Loquat Cultivars of DNA polymorphism bands of a spectrum in different varieties is selected to distinguish, until all kinds to be identified are only split up into separately." tree-shaped differentiate figure " is the result that primer after PCR-based amplification and polymorphism bands of a spectrum count, it is using primer as node, be the differentiation result of polymorphism bands of a spectrum to different varieties of this primer amplification after node, comprise the group distinguished or the kind directly distinguished.Utilize 5 pairs of EST-SSR primer pairs, 18 Loquat Cultivars to increase, can draw out complete tree-shaped discriminating figure (as shown in Figure 1), concrete differentiation step is as follows:
(3a) EST-SSR primer EJ-38 is utilized to increase (see Fig. 2) to loquat DNA:
All there is characteristic spectrum belt at 223bp, 227bp, and 245bp atypism bands of a spectrum, 1. ' large five-pointed star ', 13. ' six points of kinds ', 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ' three Loquat Cultivars is divided into A group; All there is characteristic spectrum belt at 227bp, 245bp, at 223bp atypism bands of a spectrum, 3. ' Ninghai is white ', 4. ' on small stream white sand ', 6. ' water chestnut kind ', 7. ' hat beautiful ', 10. ' white jade ', 14. ' Bai Maowu ' six Loquat Cultivars are divided into B group; At 223bp, 227bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 245bp, be numbered 5. ' Luoyang is blue or green ', 9. ' blue or green kind ', 11. ' sweet kinds ', 15. ' large rose-red robes ', 18. ' mound ' five Loquat Cultivars and be divided into C group; At 223bp, 245bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 227bp, be numbered 8. ' egg albumen ', 16. ' Chinese white poplar pier ' two Loquat Cultivars is divided into D group; At 223bp, 227bp, 245bp all atypism bands of a spectrum, be numbered 2. ' large rooms ', 12. ' red late ' two Loquat Cultivars is divided into E group.
(3b) utilize primer EJ-25 to carry out pcr amplification further and carry out PAGE gel detection, distinguishing the Loquat Cultivars in A, B, C, D, E five groups according to the characteristic spectrum belt occurred at 160bp, 210bp, 216bp and 220bp:
For C group, have characteristic spectrum belt at 220bp, at 160bp place without characteristic strip, kind 11. ' sweet kind ' is identified out by differentiation; And have characteristic spectrum belt at 210bp, at 160bp place without characteristic strip, kind 18. ' mound ' identified go out, its excess-three kind 5. ' Luoyang is blue or green ', 9. ' blue or green plant ', 15. ' large rose-red robes ', concrete schematic diagram is see Fig. 3.
(3c) utilize primer EJ-20 to ' Luoyang is blue or green ' in C group 5. further, 9. ' blue or green plant ', 15. ' large rose-red robe ' three Loquat Cultivars increases, characteristic spectrum belt is had at 200bp, kind 15. ' large rose-red robe ' is out identified, kind 5. ' Luoyang is blue or green ' and 9. ' blue or green kind ' then continues qualification, sees Fig. 4.
(3d) utilize primer EJ-16 increase to kind 5. ' Luoyang is blue or green ' and 9. ' blue or green plant ' and carry out PAGE detection, occurring characteristic spectrum belt at 172bp place, is kind 9. ' green grass or young crops kind '; Without this specific band is then kind 5. ' Luoyang is blue or green ', and see Fig. 5, so far all Loquat Cultivars of C group are also all out identified.
All the other groups are also identified according to aforesaid method, until all kinds are identified one by one in group.
(4) for guaranteeing the accuracy of the above results, the qualification of any two kinds can also be carried out according to the information in Fig. 1.When needing to inquire about the discrimination method of any Loquat Cultivars and required primer, only need to find corresponding kind to number in tree-shaped discriminating figure branches end, forward trace.
