——4— Jiangsu Agricultural Sciences can find more markers in the second issue of 2003, but the disadvantages are that RAPD markers are mostly phenotypic, and it is impossible to distinguish whether genotypes are homozygous or heterozygous, and each marker provides less information and poor repeatability. RAPD markers are usually repetitive sequences. If they are not repeated sequences, they can also be transformed into RFLP markers to further detect the results of RAPD analysis. 1, random primer (10mer) (5umol/L): buy the finished product.
2, Taq enzyme: buy finished products.
3. 10xPCR buffer: See Chapter 8 for the formula.
4. Magnesium chloride: 25 mmol/L 。
5.dNTP: 2.5mmol/L each. 1. In the 25ul reaction system, add
Template DNA 1ul (50ng)
Random primer 1ul (about 5pmol)
10xPCR buffer 2.5 microliters
Magnesium chloride 2ul
dNTP 2ul
Taq enzyme 1 unit (u)
Add ddH2O to 25ul.
Mix well and centrifuge slightly, and add a drop of mineral oil.
2. Pre-denature at 94℃ for 2 minutes in a PCR instrument heated above 90℃, and then cycle: 94℃ 1 min, 36℃ 1 min, 72℃ 1 min, * * 40 cycles.
3. After circulation, store 10 min at 72℃ and 4℃.
4. Take PCR products 15ul and 3ul loading buffer (6x) to electrophoresis on 2% agarose gel, and the voltage is stable at 50- 100V (low voltage, neat band pattern and high resolution).
5. After electrophoresis, observe and take photos.
[Note] 1. Generally, there are 5- 15 RAPD bands with the size of 0. 1-2.0kb in electrophoresis.
2. Specific DNA bands can be cloned and used as new molecular markers. Principle RAPD technology is based on PCR technology. It uses a series (usually hundreds) of different oligonucleotide single strands with randomly arranged base sequences (usually 10 bp) as primers, and amplifies the studied genomic DNA with a single primer. The template DNA is denatured and melted at 90-94℃, and then annealed at a lower temperature (36-37℃). At this time, the single-stranded template will have many sites complementary to the primer, and at 72℃, the double-stranded structure will be formed by chain extension to complete DNA synthesis. By repeating the above process, amplification products with different fragment sizes can be produced, and many different bands can be obtained by electrophoretic separation and color development, from which characteristic bands can be screened out. The polymorphism of amplified product fragments reflects the polymorphism of genomic DNA. If DNA fragments are inserted, deleted or mutated in these regions, the distribution of these specific binding sites may change accordingly, and the molecular weight of PCR products will increase, disappear or change. Therefore, the polymorphism of genomic DNA in these regions can be detected by detecting PCR products. When conducting RAPD analysis, the number of available primers is very large. Although the detection area of genomic DNA polymorphism is limited for each primer, the detection area can cover almost the whole genome by using a series of primers. Therefore, RAPD can detect the polymorphism of the whole genome DNA.
The advantages of RAPD technology are: ① no isotope is used, which reduces the harm to the health of workers; ② DNA polymorphism of species can be analyzed without any molecular biology research; ③ The purity of template DNA is not high; ④ The technology is simple, and there is no need to clone DNA probes and perform molecular hybridization; ⑤ High sensitivity and rich polymorphism; ⑥RAPD primers have no strict species boundaries, and the same set of primers can be applied to the study of any organism, so it is universal and universal.
However, RAPD technology is easily influenced by various factors. No matter the quality and concentration of template, short primer sequence, the number of cycles of PCR, the complexity of genomic DNA, technical equipment, etc. This may be the direct reason for the poor repeatability of RAPD technology. At present, the stability of the reaction is improved from the following aspects: ① standardized operation, consistent composition of the reaction system, and standardized RAPD reaction as much as possible; ② Improving the resolution of amplified fragments; ③ The stability and reliability of the reaction can be improved by transforming RAPD markers into SCAR markers and then performing routine PCR analysis.
