Journal of Aquaculture Research & Development

Comparative Analysis of Genetic Diversity in Natural Population of Labeo rohita and Cirrhinus marigala by Using Species Specific Molecular Markers

Faisal Tasleem^1*, Shahid M¹, Hina Fatima² and Numan Gulzar M^{2 1}Department of Zoology, University of Agriculture, Faisalabad, Pakistan
²Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan
^*Correspondence: Faisal Tasleem, Department of Zoology, University of Agriculture, Faisalabad, Pakistan, Tel: +92-3039889132, Email:

Received: 23-Jul-2022, Manuscript No. JARD-22-17546; Editor assigned: 25-Jul-2022, Pre QC No. JARD-22-17546 (PQ); Reviewed: 08-Aug-2022, QC No. JARD-22-17546; Revised: 21-Sep-2022, Manuscript No. JARD-22-17546 (R); Published: 28-Sep-2022, DOI: 10.35248/2155-9546.22.13.718

Introduction

Fish is an excellent source of good quality meat, vitamins, poly unsaturated fatty acids and minerals. Fish meat is easily digestible. It is a renewable resource of income and nutrition for a country [1]. Fisheries sector provides jobs and investment opportunities to lot of concerned peoples [2]. Pakistan export fish and fish products to more than 50 countries especially European union countries, China, Malaysia, Japan, Singapore, Sri Lanka, Hong Kong, Saudi Arabia and S. Korea and earns 232.5 million Dollars annually [3]. More than 24,600 fish species are found in Pakistan [4].

Labeo rohita is known as rohu in Pakistan. In Latin, it is given the name of Labeo due to having large lips. It has arched head with silvered colored cyprinid shape body. It may gain 50 kg body weight with 2 meter body length. It is omnivorous and breeds during moon soon season [5].

Similarly, Cirrhinus marigala is commonly known as morakkha and naini in Pakistan. This is also member of Indian major carps which belongs to genus Cirrhinus. The body of this specie is somewhat compressed, oblong and large with round nose, broad mouth, sharp lower lips and fringed upper lips [6]. On the palate there are various fingers like projection.

Indus, Chenab, Jhelum, Ravi, Beas and Sutlej are more famous rivers of Pakistan providing superlative vegetative, nutritive and climatic zones to lot of fish species [7]. Instead of tremendous scope, potential and earning through fishery sector, studies on genetic improvements and managemental practices are not found in literature. This is the reason that this study was designed for the evaluation of genetic potential and genetic variations of two (Cirrhinus marigala and Labeo rohita) species.

Both these species (Labeo rohita and Cirrhinus marigla) are well known table fish of South East Asians countries especially in Pakistan, India and Bangladesh [8]. This is the reason that these species are commonly cultured in the private and public hatcheries of these countries [9]. Labeo rohita is one of the top ten fish species being cultured worldwide [10]. In order to meet the fish meat requirement of human population, we should enhance the breeding and rearing programs of these species. In Pakistan, about 99% seed of Labeo rohita is provided by public and private hatcheries [11].

There are many factors such as diseases, agricultural pollutants, overfishing, poor quality brooder seed, Dam’s construction, inbreeding, inappropriate management etc. which affects the growth and survival of fresh water species very badly [12]. Ii is necessary to overcome these factors for the highest yield of fish. By understanding the genetic makeup of fishes, provision of better guidelines to farmers and via better policies we can enhance the production of fishes. According to the conservation biologist, in altering environment, restocking and genetic variations are major factors for controlling the fish production [13].

Living organisms ensure there survivability by passing through various evolutionary process [14]. Genetic variation is one of the evolutionary steps which enhance the survivability of living things even in changing environments [15]. Population genetics is helpful for understanding the evolutionary process of fish [16].

Genetic study of any species can be conducted by using different types of genetic or molecular markers. Simple Sequence Repeat (SSR) are one of the molecular markers which are widely used in genetic study due to their multi-allelic nature, co-dominance, relative abundance, hyper variability, relative abundance, reproducibility, high throughput genotyping and fixed location on the chromosomes [17]. Due to having multiple allelic natures, SSR markers reveal exact genotypic variations between samples of even same specie [18]. On the basis of such properties, species specific SSR markers have been used in this study to check the extent of genetic variations among the individuals of Labeo rohita and Cirrhinus marigala.

Materials and Methods

Sampling of fish for DNA extraction

Ten samples of Cirrhinus marigla and ten samples of Labeo rohita were collected from three areas (Head Trimmu, Head Muhammad Wala and Head Punjnad) of river Chenab.

The detail of Cirrhinus marigala and Labeo rohita samples codes along with their collection sites have been summarized in Tables 1 and 2 respectively. After catching the fish samples from running water of river Chenab with the help of net, they were packed in zip lock polythene bags and transferred to the University Lab the on the same day where they were stored at -20˚C in the freezer.

Table 1: Sample codes of Cirrhinus marigala along their site of collections.

Table 2: Sample codes of Labeo rohita with their sites of collection.

DNA extraction

DNA from the dorsal muscle of each fish sample was extracted with slight modification in Natalia Bello, 2001 method. In this method a 2 cm-3 cm piece of meat was excised and chopped with surgical blade. After digestion of meat with the help of lyses buffer and proteinase K, precipitation of DNA by Phenol: Chloroform: Isoamylalcohol and purification of DNA by chilled ethanol and 2M NaCl solution was done. The quality of DNA was checked by gel electrophoresis under UV Trans illuminator and quantity of DNA was checked by spectrophotometer (Perkin Elmer Ltd. UK).

