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Genic differentiation in natural populations
of Indian Mus musculus |
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Principal Investigator : Rajesh K Anand Collaborators The project aims to analyze natural populations of Mus
at genetic level. The main objectives of the project are i) the genetic
characterization of species and subspecies of Indian wild mice, ii)
establishment of new wild derived inbred strains and iii) susceptibility/
resistance of wild mice to infectious diseases. Genetic
characterization of Indian wild mice populations During this period, a new
category of highly polymorphic marker, microsatellites developed recently for
purpose of gene mapping were tried for characterization of Indian Mus species.
These markers have numerous advantages over biochemical or immunological
markers. These are tandem repeats of simple sequence that occur abundantly and
at random throughout most eukaryotic genomes. Since they are usually less then
100 bp long and are embedded in DNA with unique sequence, they can be
amplified in vitro using the PCR. They are easy to characterize and display
considerable polymorphism due to variation in the number of repeat units. This
polymorphism is sufficiently stable to use in genetic analyses. Partial inbred
wild house mice trapped earlier by NII’s Animal Facility from houses and
shops of Indian locations of Jalandhar (JAL), Delhi (DEL), Ghaziabad (GHZ),
Ranikhet (RK), Calcutta (CAL), Pachmarhi (PM), Coimbatore (CO) and Pune (PU)
were used for the present study. The randomly bred aboriginal mice populations
(M.terricolorIII, M.saxicola and M.platythrix) were also used as
representatives of Mus species. Inbred laboratory mice strains C57BL/6ByJ
(BL/6J) and DBA/2J (D/2J) were used as controls. 5 Microsatellites (D1Mit 308,
D4Mit 15, D4Mit 236,D5 Mit 79 and D6 Mit 102) randomly selected from a stock
of 6000 markers developed at the White Head Institute/MIT Genome Center with
an average spacing of less than 1 cM were used. Microsatellite reaction
was carried out in a total of 20 ml volume containing DNA template, PCR
buffer, H2O MgCl2 dNTP’s, forward and reverse primers with Taq polymerase (Gibco).
The reaction mixture was heat denatured at 95°C for 2 minutes. Various
conditions of amplifications were tried but the best amplification was carried
out for 35 cycles of denaturation (94°C, 45 sec.), annealing (57°, 45 Sec)
extension (72°C, 1 minute) and final extension (72°C, 6 minutes) by using
Perkin Elmer Thermal Cycler (DNA thermal cycler 480). 20 ul of amplified PCR
products was loaded on 2% Agarose gel and electrophoresed at 100 V for 2-3
hour. The allele size of the microsatellite was calculated by comparing the
fragment size with DNA marker (X 174 digested with Hae III (BRL). Various
parameters of genetic diversity within and among the wild mice populations
were statistically analyzed by using POPGEN 32 Software. To estimate the
phylogenetic relationships PHYLIP package (version 3.572) was used for
preparing gene frequencies matrix. 100 replicates of the original data set
were constructed by bootstrap resampling which were analyzed by maximum
likelihood procedure using CONTML package. CONSENSE was used to generate a
consensus tree. As far as polymorphism (% P) is concerned, Indian Mus musculus
populations from Coimbatore (CO) and Ranikhet (RK) showed 100 % polymorphism
at all the microsatellite loci tested followed by Delhi (DEL) and Ghaziabad
(GHZ) mice with 80% polymorphism and 40% in Pachmarhi (PM) and Pune (PU) mice.
The other populations of Indian Mus species as well as control inbred mice
showed 0% polymorphism indicating no variability in their genome with a
homogeneous genetic makeup. All Indian Mus musculus populations had a higher
value of Shannon’s informative index (I) meaning there was high level of
allele sharing. The maximum allele sharing (1.09) were seen in Ranikhet (RK)
population followed by Delhi (DEL) (0.80). The lowest value was observed in
Pune (PU) (0.32). In contrast other Indian Mus species and controls had I=0.00
indicating uniform population without allele sharing. A maximum of 9 alleles
were seen in Pachmarhi (PM) at D6Mit 102 locus whereas other loci produced 3-5
alleles. The allelic profiles within the population seemed to be population
specific. Majority of these mice had homozygous genotype; few mice were seen
with co- dominant expression of 2 alleles. Some populations showed null
allele, which could be due to mutations in the flanking region. Some of the
amplification reaction showed shadow bands due to heteroduplex events. High
value of F(ST) [0.89] was seen with D4Mit15 followed by D5Mit79 [0.72]. D6Mit
102 had a low F(ST) value with a higher gene flow rate. Low level of
heterozygosity seen in Pachmarhi (PM) and Pune (PU) mice could be linked with
the increased likelihood of population bottlenecks and absence of gene flow
due to geographical isolation. Nei’s genetic distance ranged from 0.00-4.11.
The most divergent mice population was seen to be Coimbatore (CO). Populations
from Jalandhar (JAL), Pachmarhi (PM), Calcutta (CAL) and Ghaziabad (GHZ) with
a genetic distance of 3.78 were genetically quite apart. To check the strength
of cluster analysis, parsimony was done. The most parsimonious tree differed
to some extent as compared to cladogram. This tree needs to be reconfirmed by
using at least 500-1000 times replicates and testing mice populations with
another 10-20 microsatellites, which may provide a clear picture of their
genetic relatedness. Derivation of new
Indian inbred strains of wild origin During this period, Delhi
(DEL) mice reached 25 generations of sibling mating followed by Pachmarhi (PM)
mice which are currently at 19th generation. Gwalior (GW), Himachal (HP) and
Bikaner (BIK) mice are slowly breeding. Inbreeding depression could be the
only reason for this performance. In addition, random mated stocks of some
localities were also raised for experimental studies. Publications Reviews/Proceedings 1. *Singh M, Gulati N, Mandal A, Totey S, Anand
R, Reddi H, Panda S, and Kumar V (2000) A tumor progression transgenic mouse
model of hepatocellular carcinoma. In: Advances in animal virology (Eds:
Jameel S and Villarreal LP), Science Publishers, Inc, Enfield, NH, USA 253-269
(*in press last year, since published). |