Starling (sturnus roseus) is a migratory bird of Pakistan. In the last three decades, there has been a significant decrease in the number of migration including sturnus roseusfrom Siberia. Its survival is now endangered in Pakistan. The aim of this study was to analyse COI gene to generate barcode data on starling in Pakistan. The reason behind the selection is that there were no molecular base data on Rosy starling on CYTB gene.The present study have been also focused on the generation of molecular data for CYTB gene of starling to make easier the study of genetic diversity within specie.Feather samples of 15 starling were collected from different areas of district Sargodha.To carry out the identification by using the nucleotide sequence analysis genomic DNA was extracted from feather follical by Russel and Sambrook method.DNA sequence analysis of CYTB ,COI gene were performed to identify the species.The similarity index from BOLD and BLAST were used to confirm species identity. The species were identified as sturnus roseus. The maximum parsimony and maximum likelihood analysis were performed to find the phylogenetic relationship by using MEGA X and Bioedit software. The results of this study indicate that molecular method play a vital role for the identificaion of species.Molecuelar approach also estimate the nucleic divergence with in a species.Futher study of barcode is needed in resolving the identification issue of starling in Pakistan. The genetic strategy is excellent shorthand for building a Starling database in Pakistan. In order to improve diversification of starling in Pakistan. This result will be first ever data to be submitted on CYTB gene of Rosy starling in NCBI BLAST. It will be helpful to improve genetic diversity of starling regarding conservation and management program.
Research Article
Mitochondrial COI and CTY B base study on genetic diversity of starling in Sargodha, Pakistan
https://doi.org/10.21203/rs.3.rs-3249550/v1
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DNA barcoding
starling
phylogeny
Cytochrome c Oxidase I gene
Cytochrome b gene
With large populations on five continents, the Rosy Starling is one of the most common and widespread bird species. It is native to Europe and Asia, but has been spread to three other continents with success (Feare et al. 1992). Migratory populations that breed in Siberia and Turkestan winter in Pakistan and India through the Central Asian Flyway. Rosy Starlings are seen across Pakistan from late October to early April, except in high-altitude, snow-covered areas. The greatest threat to Rosy Starling wintering populations in Pakistan may be illegal, unregulated hunting. Wildlife legislation must be adhered to to the letter. More research into the effects of pesticides and fungicides on rosy Starling populations in wintering areas is also required. The starling is a migratory bird that prefers stepped and open farmland. Mitochondrial DNA is use to study population size of a specie and evolutionary history of specie (Jiggins et al; 2005).Mitochondria have small amount of DNA inherited from mother.It have 37 genes which are necessary for it.Some of them use as markers.Mitchondrial DNA show high mutation rate it show ten times more mutation than
nuclear DNA so that make it the reason of evolution .Birds mtDNA show less variation as compare to mammals so it can predict gradual changings and process of organic evolution (Wright 1931).
Mitochondrial DNA can also use as a specie identification tool because it is specific in each specie so we can discover new species and also can make a evolutionary link of specie with others species.Mitochondrial DNA is also use in research because it can collect in some quantity it does not show complications than nuclear DNA.Nuclear DNA have exons which make it difficult to replicate.On the other hand mitochondrial DNA is exon free so it will be easy to make copies of it.
The principal phylogenetic investigation of higher classes of flying creatures dependent on DNA grouping information showed up in 1991 (Edwards et al. 1991). In the field of avian phylogenetics, the number and scale of DNA arrangement datasets has steadily increased. The primary investigations of complete mitochondrial (mt) genomes of ostrich and rhea in the late 1990s provided a definitive cut off for relative mitochondrial DNA (mtDNA) sequencing. While avian phylogenetics had recently used close protein sequencing (Stapel et al. 1984. The current investigation will zero in on the profound investigation of the barcoding, sequencing hereditary fluctuation of starling. The assessment will intend to accomplish the definitely center around hereditary decent variety of starling.The whole assessment will depend on the sub-atomic groupings for instance the use of DNA arrangements.The Mitochondrial DNA particularly COI locale will chose on the grounds that one of most rationed mitochondrial protein-coding qualities in creatures and hence shows a superior phylogenetic signal.COI as a barcoding marker has been utilized effectively in numerous creature taxa.Mitochondrial qualities are additionally assume a function in the foundation of various degrees of phylogenetic connections.
