Principle of Simple Collection Repeats (SSR) and Their Application in Flower Breeding
Since life commenced on the planet about 3 billion years back, the biodiversity of the plant life on our world have been through numerous natural selection and evolutionary changes, producing a abundant and diverse ecosystem helping life in a variety of geographical locations and climates. Furthermore, when people have arisen as a superior species, various vegetable species were domesticated into crops to create various valuable resources. For example, whole wheat and pea had been domesticated as soon as the Neolithic Age groups, forming the foundation of agriculture in the centre East, North Africa, India, Persia and European countries. Since vegetation like rice, whole wheat, corn, pea are important staple food, while cotton gives textile and clothing, these plants are very important for food security and economy via trading. Hence various efforts to really improve these crops have been done since the ancient days and nights via crossbreeding and selective breeding, yielding better types of the vegetation we realize of today.
However, it was not until the 17th century that the word taxonomy has been presented by Carl Linnaeus, which systematically categorizes and file living organisms scientifically. Hence, the ancestry and root base of the vegetation we could planting today are not clearly characterized and remains shrouded in mystery. Still, classical flower breeding procedures still progressed, that involves collection of desired traits predicated on the observable phenotypical characteristics, or morphological markers. This method of selective breeding, however, has some flaw and weaknesses. It is because molecular markers are often masked by environmental factors, or are provided only during late stages of expansion, and are occasionally interpreted differently by different individuals. Today, breeding of plants are facilitated by more organized marker helped selection methods such as RAPD, RFLP, AFLP, SNP, SSR and ISSR. These methods exploit inherent molecular markers within the genome of plant life to give equivalent, regular, DNA-based polymorphic patterns unaffected by environmental or other external factors.
Simple Collection Repeats (SSR), also called short tandem repeats (STR), or microsatellites, is one of the molecular markers popular today. As a kind of variable quantity tandem repeat, its rule revolves around repeats of models of dinucleotide, trinucleotide or sometimes tetranucleotide repeats. The polymorphism itself comes up due to different number of these repeating nucleotide models, ranging from 3 to 100 times depending on the species. In vegetation for illustration, AT dinucleotide repeats are very common. The AT dinucleotide will occur in alleles, and the amount of AT microsatellite repeats can differ between your alleles. These short repeating locations typically take place in the non-coding parts of the genome known as the introns. Hence, they often do not cause mutations or diseases. However, there are trinucleotide SSRs that are positioned in the coding exons, they can can be found because they do not alter the reading frame of the sequence. In fact, according to Marcotte (1998), 20% to 40% of the proteins in mammals contain duplicating sequences of amino acids brought on by SSRs. In rare cases, SSRs in the coding region likewise have been recognized to cause diseases. For example, Huntington's disease occurs when CAG trinucleotide repeats course beyond normal duplicate number. SSRs can even be classified into 3 types besed on the way in which they are organized, namely simple, ingredient, and interrupted SSRs. As the name suggests, simple SSRs are simply just an uninterrupted portion comprising one kind of repeating motif, whereas chemical substance SSRs entail 2 or even more different kinds of repeating motifs. Finally, interrupted SSRs have arbitrary nucleotides sandwiched between your repeating devices, interrupting the SSRs.
Figure 1. An illustration of microsatellites of AT dinucleotides. The polymorphism occurs when the top series has 7 repeats of the dinucleotide while the lower strand has 9 repeats.
Figure 2. An illustration talking about how simple, mixture, and interrupted SSRs are arranged.
The breakthrough of SSRs actually dates back to the 1960s. It came about when simple repeats spread in eukaryotic genomes were discovered through density gradient centrifugations of arbitrarily sheared genomic DNA. Satellite peaks were within these sheared DNA, and sequencing revealed duplicating motifs of varying length in these sequences, ranging from a single bottom part to several thousand bases. Later, satellites with 10 to 30 repeating motifs were isolated, and these were called minisatellites. It was until 1982, when Hamada and his colleagues demonstrated the lifetime of dinucleotide poly-CA repeats in eukaryotic genomes, only then the term microsatellites was coined. Since the repeating motifs are usually just one to four bases long, they can be then simply called simple series repeats, SSR. Eventually, it was exhibited that SSR polymorphisms could be easily found by using PCR with 2 primers flanking the SSR region. This discovery in turn resulted in the development of several SSRs in a variety of species and their relationship to web page link various loci in chromosomes. The initial evidence of the incident of SSRs in crops was shown when phage libraries of tropical tree genomes was screened via hybridization with poly G-T and poly A-G oligonucleotide probes. This acquired shown that SSR is also significantly abundant within a wide array of planr genomes. Since then, SSRs have been implemented as a favorite molecular marker choice for flower genetic evaluation and inhabitants studies.
