Molecular Identification and Genetic Diversity of Notopterus notopterus (Pallas, 1769) Using Mitochondrial COI Gene and RAPD Markers in Riverine Water of Pakistan

Waqas Ahmad*, Muhammad Naeem

Institute of Zoology, Bahauddin Zakariya University Multan, Pakistan.

Abstract | Identification and genetic diversity is important to explore biodiversity and conservation of species. Here we identified a commercially important fish Notopterus notopterus and analysed its genetic diversity. For the purpose of identification 15 specimens of N. notopterus were collected from Satluj, Ravi, Indus, Jehlum and Chenab rivers of Pakistan and barcoded with mitochondrial COI gene. BLAST analysis confirmed 100% identity of Notopterus notopterus with reference sequences belonging to NCBI GenBank database. Genetic diversity was analysed by using PCR amplification of 75 specimens of N. notopterus with five RAPD markers. RAPD markers data indicated high genetic diversity 0.5832 in Satluj while lowest genetic diversity 0.2173 in Chenab. High genetic polymorphism 60.52% observed in Satluj while lowest genetic polymorphism 23.07% observed in Chenab. Shannon’s Information Index (I) found 0.6281 in Satluj while 0.3162 in Chenab. Nei’s genetic diversity (h) among five riverine population was observed high in Satluj 0.5832 while lowest in Chenab 0.2173 which reflecting high genetic diversity in Satluj while lowest genetic diversity in Chenab. UPGMA dendrogram revealed that Indus and Jehlum riverine populations, and Satluj and Ravi populations of N. notopterus has close genetic similarity. Maximum likelihood tree revealed that N. notopterus identified from Pakistan has close genetic link with N. notopterus reported from Indo-Myanmar and India. Present study successfully reported molecular identification and genetic diversity of N. notopterus. Moreover, low genetic diversity reported in Chenab is matter of concern for fish resource managers and need to take constructive steps for its conservation.

Novelty Statement | DNA barcode base molecular identification and genetic diversity of N. notopterus evaluated from Satluj, Chenab, Jhelum, Ravi and Indus River of Pakistan through mitochondrial COI gene and RAPD markers reported first time from Pakistan. Low genetic diversity reported in Chenab River matter of concern for fish resource managers and need to take constructive steps for its conservation.

Article History

Received: October 25, 2023

Revised: November 05, 2023

Accepted: November 17, 2023

Published: November 30, 2023

Authors’ Contributions

WA collected samples, conducted experiments, data analysis and typing. MN revised the manuscript. WA designed experiment and wrote the article.

Keywords

DNA barcoding, Molecular identification, Genetic divergence, Mitochondrial COI, RAPD markers, Notopterus notopterus

Copyright 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Corresponding author: Waqas Ahmad

[email protected]

To cite this article: Ahmad, W. and Naeem, M., 2023. Molecular identification and genetic diversity of Notopterus notopterus (Pallas, 1769) using mitochondrial COI gene and RAPD markers in riverine water of Pakistan. Punjab Univ. J. Zool., 38(2): 181-190. https://dx.doi.org/10.17582/journal.pujz/2023.38.2.181.190

Introduction

Fish identification is critically important for conservation and management of fisheries. DNA barcode base molecular identification is an efficient approach of species identification (Suresh et al., 2022; Sajjad et al., 2023) as compare to traditional identification method. Traditional identification methods are less effective and produce ambiguities in identification of morphologically similar species (Naz et al., 2023). Mitochondrial COI gene particularly used for identification of species due to its high mutation rate and nucleotide variability. Mitochondrial COI gene is maternally inherited and quick enough to distinguish closely related morphologically similar fish species (Habib et al., 2021).

Genetic diversity data of natural populations of fish is essential to observe the genotypic variation of species. Genetic drift, mutation and migration are main causes of genetic variations in fishes (Zhao et al., 2011). Different methods of biotechnology used to analyse genetic diversity in freshwater fish populations (Yousefian et al., 2011). DNA markers universally used for evaluation of genetic diversity of freshwater fishes. Randomly Amplified Polymorphic DNA (RAPD) markers significantly used for analysis of genetic diversity (Popoola et al., 2014). The proper management is necessary to maintain genetic stock of fish in freshwater reservoirs which achieved by analysing genetic diversity (Zhao et al., 2011; Carlson et al., 2015).

Notopterus notopterus (Pallas, 1769) belongs to family Notopteridae found in Pakistan, Bangladesh, India and Indo-Myanmar. N. notopterus is a commercially important fish used in both fresh and dried forms for human consumption in Asia (Abbas et al., 2013). In Pakistan, the population of N. notopterus is declining quickly due to uncontrolled fishing, habitat destruction and water pollution (Abbas et al., 2013). However, genetic study on N. notopterus is crucial for its conservation.

