bài giảng chọn giống lúa sử dụng chỉ thị phân tử khái niệm và ứng dụng thực tế, Application of molecular markers in rice, Application of molecular markers in rice ,Application of molecular markers in rice
Application of Molecular Markers In Rice Classification of main molecular markers Types of molecular markers Based on DNA hybridization Based on PCR amplification Random primer Specific primer Single nucleotide polymorphism RFLP RAPD 、 AFLP AP 、 PCR 、 ISSR SSR 、 STS 、 SCAR 、 CAPS SSCP 、 TGGE 、 DGGE SNP Technologic Comparison of Main Molecular Markers Markers Based on PCR or not polymor phism hereditability repeatability Automatizatio n cost RFLP No Low/me dium Co-dominance High Low High RAPD Yes Medium/ dominance high Low Medium Low SCAR/C APS Yes High Co-dominance High Medium Medium AFLP Yes High dominance High Medium/high Low SSR Yes High Co-dominance High Medium/high Low ISSR Yes High dominance High Medium/high Low STS Yes High Codominance /dominance High Medium/high Low SRAP/ES T Yes Medium Co-dominance High Medium Low IRAP/RE MAP Yes High Co-dominance High Medium/high Low SNP yes Very Co- high high low Demands of ideal molecular markers Ideal molecular markers must meet the following demands: (1) Have high polymorphism; (2) Codominance heredity, meaning that they can distinguish heterozygous and homozygous genotype in diploid; (3) Can distinguish allele; (4) Spread all over the genome; (5) well-distributed in the genome except special sites; (6) Have no pleiotropism; (7) Have a simple and fast detection method （ e.g experiment procedures are easily automatized （ ; (8) Have the development cost and use-cost as low as possible; (9) Have excellent repeatability between laboratories （ available for data exchange （ RFLP (Restriction Fragment Length Polymorphism) RFLP has been widely used in genetic map construction of genomes, gene mapping （ as well as biological evolution and classification study RFLP is based on different species (individuals) whose genomes have endonuclease restriction sites of the base mutation, or restriction sites between the bases of the insertion or deletion which results in restriction fragment size changes Such a change can be carried out through specific detection probe hybridization, which can discern differences of varieties (individual) in the level of DNA (ie, polymorphism) and multiple probes can be established by comparison of the evolution of biological relations and classification The used probes come from the same species or different species of cloned genomic DNA and are located in different chromosomal loci, which can be used as a molecular marker to build molecular map When a trait (gene) coseparates with the molecular markers, it indicates that the trait (gene) links to the molecular markers The exchange value between the molecular markers and traits means the distance of the target gene and molecular markers Then genes can be located in the molecular genetic map After different restriction endonucleases cut the genomic DNA, there will be different types of cutting fragments Therefore, restriction enzymes and molecular markers can be combined to study Common restriction endonucleases are Hind Ⅲ, BamH Ⅰ, EcoR Ⅰ, EcoRV and Xba Ⅰ The more molecular markers can build a more saturated map Construction of saturated RFLP map is one of the main objectives of the study Characteristics of RFLP RFLP markers are stable and reliable and the detection is never influence by environmental conditions and the impact of developmental stages RFLP markers are codominant among alleles There is no epistatic effect between the non-alleles RFLP markers derive from genomic DNA of its own variation and are virtually unlimited in the number The research of identification would not be desirable for a large number of individuals(>200) because the current RFLP technology is still expensive and laborious with the complicated operation and requires a large quantity of DNA samples RAPD ( Random amplified polymorphim ) • RAPD technology is built on the basis of PCR technology It is using a series of (usually hundreds of ) different base sequences These sequences use random single-stranded oligonucleotide (usually 10 polymer) as primers for the study of genomic DNA for PCR amplification Through polyacrylamide or agarose gel electrophoresis separation and EB staining or autoradiography to detect radioactive PCR products of polymorphic DNA fragments, the PCR products reflect the polymorphism of DNA fragments corresponding to genomic region of DNA polymorphism Characteristics of RAPD ： RAPD technique is simple and easy to grasp compared to RFLP It not onlyovercomes the shortcomings of dificient isozyme loci but also avoids the disadvantages of RFLP and the complicated operation What’s more, it doesn’t need the isotopic molecular hybridization, so the general laboratory can use this technique Most RAPD molecular marker are dominant markers, only a small number can be developed into codominant markerd Therefore, information provided is limited and covers up the dominant homozygote and heterozygote distinction Meanwhile, markers themself are completely dominant RAPD technique has a high false positive rate, if the experimental condition is not very stringent, there will be a higher false positive rate; RAPD technique demands a high purity and is sensitive to DNA reaction condition with a bad repeatability; For the markers attained by experiments, they must be transformed into other kinds of markers to suceed to map genes and enrich the genetic map Liangyoupeijiu RM263 zhuliangyou30 RM22 Equipments DNA extraction Cell disintegrator, freeze centrifugal machine etc DNA amplification PCR plates, PCR amplification machine etc Gel electrophoresis Gel, electrophoresis system etc Imaging system UV gel imaging system ： ： ultra-pure water instrument 、 、 cooling chamber (3)Automatic high speed refrigerated centrifuge 、 、 PCR amplification machine ： ： electrophoresis system 、 、 UV gel imaging system • Instructions for Indentifying the Purity of Hybrid Rice Seeds by SSR Markers • DNA extractrion • Seeds of a rice variety are planted in a germinating box, placed in the incubator, with a temperature of 32 ℃ When the seedlings are coming out, cut and put them into a microwell plate, one seedling in one microwell • Add 40 uL buffer solution to each microwell and then put the microwell plate in a boiling water bath for exactly 30 s Then draw the plate out and add 60 uL buffer solution to each of the microwell, leave the plate in a boiling water bath for exactly and then remove and place the plate on ice • PCR reaction • PCR reaction is conducted in total volume of10 uL, with 10 ng of template DNA, pmol of the primer, 1nmol of dNTPs, （ U of Taq polymerase and uL of 10x buffer[100 mmol Tris-HCI (pH （ 3) （ 500 mmol KCl （ 15 mmol MgC12 and 0.01 % gelatin] PCR reaction system tube 50 tube 100 tube 10x buffer 50 100 dNTP(2Mm) 0.5 25 50 Taq gold(2.5U) 0.08 H2O 7.22 361 722 Primer(5uM) 0.2 10 20 DNA 50 100 Total 10 500 1000 • PCR procedure • PCR is performed in 96 microwell plates using the following procedure （ 94℃ for min, then 35 cycles of 94 ℃ for 15 s, 55 ℃ for 15 s and 72 ℃ for 30 s, and a final extension step of at 72 ℃ • Electrophoresis detection • The PCR products are analyzed by running on （ agarose gel and • electrophoresed in 1x TAE at 120V for 30 （ 45 Then the amplified products are detected by BIORAD Gel Imaging System （ ... basis of SSR markers Its basic principle is that in the SSR of the 'or 3' end to base are anchored in the form of purine or pyrimidine Then amplify DNA sequences Repeat sequence and anchoring... there will be more and more SCAR markers to be developed, which will play a huge role in the molecular markerassisted breeding Application of Molecular Markers in Molecular Biology For species-specific... specific size of cloned fragments This molecular marker-specific DNA markers after transformation is called SCAR • SCAR marker is a kind of dominant marker in the form of existence of amplified