Insect Microsatellite Database

Microsatellites are simple sequence repeats that exhibit complex patterns in their frequency of occurrence, genomic distribution, mutability, function and evolution. Apart from being the source of popular genetic markers, microsatellites per se have attracted a lot of attention with respect to their origin, distribution, expansion, mutation, and disintegration. Questions are also asked about the functional role of microsatellites in particular and biological significance of the microsatellites in general.

Genetic studies and whole genome sequence analysis have established non-random distribution, variability and high mutability as characteristics of microsatellites as listed below. Evidences are accruing, which support the role of microsatellites in gene regulation, transcription and protein function. Existence of qualitative and quantitative differences between microsatellites of different genomes and their role in adaptive evolution have also been theorized. However, such studies require information on type (mono to hexa), motif (GC%), abundance (motif preferences), frequency, distribution (linkage group-wise and chromosomal position), location (exon, intron, regulatory element, transposon), nature (perfect, imperfect and compound), and copy number (existence of paralogs) etc. of microsatellites not only on a whole genome basis but also as a comparative analysis of multiple genomes defined by phylogeny.

Insects have long exhibited the greatest genetic diversity on earth that has puzzled mankind. Biologists have relied on insects to unravel many fundamental tenets of biology. Whole sequence genomes of insects have lived up to the reputation and have thrown immense variability in size and genome organization. Among others, we have five fully sequenced genomes of Drosophila melanogaster (as a model organism it provides maximum annotated data), Anopheles gambiae (another Dipteran but economically highly important as a vector), Tribolium castaneum (relatively older insect order of Coleoptera), Apis mellifera (relatively a recent insect order, Hymenoptera) and Bombyx mori (a Lepidopteran, members of which are crop pests). Using five fully sequenced insect genomes; following questions may be addressed:

InSatDb, with an interactive interface, allows users to obtain genome level information on frequency and distribution of microsatellites motif-wise or across-the-board in a single genome or for comparative genomic analysis. One can access microsatellite cluster (compound repeats) information, and particulars of the microsatellites with common flanking sequences (microsatellite family). Following section gives a flavour of the types of analysis that can be carried out using the data obtained from InSatDb.

CHARACTERISTICS OF INSECT GENOMES

FREQUENCY OF MICROSATELLITE OCURRENCE

Distribution of lengths of microsatellites in relation to their GC content is uniform across five insect genomes
Average length of the microsatellite across GC content is 37±9 bp
Between 0-5% GC content, microsatellites tend to be longer than 60 bp
In Anopheles, very long microsatellites (104, 167 and 457 bp) were found to be either AT rich content (1 and 2 GC %) or GC rich (97%) respectively
In Bombyx, between 48% to 89% GC, average size of microsatellites is significantly large (48 bp) due to longer tetra and hexa repeat microsatellites

Does GC content influence occurrence of imperfections in a microsatellite?

The relation between GC content of the microsatellites and imperfections reflected the number of microsatellites with the said GC content
40-60% GC range has as many as 53% Anopheles microsatellites, which are mainly dinucleotide repeats
40-70% GC range has as many as 45% Drosophila, microsatellites, which are mainly dinucleotide repeats
1-12% GC range has as many as 43% Apis microsatellites, which are mainly mono and dinucleotide repeats

Compound microsatellite analysis (length)

Red dotted lines indicate genome GC%
GC values are of microsatellite (m) and intervening (i) sequences

Combination of compound repeats

Top five compound repeats (in number)

Bombyx mori		Apis mellifera		Anopheles gambiae		Drosophila melanogaster		Tribolium castaneum
254	tri-di	1499	di-di	1431	di -di	522	tri-tri	122	tri-tri
240	mono-tri	1112	di-tri	1256	tri-tri	362	di-di	51	tetra-tri
226	tri-tri	1107	di-mono	815	di-tetra	222	tri-di	43	tri-tetra
185	mono-mono	1055	mono-mono	791	di-tri	209	di-tri	37	mono-tri
158	tri-tetra	1051	tri-di	277	di-tetra	116	mono-di	36	mono-mono

Microsatellites occurring contiguously account for nearly 3.2% in the insect genomes analysed
On account of high density of microsatellites, Apis has higher number of compound loci (6.12% of the total microsatellites)
Length of intervening sequences is greater than that of microsatellites

Do longer microsatellites tend to have greater decomposed repeat sequences?

Shorter microsatellites not only predominate microsatellite population in the 5 insect genomes, they also seem to possess higher number of imperfect bases
Nearly 90% of the microsatellites fall within 50bp size, which constitute the bulk of imperfect microsatellites
Majority of imperfect microsatellites in this range are dinucleotide repeats in Drosophila, Apis and Anopheles; tri- and tetranucleotide repeats in Bombyx and Tribolium
Higher frequency of imperfect microsatellites are present in Drosophila and the least are found in Anopheles
Beyond 100bp, microsatellites remained more of perfect types

Microsatellite Families

100bp of 5' and 3' flanking sequences of each microsatellite were extracted. The sequence match was done by performing all versus all blast with parameters e=1, match=95% and alignment length= 85%. Grouping of microsatellites based on sequence match (both +/+ and +/- alignments) was done using a perl script.

Examples of microsatellite families of different species.

Bombyx mori (click on the image to enlarge)

Drosophila melanogaster (click on the image to enlarge)

Anopheles gambiae (click on the image to enlarge)

Apis mellifera (click on the image to enlarge)

Tribolium castaneum (click on the image to enlarge)

ORIGIN OF MICROSATELLITES

Neutrality of microsatellite origin

Microsatellites in insects are AT rich (on an average 23.4% GC)
However, they exist within regions that are not always AT rich
In Tribolium and Apis, the microsatellites exist in regions that have GC content less than that of the genome; in Bombyx, Drosophila and Anopheles, GC content of the flanking regions is greater than that of the genome
In Drosophila and Anopheles, average GC content of the microsatellites is almost equivalent to that of the genome

Does GC content influence microsatellite expansion?

Microsatellite expansion takes place during DNA replication, repair or recombination and hence GC content could have a bearing on the expansion process
Among 5 insects, there was no linear correlation between GC content and the average number of repeat units
There are three peaks: 30% of Tribolium microsatellites (at 5-20% GC, nearly half of which are mono repeats); about 7% of Bombyx microsatellites (at 45-65% GC, more than half are dinucleotide repeats); Bombyx microsatellites (at 75-95% GC, half are trinucleotide repeats)

Do imperfections occur all along the microsatellite?

Existence, expansion and replication of a microsatellite depends upon boundary delimitation
Imperfect repeat units originate because of substitutions and indels
Analysis of 5 genomes suggests that interruptions, if at all, occur mainly in the middle region of the repeat sequence and the ends seem to be selected against decomposition
Anopheles has the least non-consensus bases in the microsatellites. Though Triboium has the maximum imperfect bases throughout, at the 5’ and 3’ extremes they are negligible

DISTRIBUTION OF MICROSATELLITES

Availability of physical map of the chromosomes facilitates visualising microsatellite distribution at a macro scale.

Legend
X axis: size in basepairs Y axis: Frequency per 1000 bp window
Green bar: Genes(Genescan predictions), Red bar: Microsatellites (Tandem Repeat Finder predictions), Black bar: Repetitive elements (Repeat Masker predictions)

Occurrence of microsatellites in Introns and Exons

	Bombyx	Apis	Drosophila	Anopheles	Tribolium
% of microsatellites on exons	1.3	2.2	7.1	6.7	3.2
% of microsatellites on introns	27.3	16.01	15.45	19.82	18.73