Comparative Analysis of Predicted SSR Sequences and CpG Islands to Discover Evolutionary Relics of Sex-chromosomes in Divergent Animal Species

In mammals, the sex chromosomes are generally dimorphic. The X chromosomes are usually of large size and gene-rich while Y chromosomes are comparatively of smaller in size and heterochromatic in nature and are almost completely different but they at small homologous region (pseudo autosomal region) they paired with each other. Genetic markers such as CpG and microsatellites plays important role in evolution of sex chromosomes. Many biological processes significantly affect the functionality of DNA. One such process is methylation that is involved in X-chromosome inactivation (XCI) especially at promoter-proximal regions that are enriched with CpG islands (Duncan et al., 2018). The Y chromosome accumulates repeat sequences that are epigenetically repressed, results an epigenetic dispute with Y gene expression and hence possible accelerates the Y chromosome degeneration. Ageing causes the loss of Y heterochromatin, which activates transposable elements and reduces male lifespan. In placental mammals namely eutherians and marsupials X-chromosome inactivation has evolved via two different non-coding RNA molecules (Muyle et al., 2021). 
       
Both SSRs and CpG islands are present in most of the organisms and are key elements in structural organization of genomes and their function and may be related with disease states, their systematic analysis has not been reported. The study of repeat density and its distribution pattern in the genome is expected to help in understanding their significance. The accumulating evidences suggested that SSRs plays role in gene expression regulation (Kunzler et al., 1995). In the present study, in silico mining of  the nucleotide motifs (SSR regions and CpG islands) has been targeted in the entire genome to explore the evolutionary relics of sex-chromosome constitute in divergent species of animals. The accessibility of complete genome sequences for many organisms through nucleotide databases has made it possible to carry out genome-wide analysis. In silico comparative analysis of DNA markers may be helpful in understanding their role and abundance in the coding, as well as non-coding, regions of the genome may give us some clue to the function of SSRs in gene regulation.

The nucleotide sequences of sex-chromosomes of twelve selected mammalian species, namely, Gallus gallus, Meleagris gallopavo, Anopheles gambie, Drosophila melanogaster, Callithrix jacchus, Chlorocebus sabaeus, Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, Bos taurus and Sus scrofa were downloaded in the Fasta format from the nucleotide database of National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/genome). The downloaded sex chromosomes were classified into five groups according to their order type (Table 1).
 

 
Microsatellite prediction
 
Microsatellite prediction was done with a Perl-based MISA (Microsatellite analysis) tool accessed under (https://webblast.ipk-gatersleben.de/misa/). Fig 1. showing the flowchart of microsatellite prediction.
 
 
 
CpG island prediction
 
For the prediction of CpG islands, we assumed a minimum length of 200 nt, the minimum content of C+G 55% and the ratio between the frequency of observed and expected CpG sites to be at least 0.65. The downloaded chromosome sequences were subjected to notepad++ for further modification. Then the sequences were subjected to Perl code for predicting the statistical data. The statistical data were subjected to the R-programming environment for further cleaning and getting the predicted data. Fig 2 showing the flowchart of CpG island prediction.
 

Sex chromosomes of different animal species viz. species belonging to ruminants, other mammals, avians, etc were analyzed for the distribution of microsatellites and CpG island.
 
Microsatellite prediction in the avian group
 
W chromosome
 
MISA has predicted higher monomeric and lower hexameric SSRs in W chromosomes of Gallus gallus and Meleagris gallopavo species (Fig 3). 
 

 
Z chromosome

Gallus gallus has higher numbers of all predicted SSRs based on the Z chromosome and sums to 291 while Meleagris gallopavo comparatively has only 58 SSRs (Fig 4). 
 

 
CpG island prediction in the avian group
 
W chromosome
 
Gallus gallus has a greater average island length of 569.12, Variation in island length is more as compared to Meleagris gallopavo (Table 2).    
 

 
Z chromosome
 
Gallus gallus has a greater average island length of 743.57, Variation in island length is more as compared to Meleagris gallopavo (Table 3). 
 