According to best of our information, DNA barcode base molecular identification and genetic diversity of N. notopterus was not studied yet from Pakistan. Therefore, to fill this gap, we identify Notopterus notopterus fish and evaluated its genetic diversity from five main rivers of Pakistan. Moreover, morphological and meristic characteristics of N. notopterus were also studied in present study.

Materials and Methods

Collection of specimens

A total of 90 specimen of Notopterus notopterus fish were collected during the year 2020-2021 from Chenab, Indus, Jhelum, Ravi and Satluj rivers of Punjab, Pakistan (Figure 1). Among 90 collected specimens, N. notopterus 15 specimens used for molecular identification while other 75 N. notopterus specimens used for genetic diversity analysis. Sampling location and river wise number of specimen collection provided in Table 1.

 

The collected fish euthanized (kill) by immersing in concentrated Tricaine methane sulfonate (MS-222) 250 mg/L solution for 30 min (Popovic et al., 2012). After exposure, fish lose consciousness and stop breathing. Then fish specimens stored at -4°C for further studies.

Then specimens were analysed in the Laboratory of Fisheries, Institute of Zoology, Bahauddin Zakariya University Multan, Pakistan.

Morphometric study

Morphometric and meristic characters of collected specimens accomplished with the standard keys used for N. notopterus by Talwar and Jhingran (1991). Total body length, stranded length, head length, fins length and meristic features measured and counted followed Rahman (1989) and Galib et al. (2009).

 

Table 1: Detail of N. notopterus specimen collection.

N. notopterus specimens collection detail
Population	Specimen collection point	Longitude/ Latitude	Specimens used for identification using mitochondrial COI		Specimens used for RAPD diversity
River Chenab	Marala Headworks	74° 27' 51.5"/32° 40' 22.4"	3		15
River Indus	Taunsa Headworks	70°50' 57.1"/ 30° 30' 46.1"	3		15
River Jhelum	Rasul Headworks	73°31' 15.1"/ 32° 40' 49.0"	3		15
River Ravi	Balloki Headworks	73° 51' 44.7"/31° 13' 13.5"	3		15
River Satluj	Sulaimanke Headworks	73° 51' 58.6"/30° 22' 40.3"	3		15
Total				15		75

 

Table 2: Mitochondrial cytochrome oxidase I (COI) and RAPD primers used for PCR amplification.

Detail of mitochondrial cytochrome oxidase I (COI) and RAPD primers
Primer	Primer sequence	GC percentage	Number of fragments
Mitochondrial cytochrome oxidase I (COI) gene
FishF1-COI	5′-TCAACCAACCACAAAGACATTGGCAC-3′	46%	530 base pairs
FishR1-COI	5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′	48%	530 base pairs
Randomly amplified polymorphic DNA (RAPD) markers
OPY-02	CATCGCCGCA	70%	04-11
OPY-05	GGCTGCGACA	70%	03-10
OPY-07	AGAGCCGTCA	60%	04-11
OPY-11	AGACGATGGG	60%	05-08
OPY-16	GGGCCAATGT	60%	05-10

 

DNA extraction

50 mg fish fin tissue sample removed from each specimen. Then DNA of 90 specimens extracted with Phenol chloroform DNA extraction technique followed Garg et al. (2014) and Chowdhury et al. (2016). The isolated DNA was stored at -20°C until further analyses.

PCR amplification of mitochondrial gene and RAPD markers

PCR amplification of mitochondrial gene successfully completed by using the primers FishF1 COI and FishR1 COI followed Shen et al. (2016) and Naeem and Hassan (2019). The FishF1 COI and FishR1 COI primer sequences and GC percentage detail provided in Table 2. Total PCR reaction solution volume was 25µl for each PCR reaction, containing 1.5µl DNA template, 12.5μl PCR Master Mix (TaqNova-Red BLIRT S.A.), 0.1μl F1 and 0.1μl R1 CO1 primer concentrations with 10.8μl nuclease free water used. The PCR thermal cycler conditions were set as the initial denaturation for 2 min at 95°C followed by 35 complete cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 54°C and extension for 1 min at 72°C. Final extension was set for 10 min at 72°C (Ghouri et al., 2020). The success of PCR amplification checked by running the PCR products on 2% (w/v) agarose gel.

