*4.7.1 Genetic differentiation (*FST*) and geographical distance (KM)*

The pairwise population differentiation (*FST*) across all screened microsatellite loci among the *H. molitrix* populations was assessed using the windows-based software FSTAT. For the majority of the population pairs in this investigation, *FST* was statistically significant (p < 0.05) revealing genetically nonhomogenous groups. For the majority of the stocks in this investigation, the noteworthy findings relating

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

#### **Figure 8.**

*Comparative distribution of inbreeding coefficient (FIS) at different SSR loci in* H. molitrix *populations.*

population genetic differentiation revealed genetically nonhomogeneous groups (**Table 11**). A moderate level of *FST* was indicated by the pairwise estimates of *FST.* Highest level of *FST* was found 0.1033 in the population-pair of TH-KH, while the least 0.0120 between the population-pair of MH-QH in this study (**Table 11**, **Figure 9**).

**Table 12** depicts the pairwise geographical distance (KM) between H. molitrix populations. The population pair MH-TH had the greatest geographical distance, while the population pair KH-QH had the smallest geographical distance. **Figure 9** depicts a graphical comparison of population genetic differentiation and geographical distance among the various *H. molitrix* populations studied. The graph demonstrated a direct relationship between geographic distance and population genetic differentiation. The genetic differentiation in populations was observed to increase as geographical distance increased.

#### *4.7.2 Genetic distance (GD) and genetic identity (GI)*

The genetic distance (GD) and genetic identity (GI) were calculated using the windows-based software TFPGA, based on allele frequency data from all of the


#### **Table 11.**

*Pairwise population differentiation (*FST*) between natural populations of* H. molitrix.

#### **Figure 9.**

*Correlation of geographic distance (GD) and population genetic differentiation (FST) values among* H. molitrix *populations.*


#### **Table 12.**

*Pairwise geographical distance between natural populations of* H. molitrix.


#### **Table 13.**

*Pairwise Nei's unbiased genetic distance (*GD*) between populations of* H. molitrix.

studied populations. Only two population pairs yielded statistically significant results (P < 0.05). The highest GD was 0.2943 between the TH-KH population pairs, while the lowest was 0.0289 between the MH-QH population pairs (**Table 13**). Similarly, the population pair QH-MH had the highest GI value of 0. 9715, while the population pair TH-KH had the lowest GI value of 0.7451(**Table 14**).

**Figures 10** and **11,** respectively, show a graphical comparison of geographical distance versus genetic identity and genetic distance versus genetic identity in the *Genetic Assessment of Silver Carp Populations in River Chenab (Pakistan) as Revealed… DOI: http://dx.doi.org/10.5772/intechopen.108288*


#### **Table 14.**

*Pairwise Nei's unbiased genetic identity (*GI*) between natural populations of* H. molitrix.

**Figure 10.**

*Correlation of Geographic distance (KM) and genetic distance (GD) values among* H. molitrix *populations.*

#### **Figure 11.** *Correlation of Geographic distance (KM) and genetic identity (GI) values among* H. molitrix *populations.*

various *Hypophthalmichthys molitrix* populations. These graph revealed a negative relationship between genetic distance and genetic identity, as well as a negative relationship between genetic identity and geographical distance. The genetic identity of populations was seen to decrease as the genetic distance between them increased (**Figure 12**).

#### *4.7.3 Analysis of molecular variance (AMOVA)*

The AMOVA revealed a low variation percentage (7.81141%) between individuals within populations while the majority of variations (87.05210%) were occurring within individuals and 5.13648% variations among populations of *H. molitrix* in present study (**Table 15**).

#### *4.7.4 Gene flow (Nm)*

The gene flow (*Nm*) rate in various *H. molitrix* populations across all screened microsatellite loci was measured by using the windows-based program Popgene. The highest value of *Nm* (17.4152) was found at locus BL 14, while the lowest value (2.4769) was found at locus BL 108. BL 8–1, BL 52, and BL 123 were the remaining screened loci in this study, with *Nm* values of 3.4775, 5.5691, and 4.4293, respectively. Nm was found to be 4.5654 on average across all SSR loci (**Table 16**).

**Figure 12.**

*Correlation of genetic distance (GD) and genetic identity (GI) values among* H. molitrix *populations.*


**Table 15.**

*Analysis of molecular variance (AMOVA) for natural populations of* H. molitrix.

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


#### **Table 16.**

*Gene flow (*Nm*) for natural populations of* H. molitrix.

#### *4.7.5 Clustering patterns*

The UPGMA dendrogram was used to investigate genetic relatedness. There were two major clusters, or clades A and B, predicting the close relationship between these populations. Cluster A is divided into two sub-clusters: A1 and A2. Cluster A1 contained the riverine population of *H. molitrix* collected from KH, QH, and MH, while cluster A2 contained the population of CB. In cluster B, there was a population of TH (**Figure 13**).