Application of RAPD technology in the study of fish, shrimp and crab
2. Application of1in the study of genetic resources
At present, the genetic variation of marine shrimps and crabs is mostly detected by detecting the variation of isoenzymes, and the polymorphism of isoenzymes revealed is relatively low. Random Amplified Polymorphic DNA (RAPD) analysis requires very few samples, simple operation procedures and short experimental period, and can analyze a large number of samples. A large number of RAPD molecular markers of different species, strains, populations and even individuals can be obtained by using a set of primers, and can be systematically analyzed by computer. These characteristics of RAPD technology make it play an important role in population genetics and genetic diversity analysis of shrimp and crab.
RAPD markers can be used to study the population genetics of shrimps and crabs, detect the level of genetic diversity within and between populations, and provide reliable genetic markers for population identification. Garcia and others tried to analyze the genetic diversity among different geographical populations of Penaeus monodon by RAPD, and preliminarily discussed its application in shrimp breeding. Garcia and a 1 Sival-Warren, etc. The wild populations and different families of Penaeus vannamei bred with high health and no specific pathogen (SPF) were monitored and analyzed. It was found that the proportion of polymorphic loci was about 50%, and the proportion of polymorphic loci in a family was as high as 77%. Liu Ping et al. used RAPD technique to study the genomic DNA polymorphism of the parents and their offspring of Penaeus China coastal population. The results showed that the genetic distance between offspring was smaller than that between parents and offspring, and the degree of genetic variation was low. Qiu et al. used technology to analyze the genetic differences within and between 20 populations of China shrimp in Yantai and Long Island. The results show that there are some genetic differences among different geographical populations. And Liu et al. analyzed the genetic diversity of different geographical populations of China shrimp. The results showed that the genetic diversity of China shrimp was low. Gao Zhigan and others conducted RAPD analysis on Eriocheir sinensis populations in Liaohe River and Yangtze River. The results showed that the intraspecific genetic variation of Eriocheir sinensis was low. Among the three populations, the genetic variation of Liaohe population and Yijiang population is larger, while the genetic variation of Yangtze population is smaller. The genetic distance between populations in Liaohe River Basin is smaller than that between populations. The genetic diversity of NY population and NX population of Macrobrachium rosenbergii was studied by RAPD, and the variation of NY population was obviously smaller than that of NX population. Song and Zhuang Zhimeng studied the genetic structure of wild and cultured populations of Penaeus japonicus by RAPD technique. The results showed that the proportion of polymorphic loci and heterozygosity of wild population were significantly higher than those of cultured population, indicating that the germplasm resources of wild population of Penaeus japonicus in China coastal area were in good condition and should be protected.
A detailed genetic linkage map is not only of great significance to the basic genetic research of this species, but also helpful to the breeding research of this species. Potlethwait and Stephen used RAPD technique to draw the genetic linkage map of zebrafish. Liu applied the technology to the study of catfish gene map. Sun Xiaowen and others established the genetic linkage map of carp. There are 56 RAPD molecular markers, 26 carp SSLP markers, 19 carp SSLP markers, 70 zebrafish SSLP markers, 9 1 carp gene markers and 50 linkage groups in the map. The linkage map showed that the genome size of carp was about 5789cm.