The amplicons of randomly amplified polymorphic DNA were amplified using five primers OPA-02, OPA-05, OPA-07, OPA-11 and OPA-16 followed by Laxmi et al. (2013) for screening N. notopterus samples (Table 2). Total 25µl PCR reaction volume made with DNA template 1.5µl, Master Mix 12.5μl (TaqNova-Red BLIRT S.A.), 0.3μl primers (each) and 10.8μl sterile water. The PCR amplification conditions set as initial denaturation at 94°C for 5 min, 40 complete cycles of denaturation at 94°C for 1 min, annealing at 36°C for 1 min, and extension at 72°C for 2 min. A final extension step at 72°C for 7 min (Laxmi et al., 2013). The PCR amplicon products were checked on 1.5% agarose gel buffered with 1X TAE buffer and visualized under UV light after staining in 1μl/ml ethidium bromide. The gel containing PCR products stained with ethidium bromide to detect the polymorphisms. The amplicon bands of each gel photographed with Gel Documentation System PHOTONYX DARKROOM (NTGB/1003). DNA marker (Thermo fisher) 100bp ranging 100bp-2000bp in size as standard to determine the molecular weight of each amplification band.

Mitochondrial COI gene sequence and genetic connectivity analysis

PCR purified products of 15 fish specimens sent to First BASE Laboratories Sdn Bhd, Malaysia and barcoded successfully. The barcoded sequences aligned and trimmed to same length by using MEGA-X software (Tamura et al., 2011). N. notopterus barcoded COI sequences processed through BLAST (Basic Local Alignment Search Tool) analysis of nucleotide database of NCBI (National Centre for Biotechnology Information) and examined the accurate identity match. All barcoded sequences submitted in GenBank Database as reference.

Maximum likelihood (ML) tree used to infer the genetic link and genetic similarity among COI barcode sequences of N. notopterus populations using MEGA-X software (Kumar et al., 2018).

Statistical analysis of RAPD genetic diversity and phylogeographic relationship

POPGENE Software version 3.1 was used to analyses RAPD diversity (Souza-Shibatta et al., 2022). The genetic diversity data was analysed by calculating Nei’s gene diversity (h) (Zhu et al., 2022), and Shannon’s information index (I) followed Mukhopadhyay and Bhattacharjee (2014). UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram constructed to show the genetic link among five rivers using Past4.03 software.

Results and Discussion

Study of morphometric characteristics

Notopterus notopterus has highly compressed body. The body outline is equally convex from both ventral and dorsal sides. Min scales are present at surface of body. N. notopterus have short dorsal fin and long anal fin. The colour of body observed silvery white (Figure 2). Total body length was 19.9 cm (mean), stranded length 18.7 (mean), head length was 3.5 cm (mean) and eye size 19.9% (mean) of head length. Summary of morphological and meristic characteristics (mean) provided in Table 3.

 

Gene barcoding, nucleotide sequence analysis and molecular identification success

In the present study, fifteen Notopterus notopterus fish specimens were barcoded successfully with mitochondrial COI gene. All barcoded sequences submitted to the GenBank database. GenBank Accession of submitted sequences provided in Table 4. The barcoded sequences aligned with final sequence fragment of 530 base pairs. All specimen barcode data and associated sequence information recorded in the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST result confirmed 100% identity of Notopterus notopterus with relevant sequences of GenBank databases (Table 4).

Genetic connectivity of Notopterus notopterus fish identified from Pakistan

A tree of genetic connectivity was constructed using 15 DNA barcode sequences of N. notopterus of current study and 5 sequences of N. notopterus were taken from GenBank database of previously reported neighbouring countries; India, Bangladesh and Indo-Myanmar. Maximum likelihood (ML) genetic tree produced two distinct clusters. Cluster-I was an assemblage of N. notopterus species identified from Pakistan with N. notopterus species reported from Indo-Myanmar which revealed that both species have high genetic connectivity and similarity. Cluster-II showed assemblage of N. notopterus species identified from Pakistan with N. notopterus species reported from India. This assemblage under same cluster indicated a high genetic similarity and genetic connectivity between both species. This genetic connectivity indicated that N. notopterus fish identified from Pakistan has close genetic linkwith N. notopterus fish of Indo-Myanmar and India (Figure 3).

 

Table 3: Morphometric and meristic characteristic of N. Notopterus.

