EXPLORING FISH MISCELLANY FROM BIODIVERSITY HOTSPOT REGION OF NORTHEAST INDIA THROUGH CHROMOSOME PROFILING

Sjeveroistočne države Indije obiluju ekstenzivnim ekoklimatskim uvjetima te sadrže veliki dio indijske endemske flore i faune. Ova regija obiluje ribljom raznolikošću s mnoštvom vrsta akvarijskih riba, uključujući zebricu. Među potencijalnim ukrasnim ribama, Ctenops nobilis označena je kao gotovo ugrožena, a Danio dangila uključena je na popis najmanje zabrinutosti IUCN-a. Ovdje je poduzeto citogenetsko profiliranje kako bi se osigurali komplementarni podaci za preciznu identifikaciju vrsta i proučavanje genetskog nasljeđa, organizacije genoma i evolucije vrste. Citogenetskim analizama utvrđen je diploidni broj kromosoma 44 i 50 s formulom kariotipa 8m+20sm+10st+6t (FN=72) odnosno 20m+24sm+6st (FN=94) u C. nobilis i D. dangila. Bojanje nukleolarnih regija otkrilo je prisutnost Ag-NORs, CMA3 mjesta i signala gena 18S rDNA na jednom paru subtelocentričnog kromosoma i jednom paru submetacentričnog kromosoma u C. nobilis i D. dangila. Ovi biomarkeri važan su izvor za molekularnu taksonomiju, evolucijske/filogenetske studije i genetiku očuvanja C. nobilis i D. dangila.North Eastern states of India are blessed with extensive ecoclimatic conditions and much of India’s endemic flora and fauna. This region is rich in fish diversity with plenty of species of ornamental importance, including model species zebrafish. Among the potential ornamental fishes, Ctenops nobilis is designated as Near Threatened and Danio dangila is included in the Least Concern list by IUCN. Cytogenetic profiling was undertaken here to provide complementary data for precise species identification and to study genetic inheritance, genome organization and evolution of the species. The cytogenetic analyses revealed diploid chromosome numbers 44 and 50 with karyotype formula 8m+20sm+10st+6t (FN=72) and 20m+24sm+6st (FN=94), respectively, in C. nobilis and D. dangila. The staining of nucleolar organizer regions revealed the presence of Ag-NORs, CMA3 sites and 18S rDNA probe signals on one pair sub-telocentric chromosome and one pair sub-metacentric chromosome in C. nobilis and D. dangila, respectively. These biomarkers are an important resource for molecular taxonomy, evolutionary/phylogenetic studies and conservation genetics of C. nobilis and D. dangila

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Not AvailableThe freshwater snakehead fishes are members of the family Channidae and order Perciform, which are native to parts of Africa and Asia with widespread distributions. The present study was aimed to assess the genetic variability and molecular phylogeny among 22 species of genus Channa, viz. C. argus, C. asiatica, C. aurantimaculata, C. bankanensis, C. barca, C. bleheri, C. burmanica, C. diplogramma, C. gachua, C. lucius, C. maculata, C. marulius, C. melasoma, C. micropeltis, C. orientalis, C. ornatipinnis, C. panaw, C. pleurophthlama, C. pulchra, C. punctata, C. stewartii and C. striata, using partial 513 bp long consensus sequence of cytochrome-c oxidase subunit I mitochondrial DNA retrieved from NCBI (March, 2018). The study showed 281 haplotypes with 267 variable sites (i.e., 33 singletons and 234 parsimoniously informative) in nucleotide positions. Most of the sequences were A + T rich, except in C. argus which is G + C rich (50.25%). The NJ and MP trees showed similar topology with discrete branches, each as a distinct species of the Channa with slight differences in bootstrap values. Maximum K2P distance was found between the C. ornatipinnis and the C. melasoma. The maximum numbers of polymorphic loci were present in C. gachua.Not Availabl

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Not AvailableBAC library has always been an important genomic source for preserving the genetic material of an organism for sequencing, developing physical map as well as addressing the novel evolutionary biology questions. Here, the end sequences of BAC clones were generated using a set of primers and mapped on the genome scaffolds of C. magur using bioinformatics tool. Numbers of genes present on the clones were identified using scaffolds’ annotation information. Further, the clone insert DNA was labeled through nick translation using tetra methyl rhodamine-5-dUTP (Pool A contained 6 clones possessing 36 genes mapped on scaffold A) and fluorescein 12-dUTP (Pool B contained 4 clones possessing 15 genes mapped on scaffold B) fluorophores for probe construction and was subsequently hybridized on metaphase chromosomes using FISH. The BAC-FISH revealed the mapping of red color probe on 19th sub-telocentric chromosome and green color on 14th sub-metacentric chromosome. These findings will be useful in understanding the chromosomes organization and evolution of genomic structure as well as function of the species in comparison to its closely related and well studies species, such as channel catfish and zebrafish.Not Availabl

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Not AvailableA wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosome evolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scaleDepartment of Biotechnology, Government of India, New Delh

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Not AvailableBacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384—well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.CABin Scheme of ICAR and CABin Division of ICAR-IASRI, New Delhi