2.2 Determine the genetic relationship between species and varieties.
The traditional research methods of population genetic relationship are based on morphological, cytological and biochemical indexes, but they are greatly influenced by individuals and environment, and sometimes they cannot reflect the inherent characteristics of species themselves. The composition of species genome is reflected in RAPD map. The closer the genetic relationship, the more homologous sequences in the genome, and the more markers * * * amplified by the same primer. Therefore, the combination of RAPD technology and traditional methods is an effective means to study the genetic relationship of species. RAPD is used to analyze the DNA of different species and different populations of the same species, screen out characteristic bands, calculate similarity coefficient and genetic distance, and thus determine their genetic relationship. Li Sifa and others studied the genetic relationship of Eriocheir sinensis (Eriocheir sinensis and Eriocheir japonica) in six major river systems along the coast of China by RAPD. Two primers with population specificity were selected from 48 primers, among which the 880bp fragment amplified by Z2 was unique to the Pearl River Mouth crab and Nanliujiang crab, and the 700bP fragment amplified by Z2 was unique to the Yangtze River crab, Yellow River crab, Liaohe crab and Oujiang crab. This can be used as a molecular genetic marker to distinguish Eriocheir sinensis (Yangtze crab, yellow crab, Liaohe crab and Oujiang crab) from Eriocheir japonica (Pearl River crab and Nanliujiang crab). Two species of Eriocheir sinensis in six coastal water systems in China were classified by RAPD, and the results were consistent with those of biochemical genetic difference analysis and morphological multivariate analysis. The genetic relationship of six species of prawns was studied by Song, and 364 clear and stable polymorphic fragments were obtained by amplification with 20 random primers. According to the * * * enjoyment of the amplified fragments, the relative genetic distance index was calculated, and then analyzed by UPGMA and NJ clustering methods, and the phylogenetic tree was constructed to determine their genetic relationship. The results of cluster analysis showed that China shrimp, Penaeus penicillatus and Chienchyma japonicus had the closest genetic relationship. They get together first, then Penaeus monodon, and finally Penaeus vannamei and Penaeus japonicus. It can be seen from the results that species with similar external morphology show great similarity in genomic DNA, and vice versa. Song et al. studied the genomic DNA polymorphism of six species of shrimp, and the results showed that the genetic relationship of the six species of shrimp was basically consistent with the traditional classification results. Hao Xie studied the genetic relationship of three species of Eriocheir sinensis by RAPD, amplified the genomic DNA of 65,438+00 individuals of each species with a set of primers, and obtained a number of specific and repeatable amplified maps. Amplified band analysis showed that Eriocheir sinensis was far away from Eriocheir japonica, while Eriocheir sinensis (Hepu subspecies) in Nanliujiang was close to Eriocheir japonica.
Borrwsky et al. reconstructed the DNA fingerprint of vertebrates with random primer amplification products. Sultman et al. used DNA markers to study the phylogeny of Calliphoridae. He Shunping analyzed five species of CYPRINIDAE by RAPD and discussed the systematic position of carp. He Shunping and others obtained a large number of polymorphic DNA fragments with phylogenetic information through random amplification of CYPRINIDAE fish, and drew the phylogenetic map of representative genera and species of lower CYPRINIDAE. Shade analyzed the genetic relationship of three whitebait species in Taihu Lake by RAPD, and found that the nuclear genomes of some samples were significantly different from those of several whitebait species in Taihu Lake. Zou Shuming and others used RAPD to study the genetic relationship among grass carp, carp and carp, and the results supported the view that fish in eastern China had dual origins.
2.3 Markers of Specific Genes
Variations at the molecular level of DNA can be used as genetic markers for gene markers. Zhou Kaiya and others used RAPD to study and identify the Eriocheir sinensis population, and 200 random primers were used to analyze the population of Eriocheir sinensis in Liaohe, Changjiang and Oujiang. Primers HX0 1 and HX02 detected that all samples of Oujiang population and Liaohe population * * had amplified fragments of Hx0 1-0.4 and HX02-0.7; However, these two amplified fragments did not appear in the PCR reaction of the Yangtze population samples, so they can be used as identification markers of the Yangtze population. No markers were found to distinguish Oujiang population from Liaohe population. Hao Xie used RAPD to study the genetic relationship of three species of Eriocheir sinensis, and 25 individuals of Eriocheir sinensis, 27 individuals of Eriocheir japonica and 2 individuals of Eriocheir japonica subspecies/kloc-0 were amplified by primer OPO-05. The results showed that all individuals of Eriocheir sinensis could amplify a band with the size of about 1kb, which was quite obvious and stable, while a few individuals of the other two species could only amplify quite weak bands under the same conditions. Therefore, this band can be used as a genetic marker to distinguish Eriocheir sinensis from Eriocheir japonica and its Hepu subspecies.
