Microsatellite DNA molecular marker and its application

Microsatellite (MS), also known as Short Tandem Repeats (STR) or Simple Sequence Repeat (SSR), refers to a few nucleotides in the genome (mostly 2-4) A sequence of up to several tens of nucleotides is repeated in series for a plurality of units. The most common ones are dinucleotide repeats, such as (AC)n, (TG)n, etc. Microsatellite DNA is widely distributed in the genome of eukaryotes, and a microsatellite exists approximately every 10-550 kb. Microsatellite DNA has been widely used due to its specific PCR amplification, high polymorphic information capacity (PIC), good primer versatility, high mutation rate, and co-dominance.
1. Characteristics and classification of microsatellite DNA
Microsatellite DNA is rich in polymorphisms, mainly in the polymorphism of the number of nucleotide repeats and the nucleotide substitution polymorphism in the repeats. It is generally believed that the higher the number of repeats of a microsatellite DNA core sequence, the more the number of alleles, and the more abundant the polymorphism. Microsatellite DNA follows the Mendelian inheritance law and can be stably transmitted from the previous generation to the next generation, and the alleles exhibit codominant inheritance. In addition, microsatellite markers have the characteristics of low DNA usage, fast reaction speed, easy operation, and good repeatability.
Microsatellite DNA can be divided into three categories according to the repeating structure: perfect, single sequence unit without interruption or non-inversion; imperfect, single sequence unit interrupted or inverted; mixed type (compound), a mixture of units in a plurality of sequences with or without interruption and with or without inversion. In general, microsatellite DNA has sequence-specific conserved sequences on both sides of the repeat sequence. Therefore, PCR amplification, agarose gel electrophoresis (or polyacrylamide gel electrophoresis) and irradiation of genomic DNA are designed by designing primers. Self-developing (or silver staining) can detect polymorphisms in DNA regions with different repeating sequence numbers in simple repeat sequences, which is the microsatellite DNA molecular marker.
2. Advantages of microsatellite molecular marker methods
Microsatellites are evenly distributed throughout the genome. Studies by Winter et al. showed that microsatellite loci were widely distributed in other regions of the chromosome except for the centromere and telomere regions. Litt and Luty and Weber and May each demonstrated the use of a thermostable Tag enzyme PCR method to demonstrate that microsatellites are rich in polymorphism. The use of microsatellites as genetic markers has the following advantages over other DNA molecular markers, including RFLP, RAPD, and small satellite DNA.
2.1 Microsatellite markers are highly heterozygous
Since the number of alleles at the microsatellite locus is quite large, the degree of heterozygosity is high, and the polymorphic information content (PIC) is large, and the efficiency is higher than that of PFLP when distinguishing individuals (groups) with close kinship.
2.2 Microsatellite loci can be amplified by PCR
Since the alleles of small satellites are generally large, there are certain limitations in PCR amplification. PCR amplification using microsatellites uses a small number of samples. In addition, because the microsatellite sequence is short, even the degraded DNA may contain enough microsatellite sites for amplification, which makes the poorly preserved samples may also be valuable research materials.
2.3 Universality and conservation
The region in which the microsatellite DNA is located is relatively conserved in the genome of the organism, and the microsatellite primers of a species can be used in closely related species, which makes it possible to reduce the workload of acquiring microsatellites and speed up the work of comparative genome mapping. may.
2.4 codominant inheritance
The microsatellite DNA is a Mendelian codominant inheritance model that can distinguish between homozygous dominant individuals and heterozygous dominant individuals, which provides more information for genetic research.
2.5 Microsatellite polymorphism
Most SSRs have no function, increase or decrease the frequency of several repeats, and thus have extensive site variation among varieties, and are more polymorphic than RFLP and RAPD molecular markers.