 
Microsatellite prediction in the insect group
 
X chromosomes
 
X-chromosomes of Anopheles gambie and Drosophila melanogaster are closer in size. Anopheles gambie has a smaller sized X chromosome than Drosophila melanogaster but comparatively contains a large number of SSRs (Fig 5). 
 

 
Y chromosomes
 
Surprisingly, Anopheles gambie has only mononucleotide repeat motifs. All types of microsatellites were present in Drosophila melanogaster but they were found to be fewer in numbers (Fig 6).
 

 
CpG island prediction in insect group
 
X chromosomes
 
The average island length of Anopheles gambiae in the X-chromosome is 634.24 and Drosophila melanogaster has an average island length of 619.42 (Table 4).
 

 
CpG island prediction in insect group
 
Y chromosomes
 
The average island length of Anopheles gambiae in the Y-chromosome is 535 and Drosophila melanogaster has an average island length of 601.02 (Table 5).
 

 
Microsatellite prediction in primates
 
X chromosomes
 
X-chromosomes of animals belonging to this group have comparable sizes. All of them have a higher number of mono nucleotide repeat motifs. Homo sapiens among all these animal species contains the highest number of all types of SSRs (Fig 7). 
 

 
Y chromosomes
 
A comparable number of microsatellite motifs was found in Y-chromosomes irrespective of their chromosome sizes. The total number of microsatellites of different types follow decreasing order in the range of Mono>di>tri>tetra> penta>hexa (Fig 8).
 

 
CpG island prediction in primates
 
X chromosomes
 
Callithrix jacchus has a greater average island length of 634.76, Variation in island length is more as compared to Chlorocebuss abaeus, Homo sapiens and Pan troglodytes (Table 6).  
 

 
Y chromosomes
 
Callithrix jacchus has a greater average island length of 643.37, Variation in island length is more as compared to Chlorocebus sabaeus, Homo sapiens and Pan troglodytes (Table 7).  
 

 
Microsatellite prediction in rodents
 
X chromosome
 
Both these animal species have comparable X chromosome sizes and similarly, have higher monomeric and lower hexameric types of microsatellites. But Mus musculus significantly contains a five times higher number of SSRs than Rattus norvegicus (Fig 9).
 

 
Y chromosome
 
On the other side, the Y chromosome of Mus musculus is approximately 2 and half times that of Rattus norvegicus but both of them contain a comparable number of all types of SSRs (Fig 10). 
 

 
X chromosome
 
The average island length of Mus musculus in the X-chromosome is 588.17 and Rattus norvegicus has an average island length of 596.19 (Table 8).  
 

 
Y chromosome
 
The average island length of Mus musculus the Y-chromosome is 548.88 and Rattus norvegicus is 560.46 (Table 9).  


 
Microsatellite prediction in Even-toed ungulates
 
X chromosome
 
Different type nucleotide repeats were exceptionally higher in numbers in the Y chromosome of Bos taurus, while the microsatellite repeat motifs were comparable in X-chromosomes (Fig 11).
 

 
Y chromosome
 
SSRs mined from Y chromosomes follow the decreasing order of several repeats in terms of mon>di>tri>tetra> penta>hexa. Complex type SSRs were considerably lower in Sus scrofa (Fig 12). 
 

 
X chromosome
 
The average island length of Bos taurus in the X-chromosome is 701.35and Sus scrofa has an average island length of 580.44 (Table 10). 
 

 
Y chromosome
 
The average island length of Bos taurus in the Y-chromosome is 545.86 and Sus scrofa has an average island length of 567.28 which means Sus scrofa has a greater average island length (Table 11).
 