At present, RAPD has been widely used to identify and identify different biological species. Johnson identified zebrafish of different laboratory strains by RAPD technique. Xue Guoxiong analyzed the coverage of grass carp spawning grounds in Yangtze River, Pearl River and Heilongjiang and grass carp caught by RAPD in Taihu Lake. The results showed that grass carp populations in each river system had their own characteristic gene maps, which could be used as the basis for population identification. Shade Quanheng detected a breeding population of Oreochromis niloticus and three breeding populations of Oreochromis niloticus from Hu Xiang, the United States and Shashi, and obtained molecular genetic markers to distinguish Oreochromis niloticus from Oreochromis niloticus. Yao Jihua and others used 20 random primers to conduct RAPD detection on three crucian carp populations in Fangzheng, Pengze and Qihe areas, and also obtained the molecular markers for identification of crucian carp populations. Deng Huai and others amplified herring, grass carp, silver carp, bighead carp, crucian carp, carp, bream bream, mullet, catfish and yellow catfish by RAPD—PCR. The results show that RAPD is a very sensitive interspecific identification technique, especially suitable for the identification of fish eggs and fry. Zheng Guangming et al. extracted blood genomic DNA from mud carp and wheat mud carp raised in ponds, and used random primers for PCR amplification. By shortening the amplification time and electrophoresis time, three different species of shad were quickly identified, and a set of methods for quickly identifying fish species at the DNA molecular level was established.
2.4 Identify gender differences in the same species
There is no uniform pattern of sex chromosomes in shrimps and crabs, so it is impossible to identify sex chromosomes by routine karyotype analysis. Using RAPD technology, we can find the characteristic bands of genomic DNA of the same species with different sexes, which is helpful to study the sex control mechanism and provide the molecular basis for sex determination. The sex of Eriocheir sinensis was identified by RAPD in Tao Qiu. Among 200 primers, 17 primers amplified the difference at the population level, and 1 primer (OPM 14) amplified the difference at the individual level: males had a specific band of 800bP, while females did not. This can be used as a valuable sex identification marker.
2.5 Monitoring genetic penetration to maintain species genetic diversity.
At present, artificial propagation activities and artificial release have made unnatural genetic infiltration among different populations of shrimp and crab quite common, which should be paid attention to, monitored and collected to provide basis for protecting germplasm resources and maintaining species genetic diversity. Li Sifa and others used RAPD to study the genetic relationship of Eriocheir sinensis from six rivers in Chinese mainland. The frequency of 947bP fragment amplified by primer OPP 17 in the Yangtze River, Yellow River and Liaohe River decreased significantly from south to north, with the Yangtze River being 87.5%, Yellow River being 4 1.66% and Liaohe being 10.83%. This measure of genetic penetration can be used as a standard to distinguish Eriocheir sinensis from three water systems. Tao Qiu and others used RAPD to study the genetic diversity of three Eriocheir sinensis populations in Yangtze River, Liaohe River and Oujiang River. No characteristic bands were found among the three groups, but the differences between groups were greater than those within groups. No molecular genetic markers were found among the populations, but there were differences among the three populations, which indicated that genetic infiltration was one of the reasons for the recent mixed germplasm resources.
RAPD markers can be used in population genetics research to detect the level of genetic diversity within and between populations and provide reliable genetic markers for population identification. Bardakci et al. used RAPD technique to evaluate the genetic variation within and between populations of tilapia. Bilowski et al. conducted RAPD analysis on striped bass along the Pacific coast. Caccone et al. conducted RAPD—PCR analysis on DNA polymorphism of European sea bass. Garcia and others tried to analyze the genetic polymorphism among different geographical populations of Penaeus monodon by RAPD, and preliminarily discussed its application in shrimp breeding. Wang Ruomei and others used RAPD technique to detect the genomic DNA polymorphism of wild crucian carp and four representative goldfish. By using this technique, the genetic diversity of two artificially cultured silver carp populations in Guangdong and Guangxi was comparatively studied in Dechun Zhang, which provided a certain theoretical basis for the scientific norms of artificial propagation of silver carp. Zhang Siming and others studied the randomly amplified polymorphic DNA and genetic diversity of ACIPENSER sinensis. The results showed that the genetic diversity of nuclear DNA level in the natural population of ACIPENSER sinensis was low, which provided a theoretical basis for the monitoring and protection of ACIPENSER sinensis resources. Tuo Shi et al. used this technique to detect the genomic DNA polymorphism of shrimp population on the west coast of Korean Peninsula in China, and the results showed that the genetic diversity of this population was low. Song et al. made a preliminary study on the markers of genetic structure of wild and cultured populations of Penaeus japonicus, and thought that the application of isomolecular marker technology in marine animal breeding and marker-assisted breeding would be a strong guarantee for the healthy development of marine animal breeding.