Morphometric parameters	Notopterus notoptrus analysed	Morphological and meristic identity evidences	Identified species
Morphological characters
Body formation	Highly compressed	Highly compressed (Galib et al., 2009)	Notopterus notoptrus
Body outline ventrally	Equally convex	Equally convex (Galib et al., 2009)
Body outline dorsally	Equally convex	Equally convex (Galib et al., 2009)
Scales	Min	Min (Rahman, 1989)
Dorsal fin	Short	Short (Rahman, 1989)
Anal fin	Lengthy	Lengthy (Rahman, 1989)
Body coloration	Silvery white	Silvery white (Galib et al., 2009)
Lateral line	Complete	Complete (Galib et al., 2009)
Morphometric characters
Total length	19.9 cm	20.0 cm (Talwar and Jhingran, 1991)	Notopterus notoptrus
Standard length	18.7 cm	18.7 cm (Talwar and Jhingran, 1991)
Scales	230-235	230-240 (Galib et al., 2009)
Head length	3.5 cm	3.5 cm (Galib et al., 2009)
Body height	35.2% of Standard length and 32.1% of total length	35.5% of Standard length and 32.4% of total length (Galib et al., 2009)
Eye	19.9 % of head length	20% of head length (Galib et al., 2009)
Meristic characters
Anal fins	100-103	100-104 (Rahman, 1989)	Notopterus notoptrus
Pectoral fins	16-17	16-17 (Rahman, 1989)
Anal + caudal fins	101-107	101-107 (Talwar and Jhingran, 1991)
Dorsal fins	8-9	7-9 (Talwar and Jhingran, 1991)

 

Table 4: BLAST analysis of mitochondrial COI gene barcode sequences of Notopterus notopterus.

River name	Specimen collected	GenBank accession of submitted sequences	NCBI
			Identity (%)	E- value	Identified species name
Chenab	1	MZ328226	100	0.0	Notopterus notopterus
	2	MT434341	100	0.0	Notopterus notopterus
	3	MZ328225	100	0.0	Notopterus notopterus
Indus	1	MZ343242	100	0.0	Notopterus notopterus
	2	MZ343243	100	0.0	Notopterus notopterus
	3	MZ343298	100	0.0	Notopterus notopterus
Jehlum	1	MZ343251	100	0.0	Notopterus notopterus
	2	MZ343252	100	0.0	Notopterus notopterus
	3	MZ343254	100	0.0	Notopterus notopterus
Ravi	1	MZ329064	100	0.0	Notopterus notopterus
	2	MZ329667	100	0.0	Notopterus notopterus
	3	MZ329040	100	0.0	Notopterus notopterus
Satluj	1	MZ343295	100	0.0	Notopterus notopterus
	2	MZ342920	100	0.0	Notopterus notopterus
	3	MZ343294	100	0.0	Notopterus notopterus

 

 

 

Results of RAPD polymorphic and monomorphic DNA amplification

RAPD five markers produced a total of 66 polymorphic bands and 93 monomorphic bands among five populations. N. notopterus 15 specimens from each population amplified with five RAPD markers. A total number of 17, 23, 10, 10 and 06 polymorphic bands detected in populations of Ravi, Satluj, Jehlum, Indus and Chenab, respectively. Monomorphic bands were detected 18, 15, 19, 21 and 20 in populations of Ravi, Satluj, Jehlum, Indus and Chenab, respectively (Table 5). DNA band range varied from 3-11 and the DNA fingerprints of PCR amplified product showed the band size of DNA ranged from 300-1500 bp (Figure 4A-E).

RAPD five primers were produced polymorphic 66 (41.50%) bands among five population (75 specimens) of N. notopterus. In Ravi River population 48.57% polymorphic bands detected, in Satluj River population 60.52%, in Jehlum River population 34.48%, in Indus River 32.25% population and in Chenab River population 23.07% polymorphic bands were detected (Table 5).

 

RAPD diversity and genetic differentiation

RAPD five primers produced 159 total scorable bands consisting 66 (41.50%) polymorphic and 93 (58.50%) monomorphic bands. The pattern of genetic variation among five populations observed as Satluj ˃ Ravi ˃ Jehlum ˃ Indus ˃ Chenab from high to low (Table 5). Genetic differentiation (GST) among five populations was observed GST= 0.6160, total heterozygosity (HT) 0.6223±0.0012 and intra-population heterozygosity was observed (HS) 0.4132±0.1023.

The highest Nei’s genetic diversity (h) was observed 0.5832 in population of Satluj followed by Ravi 0.5234, Jehlum 0.4624, Indus 0.2572 and Chenab 0.2173 (Table 6). Results of Shannon’s information index (I) revealed the same pattern of genetic diversity as 0.6281, 0.5833, 0.4523, 0.3971and 0.3162 for populations of Satluj, Ravi, Jehlum, Indus and Chenab respectively (Table 6).

Analysis of genetic distances using RAPD markers

Based on RAPD data the maximum genetic distance 0.4142 found between Jhelum and Satluj while the lowest genetic distance found 0.1360 between Satluj and Ravi (Table 6).

