**Abstract**

Freshwater fish stocks are being exposed to increasing threats as a result of fisheries and aquaculture practices. Integrating genetic knowledge into fisheries and aquaculture management is becoming increasingly important in order to ensure the sustainability of species. So, I used SSR markers to evaluate the pattern of genetic variability in Silver Carp populations (175 samples) from five different sites of River Chenab, Pakistan. DNA was isolated and processed for analysis. There were no scoring errors related to large allele, no stuttering bands, and no null allele. The mean values of number of alleles, allelic richness, effective number of alleles, observed (Ho) and expected (He) heterozygosites, 1-Ho/He, inbreeding coefficient, pairwise population differentiation, and the gene flow provided data indicating loss of genetic diversity of silver carp in River Chenab (Pakistan). Reasons are overhunting, pollution, inbreeding, and poor control measures.

**Keywords:** microsatelites markers, silver carp genome, genomic analysis

#### **1. Introduction**

Fishes are the most diverse group of organisms. They are facing altered environmental conditions resulting from human activities. Freshwater fish biodiversity is progressively threatened by overexploitation, pollution, habitat loss, introduction of non-native species, and the climate change [1]. Global climate change is causing ocean acidification and rising aquatic temperatures, and it is expected to cause regional changes in salinity, dissolved oxygen supply, and circulation patterns in aquatic environments, which fishes will eventually have to cope with [2].

Aquaculture has been a key contributor to increasing food production for human nutrition and food security. In 2016, global per capita fish consumption was 20.3 kg, with fish accounting for 20% of animal protein intake for over 3.2 billion people around the world [3, 4]. Aquaculture in Asia provides for over 91% of global production, but it will need to continue to increase to fulfill the demands of a fast-growing human population [5]. In 2017, a total of 53.4 million tonnes of fish were produced. Freshwater fish species produced 83.6% of total fish production. Freshwater carps and cyprinids account for

over 53.1% of total fish production in the aquaculture industry [6]. One of the most important freshwater fish species in aquaculture is silver carp, *Hypophthalmichtys molitrix*. Silver Carp have been brought into many nations across the world for biological control (algal blooms) and aquaculture purposes. It reproduces naturally in few sites in the ecosystem, which are comparable to the original environment [7].

The conservation and management of aquatic resources are critical for the prolong usage of fisheries potential for the economic growth of farmers and fishery workers today and in the future. Fisheries and aquaculture are playing a significant role in social development by providing nutritional security for the human population and contributing to the economic improvement of farmers and fishery employees. The fishing sector also contributes foreign exchange profits, amounting to several millions of dollars. Furthermore, aquatic resources are proving to be an essential source of a variety of items with pharmacological and economic significance [8].

As natural environment provides resources for all living communities, so it is essential to protect the natural environment for the conservation and preservation of living species. Unfortunately, human activities are constantly altering aquatic ecosystems around the world [9]. This change has negative impact on fish community structures as well as in other aquatic animals and may be responsible for the extinction of many species.

Genetic diversity evaluates alternate types of genes or noncoding loci within population diversity. The evolution and adaptation of a population are linked to both heterozygosity and the total alleles present within a population. Populations facing stressful environmental conditions have reduced genetic diversity, declined population viability and high extinction likelihood [10].

Both extinction and the speciation have been an indispensable part of life since past. Present extinction rate is very high as compared with historical background. Chemical contamination of the environment has resulted in decline of many populations. Certain environmental toxicants cause reproductive destruction in wildlife. Conservation biology mainly focuses on the conservation of genetic diversity. Chemical contamination causes somatic and germline mutations, which reduce genetic diversity of populations. Chemical contamination damage is at the molecular level as well as population level, which results in loss of genetic diversity [11–13].

One of the most necessary aspects for the preservation and conservation of living species is the protection of the natural environment. This natural environment provides perfect conditions for all living communities. Human activities, unfortunately, continue to alter aquatic ecosystems all over the world. This change is thought to have a huge effect on fish community structures and other aquatic organisms, and it could lead to the extinction of a lot of species [9]. Humans are attempting to change approximately every environment at an unprecedented rate, and they may now be the most important biotic selective power on the planet [14]. The introduction of species outside of their historical ranges has also some problems along with benefits. Furthermore, anthropogenic disturbances can result in the creation of new environments that are beneficial to exotic species [15].

Commercially, about 40% of fisheries have collapsed or at verge of extinction. The cause for this is a lack of understanding about the fitness of genetic diversity. The majority of breeding programs do not sustain genetic adaptations [16]. Artificial breeding programs should be employed when populations are in danger of extinction. These programs are often employed to improve wild populations in fisheries management. These strategies may have raised stock sizes while also preserving genetic variability, lowering the risk of local extinction [17].

