1. 1 ? 1 generation molecular marker
1. 1. 1 ? RFLP tag technology.
Restriction fragment length polymorphism (RFLP) proposed by Botesin in 1980 can be used as a genetic marker, which initiated a new stage of direct application of DNA polymorphism and was the earliest molecular marker technology. RFLP is to detect the size of specific DNA fragments formed after digestion by restriction endonucleases, reflecting the distribution of different restriction sites on DNA molecules. Therefore, a slight change in DNA sequence, even a change of 65,438+0 nucleotides, can also cause the loss or production of restriction endonuclease cleavage sites, resulting in changes in the length of restriction endonuclease fragments.
Advantages: RFLP-labeled alleles have the characteristics of * * * dominance, the results are stable and reliable, and the repeatability is good, which is especially suitable for constructing genetic linkage maps.
Disadvantages: In RFLP analysis, DNA fragments of this site are needed as probes, and radioisotope and nucleic acid hybridization techniques are neither safe nor easy to automate. In addition, RFLP is not sensitive to DNA polymorphism detection, and there are many large spatial regions on the RFLP linkage map.
1. 1.2 ? RAPD marker technology.
In order to overcome the shortcomings of RFLP technology, Williams established random amplified polymorphic DNA (RAPD) technology in 1990. Because of its unique way of detecting DNA polymorphism, RAPD technology has rapidly penetrated into all fields of genetic research. RAPD is a molecular marker technology based on PCR. The basic principle is to randomly amplify DNA fragments by PCR with a random primer (8 ~ 10 base), and then separate the amplified fragments by gel electrophoresis to study DNA polymorphism. For any specific primer, there is a specific binding site in the genomic DNA sequence. Once the genome has DNA fragment insertion, deletion or base mutation in these regions, the distribution of these specific binding sites may change, resulting in changes in the number and size of amplified products, showing polymorphism.
Advantages: Compared with RFLP, RAPD has the advantages of simple technology, rapid detection, less DNA consumption, simple experimental equipment, no need for DNA probe, no need for pre-cloning, labeling or sequence analysis when designing primers, and no influence from species specificity and genome structure. A set of synthetic primers can be used for genome analysis of different organisms, and one primer can amplify multiple fragments without isotope, so it is safe.
Disadvantages: Of course, RAPD technology is influenced by many factors, and the stability and repeatability of the experiment are poor. The first is dominant inheritance, which can not identify heterozygote loci, making genetic analysis relatively complicated. In gene mapping and linkage genetic mapping, the accuracy of calculating the genetic distance between loci belongs to specificity and genome structure because of dominant coverage. A set of primers can be used for genome analysis of different organisms, and one primer can amplify multiple fragments without isotope, which is safe. Of course, RAPD technology is influenced by many factors, and the stability and repeatability of the experiment are poor. The first is dominant inheritance, which can not identify heterozygote loci, making genetic analysis relatively complicated. In gene mapping and linkage genetic mapping, the accuracy of calculating the genetic distance between loci will decrease due to dominant coverage. Secondly, RAPD is very sensitive to reaction conditions, including template concentration and Mg2+ concentration, so the repeatability of the experiment is poor.
1.3 ? AFLP labeling technique
? Amplified fragment length polymorphism (AFLP), also known as selective restriction fragment amplification 2 (SRFA), was invented by Zabean and Vos of KEYGENE Company in the Netherlands in 1993, and has applied for a patent. AFLP is a rapidly developing molecular marker technology in recent years. It connects the fragments of genomic DNA digested by paired restriction endonucleases with a linker (complementary to restriction sites), and amplifies a large number of DNA fragments through semi-specific primers with complementary 5' ends to the linker, thus forming a molecular marker technology of fingerprints. AFLP fingerprint is Mendel's dominant and recessive inheritance.
Advantages: It has the advantages of both RAPD and RFLP, and has high stability. A large number of loci can be detected in a short time with a small number of selective primers, and many loci detected by each pair of primers are more or less randomly distributed on multiple chromosomes. The number of AFLP markers on each chromosome is positively correlated with chromosome length (r = 0. 50 1), and the number of chromosomes involved in a pair of primers is positively correlated with the number of markers (r = 0. Therefore, AFLP markers covering the whole genome can be obtained by a small number of efficient primer combinations. At present, AFLP, as an efficient fingerprint technology, has played its advantages in genetic breeding research.
Disadvantages: However, some studies believe that AFLP requires high genomic purity and reaction conditions. In addition, when used for genetic mapping, the compactness of a few markers is different from that of the map. In addition, based on RAPD and RFLP techniques, scar (sequence characterization amplification region), caps (cleavage amplification polymorphism sequence) and daf (DNA am 2 amplification fingerprint) were established. The appearance of these technologies has further enriched and perfected the 1 generation molecular marker technology and increased people's research methods on DNA polymorphism.
1.2 ? Second generation molecular marker
2. 1 ? SSR marker technology.