RAPD technology is simple and fast, and there is no need to determine the sequence of the target gene in advance, so it has no species specificity. There are countless primers available, which can produce enough polymorphism. It can identify the differences between species and populations, and can also distinguish the differences between individuals. Therefore, many researchers engaged in aquatic breeding have applied RAPD technology to aquatic genetic breeding and achieved certain results. In the study of marine fish (such as Pseudosciaena crocea) and shrimp germplasm resources, because the level of genetic differences revealed by isoenzymes is very low, and RAPD can indicate DNA polymorphism better than isoenzymes, so RAPD molecular markers have broad application prospects in the genetic research of fish, shrimp and crab germplasm resources. At present, RAPD analysis is mainly used in marker identification, genetic mapping, phylogeny and evolution, genetic relationship research, population division, population genetic diversity research and so on. However, in practical application, RAPD also shows some shortcomings, such as the positive locus can not distinguish homozygote from heterozygote in offspring; Poor stability; Large variability and the like. Of course, some can be avoided. The solution is to screen single-stranded primers and optimize the reaction conditions, but the first disadvantage is inevitable. If RAPD can be combined with other molecular markers such as RFLP, AFLP and microsatellite, it will still play a better role. In short, with the continuous development and more applications of biotechnology, RAPD technology will be more perfect and more widely used.
Compared with PCR, RPAD has the following characteristics: ①RAPD uses random primers without knowing the target gene and the corresponding sequence in advance. ② The operation is simple, the experimental period is short, and a large number of samples can be screened in a short time. ③ Primers have universal adaptability and are suitable for automated operation and analysis.
Compared with RFLP, it has the following characteristics: ①RAPD analysis only needs a small number of templates, and one amplification only needs 20- 100ng, which is beneficial to genome analysis of materials with less DNA. For example, DNA analysis of pollen, protoplasm and seeds is feasible. ②RAPD markers are more random, which is also beneficial to map construction. For species with high DNA content and polyploid species, RFLP probes will hybridize with multiple fragments of polyploid, and the obtained mixed fingerprints will bring difficulties to the elucidation of other alleles. In addition, due to a large amount of DNA, it is not practical to carry out single-copy Southern hybridization, which requires at least a long exposure time, and the number of known probes is limited. RAPD has greatly increased the number of species that can construct genetic maps. (3) harmful isotopes are not needed, and the consumption of manpower and material resources is low. ④ High sensitivity. The change of individual bases in primers will cause drastic changes in amplification bands and intensities, which is incomparable to RFLP. ⑤RAPD markers can cover the whole genome, including coding region and non-coding region, and can reflect the changes of the whole genome. ⑥RAPD products amplified more than 50% bands in single copy region. After cloning and sequence analysis, it can be used as a probe for RFLP and in situ hybridization, and is widely used in gene location, cloning and assisted selection breeding. RAPD is a brand-new and effective genetic marker, which has many advantages compared with other molecular markers:
① Synthesize a set of primers, which can be used for genome analysis of different organisms. Relatively speaking, RFLP markers have race specificity, which limits their application.
②RAPD technology is simple, labor-saving and time-saving. There is no need for preliminary work of RFLP analysis, such as clone preparation, isotope labeling, Southern blotting and molecular hybridization. Moreover, RAPD detection is sensitive and convenient, and can be detected with fluorescent dyes or isotope markers, which greatly improves its analysis speed. That is to say, using sequence gel to analyze RAPD, one person can complete the analysis of 120 samples in only 36 hours, while RFLP takes at least one week.