       
CpG islands are found almost everywhere in vertebrate genomes. Even though many tissue-specific genes lack CpG islands, it is becoming clear that they do exist in all commonly expressed genes, as well as a large number of tissue-specific genes with CpG islands can be found at the 5' or 3' ends of genes. CGIs are a fragmented but unified DNA sequence family whose members serve as genomic platforms for controlling transcription at their associated promoters. These characteristics are based on common DNA sequences traits, such as CpG richness and a higher-than-usual G+C concentration (Thomson et al., 2010). In addition, SSR sequences possess most of the desirable attributes of molecular markers, including information content, unambiguous designation of alleles, neutral selectively (although they can be subjected to hitch-hiking effects), high reproducibility, codominance and fast and easy assaying of genotypes and therefore microsatellite markers or SSR have proved to be very useful for cultivar identification, pedigree analysis and the evaluation of genetic distance between organisms (Priolli et al., 2002) and genetic mapping (Yu et al., 2000). To date, most macropod microsatellites have been isolated using laboratory-based techniques, including standard bacteria screening and microsatellite enrichment libraries (Karagyozov et al., 1993; Hakki and Akkaya, 2000). These methods can be time-consuming and unpredictable, with no guarantees of obtaining the numbers or types of markers desired. These approaches are effectively random samples of the genome and do not permit the targeting of markers from particular chromosomes, or even the identification of the chromosomes of origin of known markers. Consequently, the availability of DNA sequences is now providing unprecedented opportunities to identify novel genetic markers for use.

In the present study, 12 different animal species were organized into five groups and targeted for microsatellite and CpG mining in sex chromosomes. Microsatellite data have been analyzed by considering the simple and complex repeats. Simple repeats comprise of six classes of repeats including mono-, di-, tri-, tetra-, penta- and hexamers. The density of each class of repeat is comparable across various genomic regions. However, there is often tremendous variation in density in different genomic regions among different SSR types, sometimes even in a chromosome-specific manner. Based on X-chromosomes analysis Mus musculus of primates group contains highest number of microsatellites i.e. 79146 while Meleagris gallopavo of the avian group had the least number (i.e. 58) of microsatellites. Complex microsatellites also followed same pattern of occurrence and were highest in the primates group and least in avian growing-type type SSRs were reported highest in Bos taurus of the even-toed ungulates group and lowest in Meleagris gallopavo of avian group. Based on Y chromosomes analysis Mus musculus of primates group scored highest with total of 49725 microsatellites. Anopheles gambie of insect group contained the least microsatellites with total of 4 numbers. Gallus gallus of avian group contained highest and Drosophila melanogastor of insect group contained lowest mono type microsatellites respectively. Complex type SSRs were reported highest in Mus musculus rodent group and lowest in Anopheles gambie i.e. 0.
               
Mining of CpG island in female dominant chromosomes revealed the highest numbers of 50388 in Anopheles gambie of the insect group and the least in Meleagris gallopavo of avian group with 83 CpG islands. Based on male dominant chromosome analysis (i.e. Y chromosome) CpG islands were found highest in gallus gallus of the avian group i.e. 4635 and least in Anopheles gambie of the insect group i.e. 3 respectively. It was concluded from this study that female dominant chromosome (i.e. X chromosome) contained highest number of both microsatellites and CpG islands as compared to male dominant Y chromosomes. It could be hypothesized that the female sex could be more prone to mutations and involved in evolution more importantly than males. Mutation rate could depend upon species type, age, sex of the individual, type of chromosome and type of allele loci. The knowledge obtained from this study can be used to understand various aspects and functions of genome organization, for marker-assisted selection in breed improvement, characterization, conservation and DNA fingerprinting. This analysis left a few questions, for example, why some repeats are in huge numbers and others extremely rare? What is the structural and functional basis for specific SSRs’ chromosome-specific differential abundance? To understand the genome-wide gene structural and functional studies other kinds of DNA sequences and repeats will be needed to be analyzed and evaluated.

The authors gratefully acknowledge the support provided by the Department of Biotechnology, Government of India, through the collaborative research project “Parentage Determination and Cytogenetic Profiling in Dogs” [DBT-19I].fingerprinting. This analysis left a few questions, for example, why some repeats are in huge numbers and others extremely rare? What is the structural and functional basis for specific SSRs’ chromosome-specific differential abundance? To understand the genome-wide gene structural and functional studies other kinds of DNA sequences and repeats will be needed to be analyzed and evaluated.