For the populations of *H. molitrix*, microsatellite data analyses by the STRUCTURE grouping algorithm method proposed the presence of two distinct genetic clusters. For each K value, constant results were obtained across the six autonomous runs. STRUCTURE HARVESTER admixture model inferences showed highest estimated log-likelihood mean value and delta-k value in this study. The two distinct colors of the column represent the estimated probability of belonging to two populations, and each vertical column represents one individual. Distinct colors in the same individual indicate the percentage of the genome shared with each cluster (**Figure 14**).

#### **Figure 13.**

*UPGMA dendograms based on Nei's genetic distance showing the relationship and clustering patterns between natural populations of* H. molitrix.

#### **Figure 14.**

*Genetic structure patterns among populations of* H. molitrix *as revealed by structure analysis. The two distinct colors of column represent the estimated probability of belonging to two populations and each vertical column represents one individual. Distinct colors in the same individual indicate the percentage of the genome shared with each cluster.*

#### **5. Discussion**

Fish that have been exposed to altered environmental conditions as a result of human activities. Overfishing, pollution, loss of habitat, climate change, and the introduction of nonnative species are all threatening freshwater fish biodiversity [1]. Aquaculture has played a significant role in increasing food production for human nutrition. Asia accounts for more than 91% of global aquaculture production [5]. Freshwater carps and cyprinids account for over 53.1% of total aquaculture fish production [6]. Many countries around the world have introduced silver carp (freshwater fish species) for biological control (algal blooms) and aquaculture [52].

Because the natural environment provides resources for all living communities, protecting the natural environment is critical for the conservation and preservation of living species. In comparison to historical data, the current rate of extinction of species is extremely high. Approximately 40% of commercial fisheries are on the verge of collapse due to lack of understanding of the genetic diversity [17]. Many populations have declined as a result of environmental contamination. The field of conservation biology is primarily concerned with the preservation of genetic diversity [11]. Humans are attempting to alter nearly every environment at an unprecedented rate, and they may now be the planet's most powerful biotic selector [15]. The degradation of the environment in aquatic ecosystems is increasing, which could lead to a loss of diversity, as population sizes shrink and intolerant species become extinct [53].

We used microsatellite markers to examine the genetic variability of *H. molitrix* and evaluate the genetic structure of the populations in the region in order to offer good genetic data for effective management and conservation of the species. Because of the growing interest in and attention on Silver Carp culture in Pakistan, it is critical to study several genetic features of the fish. Genetic variety is essential for adapting to environmental changes and stock improvement initiatives. More heterozygous individuals are superior then less heterozygous individuals. All of the genetic criteria used in this investigation suggested that the wild population had higher levels of genetic

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

diversity. The wild fish populations had the largest average number of alleles (*Na*), allelic richness (*Ar*), and effective number of alleles (*Nae*).

Genetic drift, natural selection, mutation, and gene flow are all factors that influence the allele frequency in a population. Allelic diversity (*Na*) and heterozygosity (*Ho* and *He*) are important for genetic variation, although *Na* is significantly more dependent on effective population size than heterozygosity [50]. As a result, *Na* is suitable for estimating genetic diversity in a population for selection, conservation, and enhancement programs [54].

In present work, the average number of alleles (*Na*) and allelic richness (*Ar*) in *H. molitrix* populations were measured as 5.60–9.00 and 5.51–5.94, respectively. The average values for an effective number of alleles (*Nae*) assessed varied from 2.9043 to maximum 4.7284. The highest value of *Ne* (6.7680) was found in a TH fish population, while the lowest (1.9647) was found in a KH population. The value of *Ne* was found to be lower than the *Na*, indicating that alleles are being lost in the populations, and it means that the frequencies of all alleles are not equal. The researchers [28] reported seven alleles in each locus on average and 1.04–4.72 average number effective alleles. The researchers [55] reported average number of allele as 9.2 at microsatellite loci in freshwater Silver Carp species. The results of this study were reinforced by the findings of Fang *et al*. (2021), who reported average number of alleles ranging from 4.19 to 6.526 in Silver Carp.

In all of the *H. molitrix* populations studied, the heterozygosity level ranged from moderate to high. In the populations, average values of observed heterozygosity (*Ho*) ranged from 0.4400 to 0.7257. The fish population from TH had the greatest *Ho* value, whereas the population from QH had the lowest, which may be due to small number of individuals, limited gene flow, and errors in reading alleles. The expected heterozygosity (*He*) average values in populations of *H. molitrix* ranged from 0.6361 to 0.7812. The fish population from TH had the greatest *He* value, whereas the population from KH had the lowest. Similarly, increased values of *He* can be attributable to the existence of null alleles at the loci studied, selection pressure on specific loci, or inbreeding when compared with *Ho* [56, 57]. 1-Ho/He averages were 0.0740, 0.0905, 0.0306, 0.7005, and 0.0452. According to [22], mean *Ho* was between 0.625 and 0.727, and mean *He* was between 0.69 and 0.784 for Silver Carp. [55] found *Ho* and *He* values for Silver Carp ranging from 0.37 to 1.00 (average 0.74) and from 0.40 to 0.93 (average 0.76), respectively.