2.5.1 Microsatellite mutation
The microsatellite mutation rate is very high, resulting in many alleles, which leads to the high polymorphism of microsatellites. It is generally believed that the microsatellite-rich polymorphism is microsatellite instability (MI). Performance. The mutation rate of microsatellites varies greatly between different species and different sites of the same species and even between different alleles at the same site. In mammals, the majority of microsatellite mutation rate is estimated at an average rate of mutation of human microsatellite every generation pedigree of 10-6-10-2 10-4; melanogaster controlled mutation rate in female line Each site is around 10 -6 . When the microsatellite is expressed in a host lacking an effective mismatch repair system, its instability is higher (5-10) × 10 3 than normal.
2.5.2 Mechanism of microsatellite mutation generation
The genetic mechanism of microsatellite mutations is still unclear. At present, most of the microsatellite instability is related to the unequal exchange in DNA recombination process or to the "sliding chain mismatch" in DNA replication.
Johnson and Pupko believe that the unequal exchange and gene conversion during DNA recombination between two chromosomes may be the main cause of microsatellite polymorphism. Levinson et al. believe that during the process of DNA replication and synthesis, local melting occurs, and the nascent chain and the template strand of the microsatellite are relatively slipped, resulting in a mismatch, which makes one or several repeating units form a ring and fails to participate in the pairing. , which led to the generation of microsatellite polymorphism.
3. Application of microsatellite molecular marker technology
Microsatellite DNA has great advantages as a genetic marker. In recent years, with the deepening of research, the research on microsatellite markers not only has important theoretical significance, but also has a good application prospect.
3.1 Microsatellite polymorphism analysis
In nature, the various genetic variations exhibited by biological individuals are essentially DNA differences, so it is more straightforward to analyze the genetic structure and genetic diversity of the population by studying DNA variation. The number of repeating units of microsatellites may not be exactly the same, thus forming a polymorphism, ie, SSR molecular markers, which may be due to the "strand slippage" effect of unequal exchange or replication between homologous microsatellites during mitosis. Caused. Microsatellite polymorphism reflects the evolutionary history of a species, and the shared allele is the oldest and most conserved in the genome of the species. Compared with protein labeling technology, population genetic heterozygosity and genetic distance estimated by microsatellite markers are significantly better than protein polymorphism markers, and microsatellite markers have more specificity than protein markers when analyzing populations and varieties with close genetic relationships. For accurate values.
3.2 Population genetic diversity
Powell et al. pointed out that microsatellites can reveal genetic diversity more than other molecular markers such as RFLP, RAPD, and AFLP. Scott et al. analyzed seven grape resources based on 16 pairs of SSR primers designed from 124 microsatellites isolated from 5,000 grape expression sequence tags (ESTS). The results show that the microsatellites isolated in the 3' untranslated region (3'U TR) have high polymorphism among varieties; the microsatellites in the 5' untranslated region (5'U TR) are species and varieties. There is a high polymorphism between the two; while the microsatellites isolated in the coding region have higher polymorphism between species and genera. Experiments by Fahima et al. confirmed that monocotyledonous and dicotyledonous chickpeas also have a tandem arrangement (GTAT) n and a simple repeat sequence (GACA) n and show a high degree of polymorphism. Plaschke et al. detected 40 European wheat cultivars with 23 SSR markers, and found 142 SSR polymorphic loci. On average, 6.2 polymorphic loci could be detected per SSR marker. Guo Xiaoping used 137 pairs of primers with amplification products to analyze two maize inbred lines B14 and B96, and found that 67 pairs of primers showed polymorphism, reflecting the difference in genetic material between the two inbred lines. Yang Guanpin used a multi-copy microsatellite DNA marker to analyze the genetic diversity of 238 cultivated rice and the dynamic changes of genetic diversity from farmer to current cultivar. A total of 16 length variation types and 32 phenotypes were detected. A higher polymorphism. Turuspekov Y et al. used microsatellite primers to analyze the genetic diversity of 18 barley varieties representing the main cultivated areas in Japan. Shannon had the highest information content in the Kanto region, which was 0.524, while the lowest content in the Tohoku region was 0.264. The genetic distance ranges from 0.105 in the Tohoku and Tozan areas to 0.88 in the Shikoku and Kyushu areas. Cluster analysis showed that most of the varieties cultivated in the same area belonged to one category, which reflected that barley cultivation in Japan was not only affected by historical kinship, but also influenced by geographical and environmental factors. Tanya P et al. used SSR to analyze the genetic diversity of 5 Korean soybean varieties, 8 Thai soybean varieties and 3 wild soybeans.
