SSR Molecular Marker Technology and Its Application in Construction of Maize DNA Fingerprint Library
Kang Lili Zhou Hongfei (College of Agriculture, Shenyang Agricultural University)
Corn is an important feed, grain, and industrial processing crop that occupies an important position in the national economy. The improvement of maize breeding methods is of great significance to agricultural development. For a long time, most breeders have used genetic markers such as easily identifiable morphological and isozymes to assist in breeding, and have achieved great success. However, the biggest deficiency of this type of marker is its limited number. It is often difficult to find markers that are closely related to the target trait, and it is difficult to apply it to breeding practice. Therefore, breeders have been hoping to find a large number of markers with high selection effects in order to increase the efficiency of breeding and the predictability of the breeding process. With the development of molecular biology, molecular markers based on DNA mutations have been developed. The most widely used are mainly RFLP, RAPD, SSR, AFLP, etc. Among them, SSR marker is a kind of co-dominant marker, which has good repeatability, high reliability, simple analysis, easy automation, high sensitivity, and ability to detect genetic variation. Strong. In recent years, SSR markers have been widely used in corn breeding.
1 SSR mark
1.1 Principle of SSR Marking
Simple sequence repeat (SSR), also known as Microsatellite DNA. Microsatellites usually refer to DNA sequences that are repeated in multiple tandems in units of 2-4 nucleotides, and a few are tandem repeats of l-6 nucleotides. The distribution of microsatellites in the genome is random, and it can exist in introns, exons, and any other region of the chromosome. In different plants, the base composition and copy number of microsatellite repeat units vary with the species, but it has been found that the sequences at both ends of the microsatellite are conserved. Therefore, primers complementary to conserved sequences at both ends of the microsatellite can be designed to amplify the repeats themselves by PCR and detect their polymorphisms by gel electrophoresis. The occurrence of SSR sequence polymorphisms is due to the different number of oligonucleotides of different genotypes in the same species and the repetitive sequence slips or unequal exchange of chromatids during chromosome replication. Therefore, microsatellites can be used as molecular markers. Due to the presence of transposons in maize, the polymorphism of microsatellites is extremely rich. Therefore, PCR amplification reactions are performed by specific primers, agarose gel electrophoresis (or polyacrylamide gel electrophoresis), and autoradiography ( (or silver staining) and GeneScan technology, etc., can detect polymorphisms in DNA regions that differ in the number of repeat units in a simple repeat sequence. This is also the case with SSR technology. One of the reasons why molecular marker technology is more suitable for corn research.
1.2 Types of SSR Markers
Microsatellites can be divided into 1 to 6 nucleotide repeat types according to the number of bases in the repeat unit, where there are 4 types of dinucleotide repeats: (AT)n/(TA)n, (AG)n/(TC) )n, (AC)n/(Gt)n, (GC)n/(CG)n; There are 10 types of trinucleotide repeats: (AAT)n)/(TAT)n, (AAG)n/( TCT)n, (AAC)n/(TGT)n, (ATG)n/(TCA)n, (AGT)n/(TAC)n, (AGG)n/(TCC)n, (AGC)n/( TGC)n, (ACG)n/(TCG)n, (ACC)n/(TGG)n, (GGC)n/(CGC)n; there are 32 tetranucleotide repeat types (Jurke, 1995). Microsatellites with single nucleotide repeats and dinucleotide repeats in all types of microsatellites occur most frequently in the genome. Different base repeat polymorphisms in the same repeat type are also inconsistent.
In addition, Weber (1990) classified the microsatellite markers into three types according to the structure of the microsatellite core sequence: (1) Perfect, referring to microsatellites in which the core sequences are constructed in an uninterrupted manner; (2) Imperfect means that there are several non-repetitive bases between the core sequences of microsatellites, but the number of repeats of consecutive core sequences at both ends is greater than 3; (3) Compound, refers to two types Or more than two tandem core sequences are separated by several consecutive non-repetitive bases, but the number of consecutive core sequences is not less than five.
1.3 Functions of SSR
The function of SSR in eukaryotic genomes has been controversial. In the plant genome, most of the tandem repeats are non-encoded, so for a long time it has been considered that SSR is a type of “junk†DNA that is dubbed as a dumb cell and has no definite physiological function. With the deepening of research, the data on SSR functions are continuously accumulating. In general, the functions of SSR mainly include the following aspects: Some SSRs (usually 4 to 7 base repeats) exist in the transcribed region of structural genes and encode amino acids; SSRs at the ends of chromosomes protect DNA integrity , to avoid the function of degradation, fusion and loss; SSR located in the DNA regulatory region has the effect of increasing or decreasing the transcription rate of nearby genes; SSR region may be the hot spot of gene recombination, and is the source of gene mutation; part of SSR has the transcription initiation complex Or activate the function of a chromosome. Therefore, it is believed that although most SSRs do not have a clear physiological function and only take some strategy to ensure their survival, some SSR sequences are indeed part of the functional genes.
At present, at least 10 more human genetic diseases are known to be associated with mutations in microsatellite sequences. The trinucleotide microsatellite sequences are usually contained in the vicinity of the genes involved in these diseases, introns, or exons. These microsatellite sequences carried in patients are often unstable and often have a longer length than normal people. long. Some traits have also been found in plants and are closely related to changes in the length of microsatellite sequences. For example, the Wx gene on rice chromosome 6 encodes a starch synthase that is involved in the synthesis of amylose in rice endosperm. There is a microsatellite sequence (CT)n in the 5′ non-transcribed region of Wx gene. The length of the sequence has significant correlation with the amylose content of different varieties. It is generally believed that microsatellite sequences may have important implications in the regulation of gene expression, chromosome structure, chromosome reorganization, origin and evolution of new genes, and so on.
1.4 SSR Marker Primer Source
There are two key factors in the success of microsatellite DNA analysis. One is the effect of PCR amplification and the other is the source of the flanking sequence primers. In general, there are four sources of primers for microsatellite amplification: one is the direct use of published microsatellite DNA primers; the second is the use of primers from closely related species; and the third is the screening of DNA sequence databases or the screening of clone libraries. Repeated DNA sequences were designed using computer software for primer design. Fourth, the best and most fundamental method was to construct a genomic library and then design primers based on the sequences of the two wings of the microsatellite locus.