UPGMA dendrogram construction for analysis of genetic connectivity among rivers

UPGMA dendrogram produced two distinct clusters. Cluster-I includes Satluj and Ravi River which indicated that populations of Satluj and Ravi has close genetic link. Cluster-II consist of two subclusters; Subcluster-I includes Indus and Jehlum, which indicated that populations of Indus and Jehlum has close genetic link while Sub-cluster-II observed independent consist of Chenab population alone (Figure 5).

Genetic relationship between populations

Nei’s co-efficient of genetic identity (I) was used to calculate the genetic relatedness among related fish species. Results of Nei’s co-efficient of genetic identity (I) of RAPD data analysis showed a distant high relatedness among Satluj and Ravi populations D= 0.2123 of N. notopterus whereas genetic distance between Jehlum, Indus and Chenab populations was observed 0.1711.

Fish Identification and evaluation of genetic diversity is vital for fisheries conservation and management (Suresh et al., 2022). Fish identification is also important for sustainability of aquatic ecosystem. Traditional fish identification techniques are ordinary because these techniques cannot identify morphologically similar species accurately (Hou et al., 2022). Therefore, DNA barcode of mitochondrial COI gene now used efficiently for identification of species (Habib et al., 2021).

Morphometric and meristic characters of N. notopterus reported in Table 3 of present study found similar with the standard characters reported by Talwar and Jhingran (1991). Total body length, stranded length, head length, fins length and meristic features also found similar to reference characters reported by Rahman (1989) and Galib et al. (2009).

In present study, fifteen N. notopterus specimens were barcoded with 530 base pairs, which confirmed the identification of fish species as Notopterus notopterus Table 4 as Naeem and Hassan (2019) used 650 base pairs for identification of species. N. notopterus fifteen barcode sequences produced in this study submitted in GenBank Database, which may use as reference for future studies. Record keeping of DNA barcode sequences is the basic objective of GenBank Database (Naeem and Hassan, 2019; Zhang and Hanner, 2012).

Figure 3 of ML tree in present study indicated that N. notopterus fish identified from Pakistan has close genetic link with N. notopterus fish reported from India and Indo-mayanmar as ML tree used by Naeem and Hassan (2019) and Mazhar and Saif (2022) to report phylogenetic relationship previously.

 

Table 6: Parameters of genetic diversity variations of N. notopterus.

Parameters of genetic diversity variations for N. notopterus among rivers				Nei’s (1978) Unbaised genetic diversity and genetic distance
Riverine population	Number of specimens	Nei’s gene diversity (h)±SD	Shannon’s information index (I) ± SD	River	Ravi	Satluj	Jehlum	Indus	Chenab
Ravi	15	0.5234±0.0112	0.5833±0.1523	Ravi	****	0.1360	0.3319	0.2192	0.3782
Satluj	15	0.5832±0.0143	0.6281±0.1842	Satluj	0.1360	****	0.4142	0.1652	0.2314
Jehlum	15	0.4624±0.0213	0.4523±0.1163	Jehlum	0.3319	0.4142	****	0.1860	0.2319
Indus	15	0.2572±0.0243	0.3971±0.1289	Indus	0.2192	0.1652	0.1860	****	0.3278
Chenab	15	0.2173±0.0135	0.3162±0.1327	Chenab	0.3782	0.2314	0.2319	0.3278	****

 

In present study, five RAPD primers were used (Table 2) and observed a total number of 159 bands as compared to Lal et al. (2006), who reported 1344 bands with five RAPD primers while Neekhra et al. (2014) used 6 RAPD primers and reported 746 amplified bands in Cyprinidae.

Figure 4A-E of current study presented DNA fingerprints of PCR amplified product band range from 300-1500 bp as Garg et al. (2014) reported genetic diversity in N. notopterus using DNA fingerprints.

RAPD analysis data of Table 5 showed the genetic diversity in N. notopterus. The number of bands produced per primer varied from 3 to 11 (Table 2). In present study, amplified DNA band size in N. Notopterus observed 100bp-1500 bp as Lal et al. (2006) reported band range of 250-2900 in N. notopterus and C. chitala. Table 5 of present study also revealed that RAPD five primers were produced 66 (41.50%) polymorphic bands of N. notopterus. In Ravi River population, 48.57% polymorphic bands were detected, in Satluj River population 60.52%, in Jehlum River population 34.48%, in Indus River 32.25% population and in Chenab River population 23.07% polymorphic bands were detected as Laxmi et al. (2013) reported the percentage of polymorphic bands to analysis of genetic polymorphism in N. notopterus.

