Bioinformatics & Proteomics Open Access Journal ISSN: 2642-6129 MEDWIN PUBLISHERS Committed to Create Value for Researchers Perfect and Imperfect Microsatellite Markers in Determination of Genetic Status of Greater Adjutant Stork (Leptoptilos dubius Gmelin) 1 2 3 Sharma DK *, Baruah C and Barman PD Research Article 1Department of Zoology, University of Science and Technology, Meghalaya, India Volume 5 Issue 1 2Department of Zoology, Darrang College, India Received Date: May 07, 2021 3Aaranyak, Beltola Survey, India Published Date: June 30, 2021 *Corresponding author: DK. Sharma, University of Science and Technology Meghalaya, Kling Road, Baridua, 793101, India, Email: [email protected] Abstract The Greater Adjutant Stork (Leptoptilos dubius), the most endangered stork (IUCN Red List criteria under A2bcd+3bcd+4bcd; Kamrup, Assam, India. An attempt was made to study the status of genetic variability in Greater Adjutant Stork revealed that C2a) losing its number by population has been confined in a small village named Dadra- Pacharia- Singimari in the district of though the Greater Adjutant Stork population is highly threatened, yet the group has been appeared as genetically stable as recorded from that of the observed heterozygosity. Therefore, it is of importance to study the distribution enrichment and polymorphism of microsatellites in the genome of the Greater Adjutant Stork. Five microsatellite markers of cross species- number of alleles varying between 2 to 9 across all loci used. The locus Ah341 was observed to have 2 alleles whilst the specific markers were deployed in this study. All the five microsatellite markers were recorded to be polymorphic with the locus Cc07 was with 9 alleles. The heterozygosity of L. dubius was observed to be high for the microsatellite markers used, with mean observed heterozygosity (Ho) of 0.752±0.09 and mean expected heterozygosity (He) was with 0.677±0.06. The overall mean expected heterozygosity was found to be marginally higher than the observed heterozygosity. The polymorphic (TG) was dominating at 20 followed by mononucleotide (A) at 12, while the percent (%) of Imperfect iteration stood at the interaction count (PIC) was calculated at 0.569±0.03. Iteration value in the consensus sequence recorded for dinucleotide highest of 14.8%. Thus, the result of this investigation has extended a clear genetic polymorphism in favour of the Greater Adjutant stork, perhaps yet to face any genetic threat. Keywords: Leptoptilos dubius (Gmelin) Perfect Imperfect Iterations; Microsatellite; Polymorphism; Genetic Status; Greater Adjutant; Abbreviations: Introduction SSR: Simple Sequence Repeats; PCR: The Greater Adjutant Stork Leptoptilos dubius, an InformationPolymerase CountChain Reactions; HWE: Hardy-Weinberg endangered recognized as declining species in number as Equilibrium; HI: Heterozygote Instability; PIC: Polymorphic Perfect and Imperfect Microsatellite Markers in Determination of Genetic Status of Greater Adjutant Bioinform Proteom Opn Acc J Stork (Leptotilos dubius Gemlin) 2 Bioinformatics & Proteomics Open Access Journal per the IUCN Red List criteria under A2bcd+3bcd+4bcd; C2a The categories of microsatellites, the imperfect and perfect Greater Adjutant stork was once very widely distributed repeatson record states as the that source the former of genetic one diversity is more stableof a species compared [38]. in[1-3] India, and South the species and South is in the East verse Asia, of butextinction. currently The available species gives the opportunity to identify the perfect and imperfect to the later [39,40]. And the application of bioinformatics tool only in Assam and Bihar in India [4-10] and a very few in birdsSouth have East beenAsian found countries in Assam, [1,11,12]. considered Of the tototal be estimatedthe global mutationIt has [41]. already been accepted that except for repeat copy global population of about 1000-1200 birds [13], about 800 number variation, a microsatellite (e.g., ATATATATAT) also has been endowed with the richest diversity of storks and ofstronghold the twenty of thisspecies stork. of Accordingstorks, eleven to Luthin are found [1] South in South Asia mutations, attains the status of imperfect (e.g., ATATATCATAT: East Asia among which eight species are reported from ATsuffers repeat from with nucleotide an insertion substitutions of C and and the insertion/deletion genomes possess India [1,14]. Seven species of storks are found in Assam a relatively small but significant number of imperfect Kamrup[15,16] ofDistrict which of the Assam, Greater India Adjutant holds the is highestan endangered number variationmicrosatellites being less[42]. prone Imperfect to slippage microsatellites mutation, allowsis critical the ofspecies. Greater The Adjutant