Mitochondrial genome is the most popular bit of eukaryotic DNA. As of now the nucleotide successions of the whole mitochondrial DNAs (mtDNAs) of bird's.These complete groupings, along with fractional arrangements and limitation compound examinations of mtDNAs from numerous different species, have brought about a definite comprehension of genome size, quality request and capacity, nucleotide succession uniqueness, and populace dissemination of variations of this surprising molecular.Despite the fact that mtDNAs of some major phylogenetic gatherings are yet to be examined, plainly all mitochondrial genomes will share various highlights for all intents and purpose that render the molecular gainful for study. Our ability to DNA barcode all of the world's birds is based on the effective amplification and sequencing of a 648 c oxidase (COI or cox 1) gene. For several geographic regions, many of the vouchered specimens needed to create a DNA barcoding database have already been collected and are available in museums and other institutions.However, many of these specimens have been preserved as dried skeletons or fixed testing skins since they are old (> years Cytochrome B is one of 37 genes of mitochondrial DNA.It is very informative for study of genetic variation and specie identification.Mitochondrial DNA is easy to study even in highly degraded sample of an animal specie.Because degraded sample have low quantity of DNA but we can be use this low quantity to study genetic diversity within a specie.Cytochrome B have high nucleotide diversity so we can use it as tool for study.we use a target region of gene for study. Due to high nucleotide divergence cytochrome B gene is prefer to study genetic divergence within a population of same specie rather than COI (Caine et al; 2006)
Cytochrome b is very useful marker because it show less variability within population and more variation between population.So a little change in sequence of this gene inwithinpopulation can predict about how population structure is change and how specie will extinct or recover from the original size (Caine et al; 2006).
Taxon sampling:
Feather samples of 15 starling were collected from different areas of Sargodha district. Extreme heat in the summers and mild cold in the winters characterise the city's environment. In the summer, the maximum temperature reaches 50 degrees Celsius (122 degrees Fahrenheit), while the lowest temperature reported in the winter is below freezing. The temperature is more favourable for migratory birds, particularly starlings, from September to March. Sample of starling were collected from sargodha region on the month of september-october in morning by using cannon nets traping technique. Morphological identification can be done by using morphological identification keys of Richard Grimmett, Tom Roberts and Tim Inskipp identification done on the base of feather colour and beak or bill shape, leg colour, adult plumage. Diverse Starling were chosen for explore. The follical from the feather was gathered.For the extraction of DNA and further strategies Collected Sample were taken to lab and immediately before it further starts to dried or decompose.DNA extraction process were start from follicle.
Table 1.1 Sampling detail of starling species used in this study including sample taxanomy, source,and conservation status.
DNA extraction, COI amplification and sequencing
The total genomic DNA of the collected specimen was extracted using a modified organic DNA extraction method. (Sambrook et al. 2001). COI gene was amplified using universal vertebrate primers,
VF1d (5′-TTCTCAACCAACCACAARGA(Y)AT(Y)GG-3′)
VR1d (5′-TAGACTTCTGGGTGGCCRAARAA(Y)CA-3′) (Cai et al. 2011).
And for cyt b gene
|
F1484B |
5„ ATCCAACATCTCAGCATGAT-3 |
|---|---|
|
R1485B |
5„TCAGTTTTTGGTTTACAAGAC-3 |
For the analysis of sample the product were obtain 1000 bp of COI gene was ampliufied by primer and results shown After confirmation of successful PCR Amplified product was run on 2% agarose gel and visualized under UV. 50 Bp ladder (Thermo scientific) was run in first lane against amplified samples
Sequence Analysis:
The sequence created by BioEdit v7.0.5 (Hall 1999) with manual proofreading software has been investigated. All sequences have been aligned to consider useful websites with ClustalW that uses MEGAX (Kumar et al. 2018). Further sequences were identified with the BOLD (Barcode of Life Data base) species identification tools and with information bases from BLAST GenBank to approve distinguishing evidence pieces. In addition, the Strong Sequence Examination Instruments have been also used to evaluate intra- and interspecific divergences. Using the Jukes-Cantor model (Jukes and Cantor 1969), a strong testing framework has been used for a neighboring Phylogenetic Tree (Taxon ID Tree) .
Successful sequencing resulted sequences of COI and cyt b gene which were readable fragments for further analyses. The almost all the sequences were 98–100% identical with their respective species. The details of BLAST results are given in table.