③ The amount of sample DNA needed for ③RAPD analysis is very small. It is only11000 ~1/200 of RFLP, which is of great benefit to early biological sampling and identification or DNA restriction.
④ Because RAPD does not need cloning when it is used to construct gene map, it can break the restriction of cloning vector and host and expand the application scope.
⑤ Each RAPD marker is equivalent to the target sequence site in genome analysis, which can simplify the information transmission process of cooperative research projects.
⑥RAPD markers can draw genetic linkage maps for those genomic regions that are difficult to distinguish by RFLP. ⑦RAPD analysis can be automated, reducing the tedious procedures like RFLP.
However, RAPD markers also have some shortcomings. For example, RAPD markers are difficult to distinguish heterozygote and homozygote genotypes, and can not effectively identify heterozygote. In addition, RAPD reaction is easily influenced by external factors, and the repeatability is not satisfactory, and so on. Therefore, it is very important to standardize the experimental conditions of RAPD. Black( 1993) summarizes some factors that affect the bibliography, band size and intensity, including PCR buffer. DNTP and Mg2+ concentration, cycle parameters (annealing temperature, temperature change time, PCR instrument), Taq polymerase source (Schierwater and Ender, 1993), DNA condition and content (Black et al., 1993). RAPD technology is a biochemical reaction involving many components, all of which are trace. Although the reaction sensitivity is high, there are many influencing factors, and some problems such as poor repeatability will occur. In order to obtain stable results, various reaction parameters must be optimized in advance, and every step in the operation must be careful to prevent mistakes.
The general reaction conditions of temperature RAPD are: denaturation 92-95℃, commonly used 94℃, 30-60s；; Extension 72℃, 60-120s; Annealing at 35-37 DEG C for 30-60 seconds. Generally, the denaturation and elongation temperature have little change, but the annealing temperature has great influence. When the annealing temperature is low, the binding specificity between primers and templates is poor, and bands may increase. The higher the annealing temperature, the higher the binding specificity of primers and templates. It has been suggested that annealing at 33-34℃ is better in the experiment aimed at finding genetic differences. In the optimization scheme, the annealing temperature can be increased to reduce the background and obtain fewer and clearer bands. The gradient rate of temperature is also very important, especially the gradient between annealing and extension. There are differences in temperature control, heating and cooling performance of various instruments. Some instruments (such as some old models) start timing before reaching a specific temperature, and some PCR instruments show a temperature different from the actual temperature. These need to be considered when designing the program and analyzing the results, so it is necessary to indicate the manufacturer, brand and specification of the instrument used. For the same batch of experiments, it is necessary to control the reaction at the same temperature and the results are comparable.
The effect of PCR amplification of reactants is restricted by many components, and the products only increase exponentially in some cycles (2), and will gradually reach the plateau. Template is a key constraint, and RAPD products depend on it. Very accurate template concentration is a very critical step in the experiment. When the concentration is too low, the probability of molecular collision is low, and the amplified product is unstable. If the concentration is too high, the non-specific amplification products will increase. More importantly, if the number of cycles is not well controlled and the primers are consumed prematurely, the 3 ends of PCR amplification products will anneal with the original template and the amplification products of each cycle in the following cycles, resulting in unequal length extension. Therefore, if the concentration of the original template is too high, the non-specific amplification product is enough to form a background, which is the reason why the high concentration template causes the product to disperse. Relatively speaking, the template purity has little influence, that is, the macromolecular substances such as protein, polysaccharide and RNA in the reaction solution have little influence. The ideal concentration of templates and primers can produce RAPD maps with rich polymorphism and clear strong and weak bands. The 3-terminal of the primer seems to be more important. In addition, the concentration of Mg+2 also affects the parameters of the reaction, including the specificity of the product and the formation of primer dimer. People have come to different conclusions in their own experiments, about1.5-4 mmol/L. In short, the concentrations of various reactants should be screened and optimized in advance.