#### *Genetic Assessment of Silver Carp Populations in River Chenab (Pakistan) as Revealed… DOI: http://dx.doi.org/10.5772/intechopen.108288*

Any measure that quantifies the magnitude of genetic variability within a population is termed as genetic diversity. It provides the insight for evolution by natural selection and influence fitness of the ecosystem as well as affects the growth, productivity, constancy, and inter-specific and intra-specific interactions. Knowledge about variation in either discrete allelic states or phenotypic features can be used in genetic diversity. Variation in phenotype or genotype (allelic states) might be neutral or nonneutral regarding fitness consequences. Molecular markers, for example, microsatellites, straight DNA sequences, AFLPs, or protein polymorphisms often indicate discrete allelic states that are thought to be neutral [18].

Microsatellites are extremely popular in population analysis because of their selective neutrality and the ease with which microsatellite-based data can be replicated and compared in other populations [19]. Ecological and evolutionary similarity drivers affect diversity in communities and populations. Random changes in composition are linked to community drift and genetic drift, migration and gene flow, individuals and species, and selection and coexistence processes (e.g., competition, predation). All these parameters non-randomly influence on allelic or species composition. Speciation and mutation also contribute to genetic and species diversity, but they are generally weak forces that impact composition only over extended periods of time [20].

The degradation of environmental in aquatic ecosystems is increasing, which can result in declines in diversity, reflecting population size reductions and the extinction of intolerant species [21].

Molecular markers are applied to analyze genetic variability in a variety of fish species, and they are significant tool for analyzing patterns of genetic diversity. Microsatellite DNA markers are the most informative and polymorphic markers that can be used to evaluate genetic variability at the molecular level [22]. Microsatellites are the marker of choice for genetic, evolutionary, and ecological research studies because of their high mutation rates, high level of polymorphism, great number, and even distribution across the genome, co-dominance, and ease of analysis using PCR. These microsatellites are also used to determine the genetic variability and structure of farmed food fish species [23].

Different kinds of polymorphic markers have been utilized to analyze genetic diversity such as protein based markers (i.e. allozymes) and DNA-based markers such as microsatellites, amplified fragment length polymorphisms (AFLPs), restriction fragment length polymorphisms (RFLPs), random amplification of polymorphic DNA (RAPD), and mitochondrial DNA (mtDNA). Microsatellites markers have been most frequently utilized in the analysis of the carp genetic diversity. The extreme success of microsatellites in the population analysis comes directly from the selective neutrality of these markers and effective replication and validity of microsatellite-based data in different populations. Microsatellites are especially helpful for the assessment of genetic biodiversity. Microsatellites, also known as "simple sequence repeats," possess 2–9 bp that are widely distributed across the genome and have a high degree of polymorphism. The majority of microsatellite loci are short and easy to amplify using PCR. SSR markers are used to construct a genetic fingerprint and to demonstrate connections between individuals. SSR markers are widely employed in fish population genetics and conservation research studies [24].

### **2. Review of literature**

David et al. [25] studied 47 microsatellite markers in carp species, *Cyprinus carpio* and *Ctenopharyngodon idella*, and observed polymorphism by applying the AFLP

(Amplified Fragment Length Polymorphism). The average number of allele was found 4.02 and mostly SSRs contain CA and CT motifs. The calculated fixation index (FST) for microsatellites and AFLP markers was 0.37 and 0.39. About half of the SSRs markers were used to genotype the grass carp. Their results indicated that grass carp is phylogenetically distinct from other populations.

The researchers [26] explored the population structure and genetic variability of two Hungarian common carp farms (80 and 196 individuals) by synthesizing primers to the flanking regions of eight microsatellites: MFW4, MFW7, MFW9, MFW13, MFW17, MFW20, MFW26, and MFW31. Samples were chosen at random from Attala, Dinnyes, Boszormeny, Bikal, Szajol, wild-Danube, and wild-Tisza. They detected 47 alleles in these groups. All these groups had similar allele frequencies, with the exception of wild carps. Private allele frequency was extremely low, with a value 0.003 to maximum of 0.027. At the Attala and the Dinnyes stock, the average *He* was 0.83 and 0.81, respectively, while *Ho* was 0.69 for both stocks. Most loci of these two populations were in disequilibrium when tested for HWE. These findings could aid in the identification of wild carp taxonomic status and genetic variability as well as their relatedness to domesticated stocks.

