**3.2 Preparations for DNA washing and precipitation**



**Table 1.** H. molitrix *sampling details.*

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


#### **3.3 DNA quantification**

In total, 0.8% agarose gel electrophoresis (ATTO Corporation AE 6220) was used for checking the isolated DNA quality. For making gel-like suspension, 0.8 g agarose was added into 100 mL 1X TAE buffer.

#### **3.4 Analysis of DNA**

Nanodrop was used for assessing the quality of isolated DNA. One microliter DNA sample was used for checking the convention of isolated DNA. The convention of isolated DNA was adjusted at 50 ng/μL for PCR by mixing the stock solution of DNA with nuclease-free water.

Calculation was done by applying the following formula:

$$\mathbf{C}\_1 \mathbf{V}\_1 = \mathbf{C}\_2 \mathbf{V}\_2$$

Where.

C1 = Stock DNA solution concentration.

V1 = Volume of DNA required.


#### **3.5 Microsatellite amplification**

Genomic DNA was PCR amplified by *H. molitrix.* Five primers, namely BL8–1, BL14, BL52, BL108, and BL123, were used taken from Gene Library (**Table 2**).


**Table 2.**

*SSR markers of* H. molitrix *with details.*


#### **Table 3.**

*Reaction mixture formation for PCR amplification.*

PCR reaction was conducted at 25 μL reaction mixture by using (Multigene Optimax, Lab Net, USA) that contained the following ingredients (**Table 3**)


### **3.6 Thermocycler conditions**



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

#### **3.7 Electrophoresis and visualization for separation of amplified products**

The PCR products were isolated on a 5% non-denaturing PAG containing 19:1 acrylamide: bis-acrylamide and visualized by silver-staining method. Electrophoresis was conducted using a SequiGen sequencing gel electrophoresis method. For the visualization of DNA bands, silver-staining method was used.

#### **3.8 Software and analytical packages**

Software and analytical packages for genome mapping were provided by [44–51].

a. FSTST (ver.2.9.3.2)

b. GENPOP (ver. 1.2)


#### **3.9 Data analysis**

Allele frequency, allelic richness (*Ar*), observed heterozygosity (*Ho*), and expected heterozygosity (*He*) were estimated with FSTAT ver. 2.9.3.2 to summarize the genetic composition of the population [44, 50]. The GENEPOP ver. 1.2 was used to test linkage disequilibrium (LD) between all pairs of luci [46]. ARLEQUIN ver. 2.000 was used to calculate deviation from HWE across each locus using the Markov-chain random walk algorithm [51]. To maintain a within-sample type-1 error rate of α = 0.05 for each locus, the statistical significance of deviations from HWE was corrected using the sequential Benferroni correction.

Inbreeding coefficient (Fis) and level of population subdivision/population over loci were determined by unbiased F-statistics by using software FSTAT ver. 2.9.3.2 [44]. Genetic divergence Fst among subdivisions for all pairs comparisons between sampling locations was deduced by calculating Weir and Cockerham's (1984) [44]. The significance of estimates of Fst was assessed using 10,000/mutations. The hierarchical partition of genetic diversity was estimate by conducting analyses of molecular variance (AMOVA) using ARLEQUIN ver. 2.000.

ARLEQUIN ver. 2.000 was used to calculate the pairwise estimates of Fst values and test their significance by bootstrapping analysis (1000 replicates) for genetic differentiation evaluation between populations. Exact tests for population differentiation (Raymond and Rousset, 1995) were conducted using GENEPOP. UPGMA dendrogram based on Nei's (1987) [50] unbiased distance was interpreted using TFPGA software.