There should be a blank control in the pollution experiment, because primers, various buffers and double distilled water will all cause pollution. Moreover, Taq enzyme may be contaminated, which brings difficulties to the analysis, so it is necessary to set up quality control to eliminate system errors. And the enzyme is optimized as much as possible.
Repetition of amplified bands In order to overcome the bad repeatability of RAPD, repeated experiments should be carried out. As a marker of DNA polymorphism, bands should be repeatable, and good repeatability is the most important selection index, but the strength of bands should not be used as the selection index. Some scholars believe that the strength of a band is related to its copy number in the genome. However, according to Thormann's analysis of cruciferous plants, the strength of RAPD products is not directly related to the copy number of the products in the genome, but related to the homology of primers and templates. When analyzing a trajectory, we should make a comprehensive analysis. If all the bands of an individual are weak, there may be something wrong with its template (concentration and molecular weight). If most of the bands of an individual are consistent with other individuals, and only one or several bands are weak, there may be a difference in the number of copies. Weak bands with poor repeatability (irregular bands) may be caused by non-specific amplification, annealing between products or other human factors, and cannot be recorded.
When studying flax rust, Ayliffe found that a band appeared in F 1 generation did not appear in parents. Further study found that this band was formed by a heteroduplex composed of two alleles, and the sequences of the two alleles were the same except for the 38bp sequence inserted in the middle. Similar problems have been found in the study of bees, and this band can be eliminated by single-stranded nuclease. It is also possible to increase the electrophoresis temperature above 60℃ and denature the heteroduplex to eliminate its influence.
According to the analysis of non-Mendelian bands, such as Hearn, 92.5% of the non-fuzzy bands showing polymorphism are Mendelian dominant inheritance, and the rest are not Mendelian inheritance, suggesting that they may be composed of different sequences. In general experiments, bands conforming to Mendelian inheritance are mostly used for analysis. In linkage map analysis, genotypic errors can be partially eliminated, that is, bands that do not appear in F 1 generation or 1: 1 mixture should be removed from their parents as much as possible.
Homologous and homologous bands Thormann labeled RAPD products as probes, and the hybridization results showed that bands with the same mobility as probe fragments did not come from different sources, but only occurred between species. The results of RFLP and RAPD markers within and between species were compared. It was found that the results of RFLP and RAPD markers were completely consistent at the intraspecific level, but they were far from each other at the above-specific level, indicating that there might be several bands with different sequences but the same molecular weight in the same electrophoresis result, which could not be detected by RAPD. This possibility is rare within species, but it is more likely between species. Castagne reached the same conclusion by comparing RFLP and RAPD.
Due to the problem of electrophoresis resolution, it is possible that amplification products in different positions of the genome will move together, that is, amplification products with different molecular weights may not separate to form bands during electrophoresis, and the genetic relationship between gel separation system and genome may increase this probability. Generally speaking, the resolution effect of polyacrylamide and silver staining is higher than that of agarose and ethyl ammonium bromide, and the use of longer agarose gel (up to 20cm) or higher concentration (2%) is helpful to improve the resolution. Of course, with the improvement of resolution and sensitivity, experimental errors are more likely to occur. Therefore, some people commented that the safest and simplest method is agarose pulse separation, focusing only on those bands with high repeatability. The polymorphism revealed by polyacrylamide and silver staining can undoubtedly accelerate the identification process of molecular markers.
The dominant marker problem RAPD marker comes from the replication of template DNA. After 30-40 cycles, the number of amplified bands can theoretically reach 2. It is impossible to judge whether the amplification sites in the original template are homozygous or heterozygous, because the amplification bands of homozygous sites are only twice as large as those of heterozygous sites, and no difference can be detected by electrophoresis. In hybridization. If one of the parents is homozygous, F 1 generation can show this band; If it is heterozygote, the band should be l: l segregation in F 1, that is, half of the offspring have the band and the other half do not. Of course, stripe analysis from multiple copy areas is more complicated.