The researchers [27] analyzed 54 primers for amplification of microsatellite loci in 84 samples of kali rohu, *Labeo dyocheilus* (Family Cyprinidae) from four rivers, namely Satluj, Jiabharali, Beas, and Yamuna. Successful amplification was observed in 15 primers pairs. Seven microsatellites, MFW1, MFW2, MFW9, MFW15, MFW17, R-12F, and Ca12, were polymorphic having three to nine alleles. *Ho* values ranged from 0.34 to maximum 0.53. Mean number of alleles was 3.42–4.71. There was found no significant deviation from HWE in allele frequencies except at locus Ca12 in the sample of Jiabharali. The reason may be the presence of null allele at locus Ca12, which was not amplified. Rest of the alleles showed highly nominate heterogeneity in all the sample sets. The identified microsatellite loci could be used in fine-scale population structure analysis of *L. dyocheilus*.

Li et al. [28] analyzed six wild populations of Common Carp (*C. carpio* L.) using 30 microsatellite loci. Different types of parameters for genetic diversity such as number of effective alleles (*Ae*), polymorphic information content (PIC), expected heterozygosity (*He*) and observed hererozygosity (*Ho*), genetic distance, and genetic similarity index were detected. There were present total 210 alleles in these six populations and 3–13 alleles were amplified in 30 loci. In each locus, the average number of alleles was seven. These six wild common carp showed high population variation. Effective alleles were ranging from 1.04 to maximum 4.72. The result indicated low-to-moderate level of genetic variability in these populations. PIC values of these *C. carpio* populations were 0.45, 0.51, 0.52, 0.56, 0.62, and 0.63, respectively. The average values of *He* were 0.51, 0.60, 0.57, 0.57, 0.58, and 0.55 respectively. On average, the number of effective alleles (*Nae*), observed hererozygosity (*Ho*), expected heterozygosity (*He*), and PIC were 2.71, 0.57, 0.58, and 0.48 respectively. Clustering result and the geographical distribution were in correlation with each other.

Wang et al. [29] developed SSR markers for common carp (*C. carpio*). Total 32 samples of common carp were collected from Dongting Lake in China. Most of the SSRs of common carp were found to consist of dinucleotide (AC/TG, AG/TC, and CG/GC) and trinucleotide (AAT and ATC) repeats. Polymorphism was observed in only 25 loci out of 60 SSRs in the common carp population under examination. The number of alleles/locus varied from three to seventeen. The value of *Ho* and *He* ranged 0.13–1.00 and 0.12–0.91, respectively. Six SSRs did not follow Hardy-Weinberg equilibrium (HWE), while the remaining 19 loci did. Mutation rates in gene-coding

#### *Genetic Assessment of Silver Carp Populations in River Chenab (Pakistan) as Revealed… DOI: http://dx.doi.org/10.5772/intechopen.108288*

sequences are lower than in non-coding genomic sequences due to evolutionary conservation. Furthermore, polymorphism was not totally determined by repeat length.

Zhu et al. [30] analyzed the genetic variability of five Silver Carp populations in the middle and lower reaches of the Yangtze River. For this purpose, 30 SSR markers were utilized and total 144 alleles were found with 1–10 alleles in each locus (average 4.0 to 4.1). In total, 83.33% (25 loci) were polymorphic. The average values of observed hererozygosity (*Ho*) and expected heterozygosity (*He*) ranged from 0.3233 to maximum 0.3511 and 0.4421 to maximum 0.4704, respectively, and the average PIC value ranged from 0.4068 to 0.4286. These populations were moderately differentiated and partially deviated from HWE as revealed by Fst value and Chi-square test, respectively. The genetic distance and genetic similarity coefficient values were from 0.0893 to 0.1665 and from 0.8466 to 0.9146, respectively.

Cheng et al. [31] evaluated genetic structure of bighead carp (*Aristichthys nobilis*) by using microsatellite markers and documented their cross-species amplification in Silver Carp (*Hypophthalmichthys molitrix*). For this purpose, 30 individuals of each species were collected from Zhangdu Lake of the Yangtze River, China. Forty-two pairs of primer development were synthesized from which only 30 produced desired products. Polymorphism was seen in 16 loci having 2–7 alleles/locus with an average value of 3.263 and the *Ho* was 0.100–0.690 with an average value of 0.392. By applying the same PCR conditions in the cross-species amplifications, it was observed that 11 out of 16 microsatellites of bighead carp fish were also polymorphic in Silver Carp fish.