The average heterozygosity (H) of alleles is the evaluation of genetic variation in population (Sahoo et al., 2023). Zhu et al. (2022) used parameter of Nei’s genetic diversity (h) to estimate a wide variety of organism’s diversity with RAPD markers. Table 6 of present study showed the highest Nei’s genetic diversity (h) 0.5832 in population of Satluj River followed by Ravi 0.5234, Jehlum 0.4624, Indus 0.2572 and Chenab 0.2173 as Laxmi et al. (2013) reported average heterozygosity in N. notopterus.

In Table 6, Shannon’s information index (I) showed the pattern of genetic diversity as 0.6281, 0.5833, 0.4523, 0.3971 and 0.3162 for populations of Satluj, Ravi, Jehlum, Indus and Chenab as Laxmi et al. (2013) reported using Shannon’s information index (I) in N. notopterus.

Fish species genetic relatedness can be determined by estimating the genetic distance and the genetic identity by Nei’s co-efficient of genetic identity (I) (Laxmi et al., 2013). Nei’s co-efficient of genetic identity (I) of RAPD data analysis showed a high relatedness among Satluj and Ravi populations D=0.2123 of N. notopterus in present study whereas genetic relatedness between Jehlum, Indus and Chenab populations was observed 0.1711 as Laxmi et al. (2013) used Nei’s co- efficient of genetic identity (I) to report the genetic relatedness in N. notopterus.

Fish genetic diversity could be due to mutations (Liu et al., 2022). Moreover, rate of species extinction is rapid in small size population (Wang et al., 2019). Therefore, low genetic diversity is consequences of limited founder stocks and inbreeding in a population (Wu and Yang, 2012). Continued and uncontrolled fishing from riverine water could intensely decrease size of effective population and a cause of losing genetic diversity (Garcia-Cisneros et al., 2017). There is no specific data about larval dispersal rate, sex ratio and sex biased fishing pressure on N. notopterus reported from Pakistan.

 

Figure 5 of current study resented UPGMA dendrogram that showed the genetic connectivity of N. notopterus populations among five rivers. UPGMA dendrogram revealed that Indus and Jehlum riverine populations, and Satluj and Ravi populations has close genetic similarity as Laxmi et al. (2013) used UPGMA dendrogram to report genetic connectivity among riverine populations.

Conclusions and Recommendations

Present study successfully reported the identification and genetic diversity of Notopterus notopterus fish from five main rivers (Chenab, Indus, Jhelum, Ravi and Satluj) of Pakistan through DNA barcode of mitochondrial COI gene and RAPD markers. RAPD markers data indicated that Satluj and Ravi produce high degree of genetic diversity while River Chenab had lowest genetic diversity. Therefore, population of N. notopterus in Chenab rivers need to be protect and conserved. Moreover, N. notopterus fish identified from Pakistan has close genetic similarity with N. notopterus fish reported from India and Indo-Myanmar. According to best of our knowledge, present study is first attempt to identify N. notopterus fish on molecular basis from Pakistan. The barcode sequences of mitochondrial COI gene of N. notopterus have submitted directly into GenBank database, which used as reference for future studies. Moreover, genetic diversity data generated from this study provide a valuable information for fisheries resource manager to take immediate steps in conservation of N. notopterus population in Chenab River to achieve sustainability.

Acknowledgements

We thank all the colleagues specifically Mr. Naveed Ahmad Khan and Abdul Rehman, who helped us in sample collection. We gratefully acknowledge Dr. Abir Ishtiaq and Prof. Shabbir Saqib, Mr. Zafar Abbas (Adv.) and Mr. Muhammad Ehan Sarfraz for their anonymous review, constructive comments on an early draft of the manuscript.

Ethics statement

All the methods carried out in line with international norms for animals. The study approved in meeting of Board of Studies (BOS) Zoology dated 06-03-2020. The Advance Study and Research Board (ASRB) issued letter in approval of study with No. Acad/Scholar’s File/582.dated 23-01-2021. Bahauddin Zakariya University Multan, Pakistan.

Conflict of interest

The authors have declared no conflict of interest.

References

Abbas, F., Hafeez-ur-Rehman, M., Ashraf, M. and Iqbal, K.J., 2013. Body Composition of Feather Back Notopterus notopterus and Rita rita from Balloki Headworks-Pakistan. J. Agri-Food Appl. Sci., 1: 126-129. https://doi.org/10.1016/j.tree.2014.06.005

Carlson, S.M., Cunningham, C.J. and Westley, P.A.H., 2015. Evolutionary rescue in a changing world. Trends Ecol. Evol., 29: 521–530.

Chowdhury, M.M., Rahman, A.S., Nahar, L., Rahman, M., Reza, A.H. and Ahmed, M.S., 2016. Efficiency of different DNA extraction methods for fish tissues: A comparative analysis. IOSR. J. Phar. Biol. Sci., 3: 11–15.