There are many non-coding repetitive sequences in eukaryotic genomes, such as microsatellite DNA with repetitive unit length of 15 ~ 65 nucleotides (microsatellite DNA with repetitive unit length of 2 ~ 6 nucleotides). Microsatellite and microsatellite DNA are distributed in different sites of the whole genome. Because the size and order of repeating units are different, the number of copies is also different, which constitutes a rich length polymorphism. In 199 1 year, Moore established SSR (simple sequence repeat) marker technology by combining PCR technology. SSR, also known as microsatellite DNA, is a DNA sequence consisting of several motifs (mostly 1 ~ 5) repeated in series, among which the most common are dinucleotide repeats, namely (CA) n and (TG) n. The core sequence structure of each microsatellite DNA is the same, and the number of repeat units is 10 ~ 60, and its high polymorphism mainly comes from series connection. Different repetitions of different genetic materials lead to high variability of SSR length, which is the basis of SSR markers. The basic principle of SSR markers: design primers according to specific short sequences at both ends of microsatellite repeats, and amplify microsatellite fragments by PCR reaction. Due to the different repetition times of core sequence, it is an effective method to detect DNA polymorphism by amplifying PCR products with different lengths. Microsatellite sequence is highly polymorphic and dominant in population, so it is a good molecular marker. At present, microsatellite markers have been used to construct chromosome genetic maps of human, mouse, rat, rice, wheat, corn and other species.
Advantages: These microsatellite markers have been widely used in gene mapping and cloning, disease diagnosis, genetic analysis or variety identification, crop breeding, evolution research and other fields. In addition, SSR markers can not only distinguish homozygotes from heterozygotes, but also have more reliable results, simple methods and time-saving and labor-saving.
2.2 ? ISSR marker technology.
ISSR is a new molecular marker technology. 1994, Zietkiewicz developed SSR technology and established anchored microsatellite oligonucleotide technology. They use anchored microsatellite oligonucleotides as primers, that is, adding 2 ~ 4 randomly selected nucleotides at the 5' end or 3' end of SSR can cause annealing at specific sites, thus leading to PCR amplification of genomic fragments between repetitive sequences complementary to anchored primers. This marker is also called ISSR.
Sequence repeats), assr (anchored simple sequence repeats) or AMP2PCR. Among the two-wing primers used, one can be an ASSR primer and the other can be a random primer. If one is an ASSR primer with an anchor at the 5' end and the other is a random primer, it is called RAMP technology [19]. The primer used for PCR amplification of ISSR2 is usually 16 ~ 18 base sequence, which consists of 1 ~ 4 base tandem repeat sequence and several non-repetitive anchor bases, so as to ensure that the primer is combined with the 5' or 3' end of SSR in genomic DNA, and the DNA fragments between SSR are amplified by PCR reaction.
Advantages: SSR is widely distributed in eukaryotes, and the speed of evolution and mutation is very fast, so ISSR2PCR with anchored primers can detect the differences of many loci in the genome. Compared with SSR-2 PCR, the primers used in ISSR-2 PCR do not need to be sequenced in advance, which is why some ISSR primers may have no matching regions and amplification products in specific genomic DNA, and are usually dominant markers, Mendelian inheritance and have good stability and polymorphism.
1.3 ? Third generation molecular marker
3. 1 ? SNP labeling technique.
Single nucleotide polymorphism (single nucleotide? Polymorphism (SNP) is called the third generation DNA molecular marker, which refers to the single nucleotide difference between different alleles at the same site, including the deletion or insertion of a single base, and more commonly, the substitution of a single nucleotide, which often occurs between purine bases (A and G) and pyrimidine bases (C and T). SNP markers can help distinguish the differences of genetic material between two individuals and are considered as the most promising genetic markers. At present, more than 2 000 markers have been located on human chromosomes and 236 SNP markers have been developed in Arabidopsis thaliana. About 30% SNP markers contain polymorphism of restriction sites.
Advantages: The best way to detect SNP is DNA chip technology. The newly reported microchip electrophoresis can detect SNP in clinical samples at high speed, which is 10 and 50 times faster than capillary electrophoresis and plate electrophoresis respectively. SNP is different from RFLP of 1 generation and SSR of the second generation in two aspects: first, SNP is no longer detected by the length change of DNA fragment, but directly marked by sequence variation; Secondly, SNP marker analysis completely abandoned the classic gel electrophoresis and replaced it with the latest DNA chip technology.
3.2 ? EST marker technology.
The expression sequence tag (EST) was proposed in 199 1 by Venter, a biologist at the National Institutes of Health (NIH). With the development of human genome project, EST technology has been widely used in the research fields of discovering new human genes, mapping human genome and identifying coding regions of genome sequences, and then it has been widely used in plant genome research. EST refers to random cloning and sequencing in cDNA libraries of different tissues to obtain partial cDNA sequences. An EST corresponds to a cDNA clone sequence of an mRNA, which is generally 150 ~ 500 bp in length and only contains the gene coding region.
Advantages: EST can represent an expressed gene of an organism at a certain moment, so it is called "expression sequence identification"; However, the number of ESTs shows the copy number of the gene it represents. The more times a gene is expressed, the more cDNA clones it has. Therefore, by sequencing and analyzing cDNA clones, we can know the gene expression abundance. At present, kits are generally used to construct cDNA libraries, and the rapid development of DNA sequencing technology makes it possible to further reduce the cost of large-scale DNA sequencing.