Wang et al. [32] analyzed the genetic viability of two Silver Carp populations by applying 39 microsatellite markers. The samples were collected from the middle and upper reaches of the Yangtze River, China. There were in total 260 alleles. The averages of number of alleles were 6.130 and 4.980, and averages of effective number of alleles (*Nae*) were 4.108 and 3.385 among the Wanzhou population and Jianli populations, respectively. The polymorphic informatics content varied between 0.077 and 0.865 (average 0.617). The average Ho and He were 0.834 and 0.775 and 0.713 and 0.623, respectively, for studied populations. There was a clear genetic differentiation between these two populations.

Li et al. [33] studied that SSR markers are significant DNA markers, which are accessible to figure out population structure. The Grass Carp populations were analyzed for genetic variability and genetic structure by using 45 polymorphic microsatellite loci. Different types of parameters for genetic diversity were measured such as number of alleles/locus (*Na*), effective number of allele/loci (*Ne*), expected heterozygosity (*He*), and observed hererozygosity (*Ho*). The values of the parameter were found to be: number of alleles/locus was 7.26, effective number of allele/loci was 4.21, *Ho* was 0.73, and *He* was 0.68. It was found that population genetic diversity is significantly affected by loci number, sample size, and polymorphism information of microsatellite markers.

Alam et al. [34] determined the genetic structure and genetic diversity of Indian major carp, *Labeo rohite* sampled from River Halda, River Jamuna, and River Padma in Bangladesh. They analyzed four polymorphic microsatellite loci. On average, there were 2.75–3.75 alleles with size ranging from 144 bp to 190 bp. The populations differed in terms of the frequency and the number of alleles, as well as observed (*Ho*) and expected heterozygosity (*He*) in the loci studied. A significant (P < 0.05) population differentiation (FST) was found between the Halda and the Jamuna population. Between the Padma and Jamuna populations, there was relatively high gene flow. The findings demonstrated a rather low amount of genetic diversity in L rohita populations in Bangladesh. The alleles Lr3 and Lr21 were present only in population of River Jamuna with the frequency of 0.04 and 0.02, respectively, so termed as private alleles for the Jamuna population. There was no allelic drop-out in the Jamuna population while highest allelic drop-out was observed in the Halda population. The values of *Ho* and *He* were ranging from 0.17 to 0.65 and from 0.24 to 0.62, respectively. Two clusters were delivered by UPGMA dendrogram. The Halda population was in one cluster, and Jamuna and Padma populations were in the other cluster.

Abbas et al. [35] utilized total nine polymorphic microsatellite markers to determine the genetic diversity and population structure of five populations of Yellowcheek carp (*Elopichthys bambusa*) in the Yangtze River basin in China. There was observed low-to-moderate genetic diversity. The number of alleles/locus varied from three to maximum eight with an average value of 4.6 alleles/locus. The values of *Ho* were 0.15–1.00. All these loci represented significant deviations (P < 0.01) from HWE. There occurred loss of heterozygosity within populations as indicated by the values of inbreeding coefficient. Lower but significant value (P < 0.01) of FST indicated genetic divergence between populations of *E. bambusa*. About 93.81% variance was within populations, and 7.05% of the total variance was among the populations as shown by AMOVA results. Mantel tests provided no evidence for an increase of the genetic differences with geographic distance. Due to anthropogenic interventions, the populations are reproductively isolated as revealed by UPGMA dendrogram. These findings could contribute to the effective management and prolonged conservation of *E. bambusa* populations.

Hulak et al. [36] studied the population structure and parameters of genetic diversity of 11 Carp populations in the Czech Republic. Mean heterozygosity was 0.584– 0.700. Mean number of alleles was 5.0–9.8. It was found significant heterozygote deficit. Most of tests of the analyzed loci were deviated significantly (P < 0.05) from the Hardy-Weinberg equilibrium. Inter-population genetic variation was 21%, while intra-population genetic variation was 79% as revealed by AMOVA. Mostly, interpopulation genetic was responsible for microsatellite loci variations.

Adams et al. [37] collected 56 samples of the Grass Carp (*C. idella*) population to study the genetic structure from Missouri and Mississippi River basins, USA. The numbers of alleles/locus were 2–8 across all the polymorphic microsatellite markers. Locus Ci04 did not produced any amplicons for all the collected samples. Average allelic diversity was low along basins of these rivers and highest in the upstream reach of the Missouri River and the Mississippi River. There was found no significant differences in levels of inbreeding between these populations. Fourteen out of 16 loci produced least values of both observed and expected heterozygosity. A significant bottleneck phenomenon was observed along the basins of both the rivers.