Galib, S.M., Samad, M.A., Mohsin, A.B.M., Flowra, F.A. and Alam, M.T., 2009. Present status of fishes in chalan Beel the largest Beel (Wetland) of Bangladesh. Int. J. Anim. Fish. Sci., 2: 214-218.

Garcia-Cisneros, A., Palacín, C., Khadra, B.Y. and Pérez-Portela, R., 2017. Low genetic diversity and recent demographic expansion in the red starfish Echinaster sepositus (Retzius 1816). Sci. Rep., 6: 33269. https://doi.org/10.1038/srep33269

Garg, R.K., Chaturvedi, D., Batav, N., Sharma, R. and Singh, R.K., 2014. Genetic divergence and gene flow in three populations of Notopterus notopterus (Pallas, 1769), Madhya Pradesh. J. Appl. Biol. Biotech., 2: 33-37.

Ghouri, M.Z., Ismail, M., Javed, M.A., Khan, S.H., Munawar, N., Umar, A.B., Nisa, M., Aftab, S.O., Amin, S., Khan, Z. and Ahmad, A., 2020. Identification of edible fish species of Pakistan through DNA barcoding. Front. Mar. Sci., 7: 554183. https://doi.org/10.3389/fmars.2020.554183

Habib, K.A., Neogi, A.K., Rahman, M., O.H., J, Lee, Y.H. and Kim, C.G., 2021. DNA barcoding of brackish and marine water fishes and shellfishes of Sundarbans, the world’s largest mangrove ecosystem. PLoS One, 16: e0255110. https://doi.org/10.1371/journal.pone.0255110

Hou, G., Chen, Y., Wang, J., Pan, C., Lin, J., Feng, B. and Zhang, H., 2022. Molecular identification of species diversity using pelagic fish eggs in spring and late autumn-winter in the eastern Beibu Gulf China. Front. Mar. Sci., 8: 806208. https://doi.org/10.3389/fmars.2021.806208

Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K., 2018. MEGA X molecular evolutionary genetics analysis across computing platforms. Mol. Bio. Evol., 35: 1547–1549. https://doi.org/10.1093/molbev/msy096

Lal, K.K., Mandal, A., Singh, R.K., Punia, P., Kapoor, D., Chauhan, U.K., Singh, S.P. and Mohindra, V., 2006. Genetic divergence in two featherback fishes, Chitala chitala and Notopterus notopterus. J. App. Ichthy., 22: 369-373. https://doi.org/10.1111/j.1439-0426.2006.00758.x

Laxmi, I.J. and Singh, Barat, A., 2013. Genetic characterization of Notopterus notopterus (Pallas) from different reservoirs of Uttarakhand using DNA (RAPD) marker. Trends Biosci., 6: 337-340.

Liu, B., Li, J., Zhang, K., Peng, Y., Liu, Y., Jin, X., Zheng, S., Wang, Y., Liu, L., Lü, Z., Zhang, S. and Gong, L., 2022. Population structure and genetic diversity in wild dotted gizzard shad (Konosirus punctatus) revealed by microsatellite markers. Front. Mar. Sci., 9: 1048279. https://doi.org/10.3389/fmars.2022.1048279

Mazhar, M.W., Saif, S., 2022. Molecular and morphological identification of fish gut microbiota at different growth stages of Labeo rohita. Clin. Med. Biochem., 8: 119-127. https://doi.org/10.21203/rs.3.rs-1554736/v1

Mukhopadhyay, T. and Bhattacharjee, S., 2014. Study of the genetic diversity of the ornamental fish badis badis (Hamilton-Buchanan, 1822) in the Terai Region of Sub-Himalayan West Bengal, India. Int. J. Biodiver., 2014: 791364-74. https://doi.org/10.1155/2014/791364

Naeem, M. and Hassan, S., 2019. Molecular approach for identification of Labeo bata based on COI gene sequence from Pakistan. Mito. DNA Part B: Res., 1: 244–246. https://doi.org/10.1080/23802359.2018.1547131

Naz, S., Chatha, A.M.M., Nazir, U., Iqbal, S. and Danabas, D., 2023. Morphological and genetic identification of head Taunsa barrage fish species. Cur. Tre. OMICS., 1: 40–59. https://doi.org/10.32350/cto.31.03

Neekhra, B., Mansoori, A.A., Verma, S., Koiri, R.K. and Jain, S.K., 2014. RAPD-PCR based biomarker study in fish species (Family: Cyprinidae) of Madhya Pradesh, India. Aus. J. Mol. Cell Biol., 1: 1003-1011.