It is still not certain how SSRs came about. However, there are several early theories that try to make clear the polymorphism. It was in the beginning thought that the difference in length of SSRs was triggered by unequal crossing over between the repeating devices during meiosis. The variation of the SSR bases on the other palm, is hypothesized to be anticipated to an activity called DNA replication slippage. Both these mechanisms have the ability to either add or take away the number of repeating products in DNA sequences, leading to varied quantity of repeating units ranging from 2 to 40. Generally, SSRs that have longer repeats could be more polymorphic that others that are shorter in length. It has also been reported that different kinds will usually preferentially harbor different SSR motifs. The reason for this however is still a mystery.
Figure 3. Diagram of the mechanism of Replication Slippage during DNA replication.
Figure 4. Diagram demonstrating unequal crossing over during meiosis.
The examination of SSRs entails amplifications using polymerase string reactions (PCR). A pair of primers flanking the microsatellite region will be used for the PCR. The primers will anneal to each aspect of the SSR region, and can eventually produce high level of PCR product comprising of the repeated SSR sequence. Recall that the number of repeating units will change between different species, hence the space of the PCR products may also be different. A couple of 2 commonly used methods to assess the PCR products. Among that involves gel electrophoresis using agarose or polyacrylamide gels. Because the PCR products are of differing length, they have different molecular weight, and can migrate at different rates across the gel during electrophoresis. Hence, they'll eventually divide on the gel, longer sequences will migrate slower and will remain at higher parts of the gel whereas sequence with shorter repeats will migrate faster towards the low molecular weight parts of the gel. This therefore yields a noticeable banding pattern, allowing the experimenter to tell apart the samples predicated on the bands they deliver. For gel electrophoresis, using polyacrylamide gels rather than agarose gels will provide better separation, allowing visualization of bands differing less than a single bottom pair. This is particularly useful in resolving SSR polymorphisms with very tiny difference in base pairs. The drawback however is, polyacrylamide is dangerous and very expensive. Thus, an alternative solution is to use agarose gels of higher focus. Another method that can be used is capillary electrophoresis. The mechanism of separation is relatively similar in comparison to that of gel electrophoresis, except that it is done in a thin capillary tube. The PCR product migrating through the capillaries will be excited via exposure to UV light, and a detector will find the signals produced by the DNA. The alerts will be viewed as a graph, with peaks indicating the detection of DNA. Enough time whereby each top is discovered will correlate to the size of the fragment. Small the fragment, the faster it migrates through the capillary, and the earlier it will be detected.
Figure 5. Gel photo depicts an example of SSR-PCR results of 20 different wheat cultivars.
Figure 6. An example of signals detected on the gene locus in Arabian Oryx using capillary electrophoresis.
Besides of the 2 2 PCR centered SSR method discussed earlier, addititionally there is another PCR amplification founded analytical method that also includes simple sequence repeats. It is called inter-simple series repeats (ISSR). ISSR evaluation is slightly different from SSR PCR, the primary difference requires the primers themselves. ISSR primers are short oligonucleotide sequences that are complementary to the duplicating SSR sequences. The binding of the primer at these regions will subsequently amplify the sequence sandwiched between your SSR repeats. The PCR products can be then segregated just as, via gel electrophoresis for visualization. The advantage of ISSR analysis is the fact no prior sequence knowledge is needed, only oligonucleotides of different repeating motifs are needed. However, the drawback is the fact molecular aspect of the polymorphism remains undiscovered unless the fragments segregated in the gel is extracted and sequenced.
We have now talked about how SSRs can be analyzed via PCR amplification using flanking primers. Just how were these primers designed? Among the approaches involves looking genome databases that exist using various bioinformatic tools. There are many fully characterized genes, completely sequenced cDNAs on online databases such as PubMed, GenBank and EMBL. A search for SSR sequences can be performed on this wealth of information, and then flanking sequences of the SSR areas can then be applied to design the required primers. Moreover, nowadays there are even custom-made online SSR search softwares made for this purpose only, such as MISA (MIcroSatellite), CUGssr, SSRSEARCH, and Sputnik. However, this computational search procedure has its limitations. When we need to do SSR analysis in a few small or less known