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Not AvailableThe chromosomal characteristics of Clarias magur were examined using conventional (Giemsa-staining, Ag-impregnation and CMA3 + DAPI fluorescence) and molecular/ FISH (18S & 5S rDNA probes + one BAC DNA probe) cytogenetic tools. The diploid chromosome number was 50 and the karyotype consisted of 14 metacentric, 20 sub-metacentric, 8 sub-telocentric, 8 acrocentric chromosomes with 84 chromosome arms without any heteromorphic pair. The C-heterochromatic blocks were located on centromeric position of 13 pairs of chromosomes. The NOR sites, visualized by AgNO3-and CMA3-staining, were situated at p arms of chromosome pair No. 21, which also corresponded to 18S rDNA site visualized by FISH. The FISH signal of ICF_001_D19 clone probe was observed on 18th chromosome pair. The findings of the present study on C. magur provided valuable markers for the chromosome identification and locations of genes of the BAC clone on the chromosome will lead to the construction of physical map of genome of this species.CABin Scheme of ICAR, implemented through ICAR-Indian Agricultural Statistics Research Institute (IASRI), New Delhi

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Not AvailableThe Glossogobius giuris (Gobiidae), tank goby a species of goby fish, is native mainly to freshwater and estuaries and has importance in the aquarium trade. It is classified as least concern under IUCN Red List. Live individuals of this species were collected from Loktak Lake in Manipur for cytogenetic investigation to reveal the nucleolar organizer region (NOR) through silver nitrate (AgNO3) and chromomycin A3 (CMA3) staining as well as DAPI staining and single-color fluorescence in situ hybridization (FISH) using 18S rDNA sequence as probe. Analysis of more than 50 metaphase chromosome complement (obtained from colchicine–potassium chloride–Carnoy’s fixation–Giemsa staining procedures) showed the presence of 46 diploid chromosome numbers with all telocentric having fundamental arm number as 46 without any heteromorphic pair. One pairs of silver-stained NORs, situated terminally on the telocentric chromosome, were observed. Similarly, one pair of CMA3-positive sites were observed on the chromosome that suggested abundance of GC-rich repetitive DNAs in this region. One pair of 18S rDNA positive sites was observed on the telocentric chromosomes using FISH. These karyological features can be useful markers in cytotaxonomy and conservation of this species.Department of Biotechnology, Ministry of Science and Technology, Government of India, for financial support through Project Sanction No. BT/13/NE/TBP/201

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Not AvailableThe snakehead murrel Channa striata, with extensive distribution in Asia, is important in both culture and capture fisheries. Wild sub-populations of C. striata from Indian waters were analyzed in the present study using mitochondrial cytochrome b gene and nuclear microsatellite loci for genetic diversity assessment and sub-population structuring with the expectations that this fish have resulted in isolated sub-populations. A total of 191 specimens were collected for the study from eight distinctly located rivers from India. The cyt b analyses showed moderate to high, while microsatellites indicated low to moderate levels of inter-sub-populations' diversity. Tapti sub-population showed high level of genetic variation in cyt b analyses but low in SSRs, with a recent bottleneck. Betwa sub-population showed minimum haplotype variation, while Brahmaputra exhibited minimum expected heterozygosity at all the loci. Cyt b gene revealed 45 polymorphic sites with overall nucleotide diversity 0.00924 and haplotype diversity 0.9301. In phylogenetic analysis and haplotypes network, Chaliyar population appeared in different lineage. Chaliyar sub-population revealed maximum mean private alleles for microsatellite loci, while Tapti showed one private allele and Brahmaputra without private allele. The results indicate a gradual loss in number of private alleles within the species, which might be a concern for its conservation in the future.Not Availabl

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Not AvailableThe connective tissue growth factor gene plays important role in several biological processes and also responsive to hypoxia stress in fishes. The freshwater fish, Labeo rohita, highly cultured in Indian subcontinent for food, is reported as hypoxia sensitive but annotation and sequences of nuclear genes were not available for this species so far in the public domain, except some transcripts. In this study, an attempt was made for isolation and annotation of the CTGF gene in L. rohita using information of zebrafish from the same family. The CTGF gene sequence was obtained by aligning assembled genome of L. rohita, (NCBI BioProject ID: PRJNA437789), with the CTGF protein of zebrafish. Eight overlapping sets of forward and reverse primers from aligned region were designed for amplification of around 600 bp long successive overlapping fragments of CTGF gene in L. rohita. Assembly and annotation of overlapping fragments confirmed a complete 2421 bp long CTGF gene sequence with a full coding region that comprised of five exons between 308 and 1921 positions. This annotated CTGF gene sequence was submitted to GenBank (Acc. No. KY940466). Characterization of CTGF will be an initiative in identification of hypoxia response genes in L. rohita which may further help in understanding the mechanism of hypoxia tolerability in this species.CABin Scheme of ICAR and CABin Division of ICAR-IASRI, New Delhi

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Not AvailableThe HMOX1 gene plays role in several biological processes and is also responsive to hypoxia stress. Freshwater carp fish, Labeo rohita, is reported as hypoxia sensitive, but the information of annotated hypoxia genes in public domain is very scanty for this species. Here, an attempt was made to isolate and characterize HMOX1 gene in L. rohita using information from zebrafish. HMOX1 gene was obtained by mapping HMOX1 protein of zebrafish over assembled genome of L. rohita. Aligned region was used for designing primers for HMOX1 amplification. Eight overlapping sets of primers were designed for amplifying ~540 bp long successive overlapping fragments. Splicing of overlapping amplicons generated 3715 bp fragment that was confirmed as HMOX1 gene having full coding region with 6 exons between 184 and 2156 bp positions. HMOX1 characterization is an initiative for L. rohita genes annotation to support the characterization of new genes in the important species.CABin Scheme of ICAR and CABin Division of ICAR-IASRI, New Delhi