Polymerase Chain Reaction (PCR)

20 ul PCR reaction mixture containing 10.75 ul PCR water, 2 ul template DNA, 2.5 ul dNTPs, 2.5 ul 10X buffer, 1 ul forward, 1 ul reverse primer and 0.25 ul Taq DNA polymerase was used for the amplification used DNA with the help of Gene Amp system (Applied Biosystems). PCR profile was set as; 1 cycle of initial denaturation at 95°C for 5 minute, 35 cycles of final denaturation at 94°C for 1 minute, annealing for 1 minute at 55°C-60°C (varies from primer to primer), initial and final extention at 72°C for 2 and 7 minutes (Tables 3 and 4).

Table 3: SSR primers information for Labeo rohita.

Table 4: SSR primers information for Cirrhinus marigala.

In this study, five SSR markers for Labeo rohita and five for Cirrhinus marigala was used which were designed and purchased from the Humanizing Genomics Macrogen Company. The detail of each primer is given below [19].

Polyacrylamide Gel Electrophoresis (PAGE)

30% polyacrylamide gel containing 11.25 ml of 30% acrylamide, 400 ul APS, 26.25 ml 1X TBE buffer, 30 ul TEMED and 400 ul APS solution was used in horizontal PAGE apparatus (Cat#MGV-220-33 CBS scientific UK) for the separation and detection of PCR amplified products. This apparatus was given current of 100 A and 200 voltage for 80 minutes. Then the gel was passed through three steps (fixation, staining and developing) of silver staining and at the end it was seen under UV trans illuminator. Make the picture of visible bands, score and apply software for genetic analysis.

Software and analytical packages

An excel sheet was made on the basis of scoring which ultimately subjected to statistical analysis POPGENE version 1.32 and FSTAT version 2.9.3.2 for measurement of allele numbers, allelic frequency, Polymorphism Information Content (PIC) and gene diversity values for each primer [20,21]. Jaccard and Dice similarity coefficient and simple matching analysis was used for the calculation of similarity index and frequency based Genetic difference was calculated by using NTSYS_PC package statistical software [22,23]. Cluster of all the samples were made by using Un-weighted Pair Group Method with Arithmetic Mean (UPGMA) in SAHN program of NTSYS PC version 2.11 J Package.

Results

Genetic diversity among the Labeo rohita and Cirrhinus marigala samples of river Chenab was evaluated by using five simple sequence repeat markers for each species. The results of genetic parameters after applying statistical analysis on the scoring sheet are shown in Table 5.

Table 5: Values of basic genetic parameters of Labeo rohita samples.

Five simple sequence repeat markers (clone Lr22, Lr30, Lr32, Lr33 and Lr38) were used for Labeo rohita samples. Different markers show different values of genetic parameters. Values of gene diversity varies from 0.1800 (Lr30) to 0.8800 (Lr38) and 0.6360 as an average value. Allele number ranges from 4.000-9.000 with 5.200 as a mean value. Values of allelic frequency varies from 0.2000 (Lr38) to 0.9000 (Lr30) with 0.4600 average value. Polymorphism information content values are best indicators as these values are proportional to genetic diversity. PIC values ranges from 0.1638 (Lr30) to 0.8680 (Lr38). All these values indicates moderate to high genetic variation between the samples of Labeo rohita specie.

Phylogenetic tree of Labeo rohita samples

Phylogenetic tree represents evolutionary relationship among the samples. UPGMA a distance based algorithm was used for the construction of phylogenetic tree. The resultant dendrogram divided ten individuals of L. rohita into three clusters and three sub clusters as shown in Figure 1.

Figure 1: UPGMA Dendrogram based on Nei’s (1972) genetic distance among the samples of L. rohita.

Cluster 1 contains LHMW7 (Labeo rohita sample no.7 from Head Muhammad Wala) which shows pair wise genetic difference 0.600-0.900 from other samples. Sub cluster 1 consists of two samples of Head Trimmu. Sub cluster 2 consists of one sample of Head Trimmu while other of Head Punjnad. Sub cluster 3 consists of LHMW6 and LHPND4. Cluster 3 consists of LHMW5 and LHPND3. Pair wise genetic similarity between these samples is 0.6000. LHMW5 is sample of Head Muhammad Wala and LHPND4 is sample of Head PUNJNAD. Despite of geographical distribution, both samples falls in the same cluster.

LHPND 1-4 (Labeo rohita collected from Head Punjnad), LHMW 5-7 (Labeo rohita from Head Muhammad Wala, LHT 8-10 (Labeo rohita from Head Trimmu) (Table 6).

Table 6: Values of basic parameters of genetic diversity.

The values of genetic parameters in case of Cirrhinus marigala are different from those of Labeo rohita samples. All these parameters also show moderate to high genetic variations among the samples of Cirrhinus marigala.

Phylogenetic tree

A dendrogam based on UPGMA divided ten samples into three clusters and two sub clusters as shown in Figure 2. Cluster 1 consists of only sample no.9 (CHT9) which was collected from Head Trimmu. Cluster 2 consists of seven individuals (CHPND4, CHT10, CHMW7, CHMW6, CHPND1, CHT8 and CHPND2). Further it is divided into two sub clusters (sub cluster 1 and sub cluster 2). Sub cluster 1 consists of two samples. One sample (CHPND4) which was collected from Head Punjnad and second sample (CHT10) was collected from Head Trimmu. Sub cluster 2 is consists of CHMW6 and CHMW7 samples. Both these samples were collected from Head Muhammad Wala and show genetic difference (0.4000). The remaining individuals of cluster 2 are CHPND1, CHPND2 and CHT8. CHPND1 and CHPND2 were collected from Head Punjnad and CHT8 was collected from Head Trimmu. Cluster 3 consists of only two samples (CHPND3 and CHMW5) as shown in Figure 2.

Figure 2: Dendrogram (Cirrhinus marigala).