Such as, utilize the inventive method to distinguish 1. ' large five-pointed stars ' and 8. ' egg albumen ', 3. ' Ninghai is white ' and 13. ' six points of kinds ', 14. ' Bai Maowu ' and 16. ' Chinese white poplar pier ', 2. these five groups of Loquat Cultivars of ' large room ' and 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ', 4. ' on small stream white sand ' and 10. ' white jade '.Can find according to Fig. 1, primer EJ-38, EJ-25, EJ-16 can be selected respectively to identify, amplification is see Fig. 6.Can find out, qualification result is consistent with the cultivar identification figure of Fig. 1.
In sum, as long as relate to the loquat of any two kinds or its combination in above-mentioned 18 kinds, Variety identification and differentiation can be completed according to above-mentioned steps.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (4)
1., based on a method for EST-SSR Marker Identification Loquat Cultivars, it is characterized in that: be used for identifying that the primer of Loquat Cultivars is the SSR primer by designing for loquat est sequence, filter out through 3 times the primer that band is clear, polymorphism is good, specifically comprise:
。
2. the method based on EST-SSR Marker Identification Loquat Cultivars according to claim 1, is characterized in that: specifically comprise the steps:
(1) the loquat DNA needing to carry out identifying, distinguishing is extracted, diluted for use;
(2) primer EJ-38 is utilized to carry out pcr amplification to loquat DNA and carry out PAGE gel detection, all characteristic spectrum belt is there is at 223bp, 227bp, and 245bp atypism bands of a spectrum, 1. ' large five-pointed star ', 13. ' six points of kinds ', 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ' three Loquat Cultivars is divided into A group; All there is characteristic spectrum belt at 227bp, 245bp, at 223bp atypism bands of a spectrum, 3. ' Ninghai is white ', 4. ' on small stream white sand ', 6. ' water chestnut kind ', 7. ' hat beautiful ', 10. ' white jade ', 14. ' Bai Maowu ' six Loquat Cultivars are divided into B group; At 223bp, 227bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 245bp, be numbered 5. ' Luoyang is blue or green ', 9. ' blue or green kind ', 11. ' sweet kinds ', 15. ' large rose-red robes ', 18. ' mound ' five Loquat Cultivars and be divided into C group; At 223bp, 245bp all atypism bands of a spectrum, only there is characteristic spectrum belt at 227bp, be numbered 8. ' egg albumen ', 16. ' Chinese white poplar pier ' two Loquat Cultivars is divided into D group; At 223bp, 227bp, 245bp all atypism bands of a spectrum, be numbered 2. ' large rooms ', 12. ' red late ' two Loquat Cultivars is divided into E group, and enters step (3);
(3) utilize primer EJ-25 to carry out pcr amplification further and carry out PAGE gel detection, distinguishing the Loquat Cultivars in A, B, C, D, E five groups according to the characteristic spectrum belt occurred at 160bp, 210bp, 216bp and 220bp:
In A group, at 160bp place without characteristic strip to occur at 210bp place characteristic spectrum belt then kind 1. ' large five-pointed star ' distinguished; If all there is characteristic spectrum belt at 160bp, 220bp, kind 13. ' six points of kinds ' distinguished; 160bp, 210bp all occur characteristic spectrum belt then kind 17. ' tip Radix Campylotropis Hirtella (Herba Myrsines Africanae) ' distinguished;
In B group, all characteristic spectrum belt is there is at 216bp, then kind 7. ' hat is beautiful ' is distinguished, and 3. ' Ninghai is white ', 4. ' on small stream white sand ', 6. ' water chestnut kind ', 10. ' white jade ', 14. ' Bai Maowu ' five kinds at 216bp atypism bands of a spectrum, enter step (4);
In C group, have characteristic spectrum belt at 220bp, at 160bp place without characteristic strip, kind 11. ' sweet kind ' is identified out by differentiation; And have characteristic spectrum belt at 210bp, at 160bp place without characteristic strip, kind 18. ' mound ' identified go out, its excess-three kind 5. ' Luoyang is blue or green ', 9. ' blue or green plant ', 15. ' large rose-red robes ' only have characteristic strip at 160bp, then enter step (4);
