Cotton is the seed fiber of the Gossypium plant of the Malvaceae family. Breeding salt-tolerant cotton varieties will be a way to effectively develop and utilize saline-alkali land and promote sustainable agricultural development. The identification of cotton's salt tolerance plays a vital role in the selection of salt-tolerant varieties. Currently, the morphological salt tolerance identification methods commonly used in production are time-consuming and labor-intensive, and are easily affected by changes in the external environment and seasonal restrictions. The SSR identification method of cotton salt tolerance lays a foundation for the molecular identification of salt tolerance of cotton seeds and provides a theoretical basis for cotton salt tolerance breeding.
It is generally believed that simple sequence repeat (SSR) or microsatellite sequences are a type of DNA sequence consisting of tandem repeats of a motif consisting of several bases (mostly 1 to 6). Its length is generally short, within 100 bp. SSR occurs as a result of DNA slippage and mismatching during DNA replication or repair or unequal exchange of sister chromatids during mitosis and meiosis. Microsatellites mainly have two nucleotides as repeating units, some microsatellites have 3 nucleotides as repeating units, and a few have 4 nucleotides or more. The most common motifs of SSR are (CA)n and (TG)n. (AT)n is the most abundant in plant genomes, and prokaryotic genomes also contain a small amount of microsatellite sequences. The variability in the number of repeats in different genetic materials leads to a high degree of variability in SSR length, which is the basis for the generation of SSR markers. The mutation rate of microsatellites is very high, resulting in many alleles and high polymorphism of microsatellites. It is generally believed that the abundant polymorphism of microsatellites is a manifestation of microsatellite instability. Microsatellite mutation rates vary widely between species, at different loci within the same species, and between different alleles at the same locus. Although microsatellite DNA is distributed at different locations throughout the genome, the sequences at both ends are mostly conserved single-copy sequences. Therefore, a pair of specific primers can be designed based on the sequences at both ends, and the core microsatellite DNA sequence between them can be amplified through PCR technology, and its length polymorphism, that is, SSR marker, can be obtained using electrophoresis analysis technology. The high degree of polymorphism of SSR markers mainly comes from the difference in the number of concatenations. Genotypes were determined based on the size of the isolated fragments, and allele frequencies were calculated.
For DNA extraction and purification methods, please refer to extraction and purification of rice genomic DNA.
A. Primer design
The premise of using SSR markers is to know the DNA sequences on both sides of the repeat sequence and design primers. Therefore, the primers used in SSR marker technology are crucial to the application of SSR marker technology. The primers of SSR marker technology have the following four sources.
a. Related literature.
b. Primers of closely related species.
c. Primers designed based on a set of sequences on both sides of the SSR locus screened from the genome library of the research group. The main methods for developing SSR primers include classical methods and microsatellite enrichment methods.
d. Database search method: Use the special SSR analysis software Sputnik and the FindPatterns program in the WisconsinGCG program package to search for DNA sequences and EST sequences in public databases such as GenBank, EMBL and DDBJ to obtain SSR sequences.
B. PCR reaction
The final volume of PCR reaction is 15 µL. Each reaction contains the following components: 45 ng template DNA, 2.25 µmol/L primers, 11.5 mmol/L MgCl, 625 µmol/L of each of the four dNTPs, 10× PCR buffer, 1.5 U TaqDNA polymerase.
Reaction parameters: first step, 94°C, 3 min; second step, 94°C, 25 s, 50-60°C, 25 s, 72°C, 45 s; third step, 72°C, 10 min.
The reactants were used for electrophoresis or stored at 4°C for later use.
After SSR-PCR amplification, the amplification products should be detected. Generally, polyacrylamide gel electrophoresis or special agarose gel is used to detect the amplification products
When using polyacrylamide gel electrophoresis to detect the amplification products, the separation effect of PCR products on denaturing polyacrylamide sequence gel is usually better than that on non-denaturing gel. Because heterozygous individuals will produce heteroduplex molecules in the later cycles of PCR, resulting in 3 or even 4 bands in the gel in the case of heterozygosity, instead of the normal two bands. The occurrence of this situation will interfere with the statistics of alleles.
After the SSR-PCR amplification products are separated on 5%-8% denaturing polyacrylamide sequence gel, the changes of 1-2 nucleotides in microsatellite DNA can be distinguished. Each PCR reaction product occupies an electrophoresis lane, and there are 25-50 sample wells when performing sequence gel electrophoresis. Therefore, each plate of electrophoresis can simultaneously analyze the precise changes of alleles in 25-50 individuals. This method is suitable for population genetic research.
After the PCR products have been separated on a gel by electrophoresis, the bands must be read and the data obtained statistically analyzed. Within a range of appropriate sizes, co-dominant inheritance of microsatellite loci should produce one (homozygous) or two (heterozygous) bands, sometimes with a few weak bands, which are false bands amplified by PCR. Longer repeats, such as three or four nucleotide repeats, produce fewer of these false bands, but longer repeats are rare and are more difficult to isolate from libraries than two nucleotide repeats. Any type of microsatellite analysis produces data on allele and genotype frequencies and can therefore be analyzed using standard population-based models of inheritance.
In SSR data processing, the information content is usually expressed as the ratio of polymorphism or polymorphism information content (PIC). As a molecular marker, the information content of SSR loci is directly proportional to the number and frequency of existing alleles in a population and directly related to the number of tandem repeat units. The range of PIC is from close to 0 (n=10) to 0.8 (n=24). The PIC of incomplete repeats is lower than expected, and the highest expected value is for the longest repeat sequences without substitutions in the middle.
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