3.3 Gene Mapping and Construction of Genetic Linkage Map
Due to the large number of microsatellite repeat units, the repeat unit fragments are short, and the degree of repetition is different. The same type of microsatellites can be distributed in different positions of the whole genome, and the random PCR markers can be anchored along the chromosomes at known positions, thus Used as a functional gene mapping. Since the microsatellite marker is derived from the sequence of the gene itself, the location of the microsatellite marker is mapped accordingly. According to Beckmman and Soller, the polymorphic Sequence-tagged Microsatellite site (STMS) provides an effective marker for gene mapping.
The genetic linkage map is a linkage analysis using genetic markers to reflect the relative relationship between genetic markers. The most widely used is the microsatellite marker. The basic principle is: based on the microsatellite locus, find a polymorphic microsatellite marker at a certain distance in the genome, when these markers reach sufficient saturation (about every 10-20cm, and cover 90 After % of the genome, any functional genes and QTLs in the genome can be found by microsatellite markers and linked to determine the location of the QTL at the map, the genetic distance between the markers, and the phenotypic effects of the QTL. Akkaga et al. integrated 121 microsatellite markers into the RFLP framework of four rice populations, with an average of one microsatellite marker every 16-20 cm. Arabidopsis, rice, tomato, potato, and corn have a genetic map of 150 kb, 300 kb, 500 kb, 1000 kb, and 1500 kb, respectively, on a genetic map. It is believed that with the further improvement of molecular genetic marker technology, especially microsatellite technology, it will further promote the construction of gene libraries.
3.4 Building a fingerprint
The microsatellite loci in the genome are similar in base composition and structure except for the number of repeats. Therefore, the core sequences of microsatellites such as (AC)n, (TG)n, etc. can be used as multi-site probes. Multiple sites are detected simultaneously in the genome. Since different individuals, varieties (lines) or groups have certain differences in the detected sites, these differences will be manifested by the presence or absence of hybridization bands by electrophoresis and hybridization, that is, microsatellite DNA fingerprints are generated. B. Beyerman et al. confirmed the significant differences in the fingerprint bands of barley and sugar beet DNA by DNA fingerprinting using simple repeats (GATA) 1 and (GTG) 5 as probes.
3.5 For germplasm identification and variety classification
Due to the re-isotopic nature of the microsatellite locus, it is easy to identify different genotypes of the same species. Guilford et al. used (GA) 15 (GT) 15 as a probe to screen apple genomic libraries, demonstrating the polymorphism of these repeats, and using three microsatellite markers to distinguish 21 apple varieties. Akagi et al. successfully distinguished 59 indica rice varieties with close kinship using 17 highly microsatellite markers containing (AT)n. Aranzana MJ used SSR to analyze 100 peach varieties, and used 32 polymorphic microsatellite loci to obtain 32 alleles, which can distinguish 78 different genotypes.
3.6 molecular marker assisted selection
The application of microsatellite markers to marker-assisted selection has great potential, and it changes the selection process for inferring genotype values ​​from phenotypic values. Compared with traditional phenotypic selection, molecular marker-assisted selection can achieve greater genetic progress, especially for low heritability traits, restrictive traits and late expression traits, which can increase selection intensity, shorten generation interval, and improve selection. accuracy. Zhang et al. used microsatellite markers to predict yield and estimate heterosis.
4. Outlook
The research on microsatellite DNA has made significant progress in recent years, but there are still many problems in microsatellite DNA markers. For example, the mutation mechanism of microsatellite DNA molecules is not fully understood, PCR primers are poorly conserved among different species, and different species are required. Specific primer design, etc., but microsatellite DNA is still the highest resolution and most revealing nuclear DNA marker in the current genetic variation. It is believed that with the deepening of microsatellite DNA research, microsatellites will have wider application in various fields.

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