In D group, at 160bp place without characteristic strip, kind 8. ' egg albumen ' is identified out by differentiation; And characteristic spectrum belt is had at 160bp place, kind 16. ' Chinese white poplar pier ' then distinguished;
In E group, at 210bp atypism bands of a spectrum, kind 2. ' large room ' is identified out by differentiation; And characteristic spectrum belt is had at 210bp place, kind 12. ' red late ' is out identified;
(4) utilize primer EJ-20 to carry out pcr amplification, and carry out PAGE gel detection,
In B group, occur characteristic spectrum belt at 200bp place, kind 3. ' Ninghai is white ', 6. ' water chestnut kind ', 14. ' Bai Maowu ' three kinds are divided into one group; Kind 4. ' on small stream white sand ' and 10. ' white jades ', at 200bp place atypism bands of a spectrum, are divided into another group, continue to enter step (5);
In C group, have characteristic spectrum belt at 200bp, kind 15. ' large rose-red robe ' is out identified, and kind 5. ' Luoyang is blue or green ' and 9. ' blue or green kind ' then enters step (5);
(5) utilize primer EJ-16 and EJ-40 to carry out pcr amplification respectively, and carry out PAGE gel detection,
In B group, utilize primer EJ-40 increase to kind 3. ' Ninghai is white ', 6. ' water chestnut kind ', 14. ' Bai Maowu ' three kinds and detect, all occur characteristic spectrum belt at 236bp, 238bp place, kind 3. ' Ninghai is white ' is out identified; Only there is characteristic spectrum belt at 236bp place, and be kind 14. ' Bai Maowu ' at 238bp place without specific spectruming belt; Only there is characteristic spectrum belt at 238bp place, and be kind 6. ' water chestnut kind ' at 236bp place without specific spectruming belt;
Utilize primer EJ-16 to carry out amplification PAGE to kind 4. ' on small stream white sand ' and 10. ' white jades ' to detect, occurring characteristic spectrum belt at 172bp place, is kind 4. ' on small stream white sand '; Without this specific band is then kind 10. ' white jade ', and so far all Loquat Cultivars of B group are all out identified;
In C group, utilize primer EJ-16 increase to kind 5. ' Luoyang is blue or green ' and 9. ' blue or green plant ' and carry out PAGE detection, occurring characteristic spectrum belt at 172bp place, is kind 9. ' green grass or young crops kind '; Without this specific band is then kind 5. ' Luoyang is blue or green ', and so far all Loquat Cultivars of C group are also all out identified.
3. the method based on EST-SSR Marker Identification Loquat Cultivars according to claim 2, it is characterized in that: in described method, to be the PCR reaction system of 20 μ l be in EST-SSR amplification: 10 μ l2 × Taq mix, the each 0.8 μ L of primer, 3 μ L DNA 30-50ng, appropriate ddH
2o to cumulative volume 20 μ L; Response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, renaturation 53 ~ 60 DEG C of 40s, 72 DEG C extend 1min, 35 circulations; Last 72 DEG C of condition downward-extension 10min; Get 5 μ L products after pcr amplification and carry out agarose 1.5% and PAGE electrophoresis detection, draw Loquat Cultivars qualification collection of illustrative plates according to the polymorphic bands of amplification.
4. the method based on EST-SSR Marker Identification Loquat Cultivars according to claim 2, it is characterized in that: in described method in EST-SSR amplified reaction, the annealing temperature of primer used is as following table:
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| 枇杷基因组SSR分子标记的分离及筛选;向素琼等;《第四届全国枇杷学术研讨会论文集》;20091231;第244、247页 * |
| 枇杷属植物等位酶遗传变异及品种基因型指纹;蔡礼鸿等;《武汉植物学研究》;20051231;第23卷(第5期);第406-416页 * |
| 枇杷种质资源SSR标记指纹图谱的构建;岳娜等;《中国农学通报》;20121231;第28卷(第16期);第186-189页 * |
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