Sahu et al. [38] studied Raho (*Labeo rohita)* from normalized cDNA libraries to investigate genetic structure and genetic diversity*.* They assembled 3631 unique sequences (709 contigs and 2922 singletons) from 6161 random clones sequences. In total, 182 unique sequences out of 3631 unique sequences (709 contigs and 2922 singletons) were found to be associated with reproduction-related gene. Polymorphism was seen in 20 loci in 36 unrelated individuals, and their allele frequency ranged from 2 to 7 per locus. From these 20 polymorphic loci, 14 loci deviated from HWE (p < 0.05). In 3631 unique sequences, AG repeats were most frequent motif. The values of expected heterozygosity *H*<sup>e</sup> and observed heterozygosity *H*<sup>o</sup> ranged from 0.109 to 0.801 and from 0.096 to 0.774, respectively. These microsatellite loci did not show any linkage disequilibrium.

*Genetic Assessment of Silver Carp Populations in River Chenab (Pakistan) as Revealed… DOI: http://dx.doi.org/10.5772/intechopen.108288*

Sahoo et al. [39] evaluated the genetic variation of *L. rohita*. Eleven microsatellites loci were used to assess the genetic diversity of three wild and one farm population of rohu. For this purpose, total 192 samples were analyzed, and the number of alleles were found to be four to maximum 23, observed heterozygosity 0.500–0.870, and expected heterozygosity from 0.389 to 0.878. At least, one locus was not in HWE in these riverine samples. Negative values of inbreeding coefficients (FIS) indicated little genetic differentiation but very high level of genetic diversity among populations. Micro-Checker did not revealed any null alleles. There was reported minimum scoring error. FST values ranged from 0.005 to 0.043. All of the rohu populations had a high allelic richness and were genetically diverse.

Tibihika et al. [40] described the genetic structure of East African Nile tilapia (*Oreochromis niloticus*) by using SSR-GBS technique. For this purpose, 2,403,293 paired reads were produced for primer design containing 6724 SSR motifs, from which 35 SSRs were developed, and only 26 produced amplified products. Fis values deviated from HWE for all 26 loci. Most loci were polymorphic and four loci deviated from HWE. PIC values for 18 loci were above 0.5 showing that they were highly informative markers and for remaining four loci, PIC values were between 0.25 and 0.5 indicating slightly informative markers.

Fang et al. [41] developed 12 SSRs for cross-species amplification in silver carp (*H. molitrix*) and bighead carp (*Hypophthalmichthys nobilis)*. The values of number of alleles (*Na*), the observed heterozygosity (*Ho*) and expected heterozygosity (*He*), and the polymorphic information content (PIC) vary from 5 to 20, 0.189–0.956, 0.177– 901, and 0.169–0.887, respectively. All these loci were polymorphic. Genetic structure was similar for population of same species and obvious genetic differentiations was present between populations from different species. Private alleles were ranging from 1 to 6 in *H. nobilis* and 3–10 in *H. molitrix* individuals.

Zhou et al. [42] studied that there are rare population genetic studies for Black carp (*Mylopharyngodon piceus*) in the Yangtze River basin and developed 31 novel microsatellite markers. Ten microsatellite markers were used to access the genetic variability of Black Carp. Mean number of alleles (*Na*) was 14, and number of effective alleles (*Ae*) was 6. Their study explained that wild populations had higher genetic diversities than cultured populations. The values of *Ho*, *He,* and PIC parameters for genetic diversity of wild populations were 0.767, 0.806, and 0.767, respectively. The values of *Ho*, *He,* and PIC parameters for genetic diversity of cultured populations were 0.730, 0.722, and 0.6731, respectively. Founder effects may be one of the most probable causes of the reduction of genetic variation in cultured populations.

Fang et al. [22] evaluated the genetic status of eight populations of Silver Carp in Jiangsu province and four populations of Silver Carp from Lower Reaches of the Yangtze River (LRYR) in China by using microsatellite loci. High polymorphism was observed between all the loci. There were in total 3–33 alleles/locus with mean value ranging from 5.727 to 14.818. The range of effective number of alleles per locus was 4.19 to maximum value 6.526. The average observed heterozygosity (*Ho*) ranged from 0.625 to 0.727, and the average expected heterozygosity (*He*) ranged from 0.69 to 0.784. Gene flow values (*Nm*) were high across all populations, ranging from 3.496 to 79.845. With AMOVA analysis, the majority of differentiation variations (95.85%) were assigned within populations, with only 4.15% existing between populations. Most populations were potentially threatened by inbreeding depression. Fst values ranged from 0.003 to 0.067, and all groups exhibited moderate genetic difference.