#### **4. Results**

#### **4.1 Genetic diversity in** *H. molitrix* **populations**

In current study, the "Micro-checker" software was applied to the genotypie data obtained for *H. molitrix* populations that showed no scoring errors related to large allele, no stuttering hands, and no presence of null alleles at all the loci. In the current study, the screened microsatellite loci in all the examined *H. molirix* populations were demonstrated to be varied. The patterns of genetic diversity fluctuate depending on the screened microsatellite locus and the studied fish population. The average allele frequency and allele size ranged from 0.003 to 0.574 and from 157 to 389 base pairs, respectively, were observed at different screene d loci in *H. molitrix* populations in the present study (**Figure 1**, **Table 5**)

#### **4.2 Allelic diversity (Na) and Allelic Richness (Ar)**

In current study, the number of alleles (*Na*) per locus extended from 3.00 to 9.00 with an average from 5.6 to 9.0, While the values of allelic richness (*Ar*) were ranging from 2.943 to 8.940 with an average value varying from 5.513 to 5.942 in various *H. molitrix* populations. The largest average value of number of alleles and allelic richness were noted in the population of CB and minimum in the population of MH. At locus BL 14, the highest values of number of alleles and allelic richness were found 8.911 in the MH population. The average values of *Na* and *Ar* in the populations of TH, MH, KH, QH, and CB were observed as 5.8 (*Ar* = 5.79), 5.6 (*Ar* = 5.513), 5.8 (*Ar* = 5.753), 5.8 (*Ar* = 5.754), and 6.0 (*Ar* = 5.942) respectively (**Table 6**–**10**, **Figures 2**–**7**).

**Figure 1.** *Map of Pakistan showing Chenab River.*


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

#### **Table 5.**

*Allele frequency and size (bp) noticed at each locus in all populations of* H. molitrix.

#### **4.3 Effective number of alleles (Nae)**

The values of effective number of alleles (*Nae*) were observed ranging from 1.9647 to 6.7680 in various studied *H. molitrix* populations. The average value of *Nae* was observed as 4.7284, 2.9711, 2.9043, 3.7013, and 2.9933 in TH, MH, KH, QH, and CB, respectively. The largest value of *Nae* was observed in population collected from TH while the lowest in the population of KH (**Tables 6**–**10**, **Figures 2**–**7**).

### **4.4 Heterozygosity (H)**

In the present study, heterozygosity (*H*) level was observed moderate to high in all examined *H. molitrix* populations. In various examined *H. molitrix* populations, the values of observed heterozygosity (*Ho*) were measured ranging from 0.3429 to 0.8571.


#### **Table 6.**

*Genetic diversity at different microsatellite loci in TH populations of* H. molitrix.


**Table 7.** *Genetic diversity at different microsatellite loci in MH populations of* H. molitrix.


**Table 8.**

*Genetic diversity at different microsatellite loci in KH populations of* H. molitrix.

The average *Ho* value was observed as 0.7257, 0.5714, 0.6343, 0.4400, and 0.6800 in TH, MH, KH, QH, and CB, respectively. The fish population sampled from QH revealed the lowest value of *Ho* and TH and CB population showed the highest value as compared with others (**Tables 6**–**10**, **Figures 2**–**7**). The values of expected heterozygosity (*He*) were ranging from 0.4981 to 0.8646 in various selected *H. molitrix* populations. The average values of *He* were determined as 0.7812, 0.6456, 0.6361, 0.7005, and 0.6533 in TH, MH, KH, QH, and CB, respectively.

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


#### **Table 9.**

*Genetic diversity at different microsatellite loci in QH populations of* H. molitrix.


#### **Table 10.**

*Genetic diversity at different microsatellite loci in CB populations of* H. molitrix.

**Figure 2.**

*Comparative distribution of number of alleles (*Na*) at different SSR loci in* H. molitrix *populations.*

**Figure 3.**

*Comparative distribution of allelic richness (*Ar*) at different SSR loci in* H. molitrix *populations.*

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

**Figure 5.**

*Comparative distribution of observed heterozygosity (*Ho*) at different SSR loci in* H. molitrix *populations.*

**Figure 6.** *Comparative distribution of expected heterozygosity (*He*) at different SSR loci in* H. molitrix *populations.*

**Figure 7.** *Comparative distribution of* 1-Ho/He *at different SSR loci in* H. molitrix *populations.*