Dadara-Pacharia-Singimari and is located just village10 km areaaway in from the imperfectfor their maintenance microsatellites in the to genomebe more [43] stable due comparedto mismatch to of motif mismatches in imperfect microsatellites is still the Greatercity of Guwahati,Adjutant Leptoptilos Assam, India dubius [17,18]. has been According adjudged to extremelyperfect microsatellite limited and [40]. their Yet, correlation the level ofwith understanding life aspects asICBP/IWRB the systematically Storks, ibises disappeared and spoonbill bird species group throughout including demands critical evaluation. its early distribution ranges and this might be the possible reasons to attract this species as the most threatened one with the extinction possibilities, needs special attention be one of the reason for no genetic variability investigation, yet certainLack of attemptsspecies-specific were mademicrosatellite on the genetic markers, variability perhaps and to a certain degree of ecological approaches are available [1,18,19]. Information related to its taxonomy, morphology this species exhibits no migratory behaviour and tends to using microsatellite markers in other birds [44,45] like use[20-25], the colonial yet without sites overwith theany years genetic like information.that of wood Sincestork oriental population of white stork [46], European white stork Mycteria americana decline[47], white of the faced Greater Ibis [48],adjutant Painted has storkdrawn [49], wider Wood attention stork to[50] this and background. in Asian woolly Hence, necked the aim stork of the [51]. present Thus, study the rapid was [26], generally response to the seasonal to analyse perfect and imperfect microsatellite markers for fluctuation like its attempt for pairing and colony formation determination of genetic status of Greater Adjutant Stork in theSo same far, no locality approach of their has nestingbeen made sites to [27]. investigate on the (Leptoptilos dubius Gmelin). The objectives were to genetic variability in Greater adjutant, which could perhaps Examine the levels of genetic diversity, be able to determine its position with the help of molecular Inbreeding status, and markers like microsatellites. Microsatellites or simple Leptoptilos dubius. Population differentiation in breeding colonies of sequence repeats (SSR) are the nucleotide tandem repeats Microsatellitesof short sequence are motifthe regions of 1-6 baseof DNA pairs that [28], contain distributed di, tri markers developed for L.dubius that have been compiled orboth tetra in nucleotidecoding and repeats. non-coding They areregions randomly of DNA distributed [29,30]. This study reports on the five new microsatellite white stork Ciconia ciconia and white heron Ardea herodias high degree of polymorphic nature, they are extensively in a series of PCR with six markers previously described for usedacross in the studies genome related of most to species evolution [31,32]. of genetic Because status of their of the levels of genetic diversity, inbreeding and population differentiation[47,52]. Hence thein breedinggoal of the colonies, present whichstudy wascould to examineperhaps microsatellite in the coding region often changes the protein be able to suggest a benchmark information to formulate a species [33]. Increase or decrease of repeat base number in conservation action plan. product [29,34], and in the noncoding regions leading to the Materials and Methods 10mutationsto 10 [35]. They are concomitant and highly polymorphic polymorphism,markers-2 [36]-6 carries they are high extensively increases usedthe mutation in studies rate related like Sampling per meiotic cycle [37]. Because of their high higher rate of mutability the microsatellite has been placed Samples for the present work had been collected from to evolution of genetic status of a species [33]. Due to its Sharma DK, et al. Perfect and Imperfect Microsatellite Markers in Determination of Genetic Status of Copyright© Sharma DK, et al. Greater Adjutant Stork (Leptotilos dubius Gmelin). Bioinform Proteom Opn Acc J 2021, 5(1): 000137. 3 Bioinformatics & Proteomics Open Access Journal the dead specimens of Greater adjutant either from adult centrifuge tube to which 500 µL of the following solution was or from the chickens at certain wetlands and from the lone garbage site of the capital city of Guwahati in the Kamrup NaCl and 0.8%-1 SDS followed by the-1 District of Assam situated between 25°43 and 26°51 N additionadded comprising of K 40 µg.mL of-1 10 proteases. mmol-L ,A Tris-HCl, veterinarian 100 mmol-Lfrom the Latitudes and 90°12 and 90°36 E Longitudes/ (Table 1)./ A CollegeEDTA, 150 of Veterinary mmol-L Science, Guwahati, India collected the total of 23 tissue samples/ were /opportunistically collected