Popoola, O.M., Fasakin, E.A. and Awopetu, J.I., 2014. Genetic variability in cultured and wild populations of Clarias gariepinus (Osteichthys: Clariidae) using random amplified polymorphic DNA (RAPD) marker. Cro. J. Fish., 72: 5–11. https://doi.org/10.14798/72.1.712

Popovic, N.T., Strunjak-Perovic, I., Coz-Rakovac, R., Barisic, J., Jadan, M., Berakovic, A.P. and Klobucar, R.S., 2012. Tricaine methane-sulfonate (MS-222) application in fish anaesthesia. J. Appl. Ichthyol., 28: 553–564. https://doi.org/10.1111/j.1439-0426.2012.01950.x

Rahman, A.K.A., 1989. Freshwater fishes of Bangladesh. 1st edition, Zoological Society of Bangladesh, Department of Zoology, University of Dhaka, Dhaka-1000.

Sahoo, B., Das, G., Nandanpawar, P., Priyadarshini, N., Sahoo, L., Meher, P.K., Udit, U.K., Sundaray, J.K. Das, P., 2023. Genetic diversity and genome-scale population structure of wild Indian major carp, Labeo catla (Hamilton, 1822), revealed by genotyping-by-sequencing. Front. Genet., 14: 1166385-93. https://doi.org/10.3389/fgene.2023.1166385

Sajjad, A., Jabeen, F., Ali, M. and Zafar, S., 2023. DNA barcoding and phylogenetics of Wallago attu using mitochondrial COI gene from the River Indus. J. King Saud Uni. Sci., 35: 102725. https://doi.org/10.1016/j.jksus.2023.102725

Shen, Y., Lihong, G., Dengqiang, W. and Xiaoni, G., 2016. DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River. Ecol. Evol., 6: 2702–2713. https://doi.org/10.1002/ece3.2060

Souza-Shibatta, L., Ferreira, D.G., Assumpção, L.d., Shibatta, O.A., Sofia, S.H., Pini, S.F.R., Silva, P.S.d., Makrakis, S. and Makrakis, M.C., 2022. Genetic diversity of the surubim-do-iguaçu, a giant catfish species threatened with extinction: Recommendations for species conservation. Diversity, 2022: 14–16. https://doi.org/10.3390/d14010016

Suresh, E., Rathipriya, A., Shanmugam, S.A., Hamsavalli, R. and Kathirvelpandian, A., 2022. Character-based diagnostic keys, molecular identification and phylogenetic relationships of threadfin breams (family: Nemipteridae) based on mitochondrial genes from the Southern coromandel Coast, India. Anim. Biotech., 34: 1-13. https://doi.org/10.1080/10495398.2022.2040522

Talwar, P.K. and Jhingran, A.G., 1991. Inland fishes of India and adjacent countries, Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi-Calcutta, 1: 64-65.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S., 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Bio. Evol., 28: 2731–2739. https://doi.org/10.1093/molbev/msr121

Wang, T., Fujiwara, M., Gao, X. and Liu, H., 2019. Minimum viable population size and population growth rate of freshwater fishes and their relationships with life history traits. Sci. Rep., 9: 3612. https://doi.org/10.1038/s41598-019-40340-z

Wu, L. and Yang, J., 2012. Identifications of captive and wild tilapia species existing in hawaii by mitochondrial DNA control region sequence. PLoS One, 7: e51731. https://doi.org/10.1371/journal.pone.0051731

Yousefian, M., Sharifrohani, M., Sahafi, H.H., Laloei, F. and Makhdoomi, C., 2011. Heritability estimation for growth-related traits in juvenile wild common carp (Cyprinus carpio L.) in the south of Caspian Sea. Iran. J. Fish. Sci., 10: 740–748.

Zhang, J. and Hanner, R., 2012. Molecular approach to the identification of fish in the south China Sea. PLoS One, 2: e30621. https://doi.org/10.1371/journal.pone.0030621

Zhao, K., Duan, Z., Peng, Z., Gan, X., Zhang, R., He, S. and Zhao, X., 2011. Phylogeography of the endemic Gymnocypris chilianensis (Cyprinidae): Sequential westward colonization followed by allopatric evolution in response to cyclical Pleistocene glaciations on the Tibetan Plateau. Mol. Phylo. Evol., 59: 303–310. https://doi.org/10.1016/j.ympev.2011.02.001

Zhu, W., Fu, J., Luo, M., Wang, L., Wang, P., Liu, Q. and Dong, Z., 2022. Genetic diversity and population structure of bighead carp (Hypophthalmichthys nobilis) from the middle and lower reaches of the Yangtze River revealed using microsatellite markers. Aqua. Rep., 27: 101377-101387. https://doi.org/10.1016/j.aqrep.2022.101377