The intraspecific and interspecific divergences were calculated using MEGAX. Intraspecific divergence ranged According to our neighbor-joining tree (Fig. 1),the overall phylogeny of starling remained same as previous studies
Sequence characteristics:
High nucleotide divergence is necessary for the of genetic divergence within a specie. CYTB gene have high nucleotide diversity so it is more preferable gene for study. It can be predict a little change in nucleotide sequences within and between a population (Caine et al; 2006).
15 starling sequences were studied in present work for the investigation of genetic variation by using CYTB gene. Samples were examined morphologically by using bird morphological key by (Richard Grimmett, Tom Robert &Tim Inskipp).Mitochondrial DNA was extracted and amplified by using universal primers.For nucleotide sequence analysis NCBI BLAST program was used to check the similarity of nucleotide sequences. Phylogenetic analysis was studied by MEGA X software for the study of evolutionary relationships.
The Hasegawa-Kishino-Yano model and the Maximum Likelihood approach were used to infer the evolutionary past [1The evolutionary history of the studied taxa is determined from the 1000 replicates of the consensus bootstrap tree[3]. Branches referring to partitions replicated with less than 50% of bootstrap replicates have collapsed. The percentage of duplicates in which the corresponding taxa were clustered in the Bootstrap test together (1000 replicates) is situated next to these branches[3]. Automatically generate the original tree(s) for the heuristic search by applying Neighbor-Join and BioNJ algorithms to a matrix from pairway length calculated with an MCL approach, and select a topology with the highest log probability value. A discrete gamma distribution was used in order to model evolutionary differences in sites (5 categories (+G, parameter = 35.5079). In this sample, there were 33 nuclear sequences. Positions of 1st+2nd+3rd codon have been included. The final datasets included a total of 1551 positions. Evolutionary studies were carried out with MEGA X.
The evolutionary analysis was demonstrated by the use of Maximum Likelihood method and General Time Reversible model. The tree have highest log likelihood is (-6730.86) . The percentage of trees with associated taxa is shown next to the branches. Initial tree(s) for the heuristic search were obtained by Neighbor-Join and BioNJ algorithms method.Matrix of pairwise distances calculated by using the Maximum Composite Likelihood (MCL) approach with selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to show evolutionary rate differences among sites (+G, parameter = 0.4468). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 25.79% sites). The analysis involved 49 nucleotide sequences. Codon positions were 1st+2nd+3rd.Total of 1143 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [2]
We conclude that mtCOI is an efficient marker for species identification as well as for studying the phylogeny of starling Researchers may use barcoding to extend their taxonomic analysis for extant organisms that aren't known correctly using morphological techniques. Furthermore, only use morphological methods of identification and avoid illegal strling hunting. The use of DNA barcoding as a species identification tool. The use of the COI gene to identify this species. According to that study, starling is a migratory bird species that comes only for a few months in Pakistan and Sargodha from Siberia to Asia by using Green Route No 4.Mitochondrial DNA is a excellent molecular marker which used to find out the population genetics and phylogenetics relationships because of Nucleotide differences can be detected by using of resistriction enzyme .Methodologies can be developed for the isoloation od DNA. Universal primar can be easily applicablefor DNA amplications. PCR methodologies have developed (patwardham et al.,2014)
The use of cytochrome b gene make capable us to study genetic variation within specie level. Mitochondrial DNA proves an excellent marker system for study of population genetics and phylogenetic relationships.Molecular analysis of starling DNA can easily evaluate the present work of understudy specie which will be reliable for further molecular analysis and will provide pathway for the utilization of the work for conservation of the specie but there is a need of more extensive sampling.
Ethical Approval
Starling heads are collected from hunters.
Competing interests
“not applicable
Authors' contributions
(I.A. carried out all molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. A.N. carried out all supervision of this research. H.T. participated in the sequence alignment. And participated in the design of the study and performed the statistical analysis and Trees. A.M conceived of the study, participated in its design and coordination, and helped to draft the manuscript. S.A All authors read and approved the final manuscript)
Funding
(No one has committed any funding
Availability of data and materials
All data for this paper are collected from different sites like identification was done from NCBI, Phylogenetic trees were created from MegaX and the rest of the data was taken from BioEdit and Mega X software.By using This data can be accessed
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No competing interests reported.