On an average, FIS values were ranging from 0.067 to 0.365 in several *H. molitrix* populations examined in this study. The highest average FIS value was found in the QH fish stock, while the lowest was found in the CB population. The value of FIS ranged between 0 and 1. Zero value indicates that there is occurring neither inbreeding nor outbreeding, which means that the population is in Hardy-Weinberg expectation or mate randomly. If FIS value is 1, it indicates the population is totally inbreeding and 1 shows the population is totally outbreeding. A negative FIS value indicated heterozygosity excess and suggested that this group does not lose heterozygosity, implying that individuals in this population are outbreed. Positive FIS readings indicate a population's homozygosity excess and significant divergence from the HWE [35].

Inbreeding, genetic drift, bottleneck effect, innate gene pool contamination by introgression, overexploitation, bio-invasion (introduction of exotic species), environmental pollution, habitat degradation, hydrological manipulations, and climate change are all factors that can cause a fish species' genetic structure to change over time. The strength of natural and human involvements determines the pattern and severity of changes. Microsatellite markers have a high resolving power, allowing them to identify very low amounts of genetic alteration caused by the different variables [58, 59].

Geographic distance separating populations has likely attracted the most attention from an environmental perspective [2]. In this work, pairwise FST estimations revealed intermediate genetic differentiation in wild populations of *H. molitrix*. A low level of genetic differentiation is indicated by an FST value of 0–0.05. If FST is 0, it means there is no differentiation or structure; if it is 1, it means fully differentiated. The lower FST value suggested that populations were of similar genetic origin and had reduced genetic integrity. Second, its possible that it is owing to the exchange of brooders between different populations. The maximum level of genetic divergence showed that these groups were of divergent genetic origin, whereas the lowest level indicated that they were of close genetic origin [47]. Hypothetically, if *Nm* is less than 1, genetic drift is assumed to be the most important mechanism in genetic differentiation. Similarly, if *Nm* is greater than 1, gene flow is the most important component in genetic differentiation [60].

The unbiased genetic distance between pairs of populations showed a lot of variance in magnitude. The values of genetic identity were shown to be contradictory to those of genetic distance. A large genetic distance indicates that both populations have a dissimilar genetic background and vice versa [61].

AMOVA is an appropriate benchmark for assessing population genetic structure and determining genetic similarity and differentiation between populations [51]. The AMOVA revealed that the majority of variation in wild populations of *H. molitrix* occurs within individuals. Clustering patterns in populations represent relationships. The genetic relationship between populations with the highest levels of genetic identity will be the closest, while those with the lowest levels of genetic identity will have the furthest genetic relationship. The UPGMA dendrogram was used to study the genetic structural patterns among populations. A close genetic link had been discovered among groups in the same cluster.

Biodiversity conservation has become increasingly important in recent years. As the human population grows, habitat loss is causing numerous animal populations to decline and potentially become extinct. Genetic variety is essential for a species' evolutionary survival. Genetic diversity levels can be maximized through effective management. Genetic monitoring programs for a fish population are necessary for an effective management approach. Molecular markers are effective tools for assessing and evaluating the genetic status of species. These markers can be used to manage pure stocks in the natural environment and to assist in the genetic conservation *of H. molitrix* species. The present data are important for considering its management and conservation. However, because there are wide regions of floodplains and river branches to design a good management policy, more research involving genetic analysis with more markers and population samples covering different wild sources throughout Pakistan is still needed.

#### **6. Summary**

The objective of the present research work entitled "Genetic assessment Silver Carp (*H. molitrix)* population in River Chenab as revealed by SSR markers" was to assess the levels of genetic diversity, population structure, and genetic differentiation among five different populations of River Chenab, Pakistan by using SSR markers. A total of 175 samples of *H. molitrix* were collected from five different sites of River Chenab, Pakistan. DNA was isolated by using the standard "proteinase-K and phenol/ chloroform" method, by following Sambrook and Russell (2001) [43], having slight

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

modifications and quantified with the help of agarose gel electrophoresis and nanodrop and separated on PAGE. Genomic DNA was PCR amplified by *H. molitrix* by using five primers. Data were analyzed by using different software including FSTAT, TFPGA, STRUCTURE, MICRO-CHECKER, POPGENE, and ARLEQUIN. The analysis of data gave following results:


So far, no research on microsatellites in this species has been reported in Pakistan. The primary goals of this work were to use latest molecular techniques to monitor the genetic status of *H. molitrix* in the River Chenab and develop strategies for successful management and protection of this vital fish resource.

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