Submitted:
24 November 2023
Posted:
28 November 2023
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Abstract
Keywords:
1. Introduction
2. Materials and Methods
2.1. Area of study:
2.2. Sample collection
2.3. DNA Extraction
2.4. Polymerase Chain Reaction
2.5. Data analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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| Study zone | Sampled individuals | Latitude | Longitude | Altitude |
|---|---|---|---|---|
| Maracayo | 15 | 8° 24' 34" N | 75° 53' 27" W | 35 mamsl |
| Patio Bonito | 15 | 8° 47' 37" N | 75° 56' 4" W | 16 mamsl |
| Santa Lucia | 15 | 8° 37' 25" N | 75° 52' 15" W | 17 mamsl |
| Marker | Primer forward 5’ – 3’ | Primer reverse 5’ – 3’ |
|---|---|---|
| mPgCIR9 | GCGTGTCGTATTGTTTC | ATTTTCTTCTGCCTTGTC |
| mPgCIR11 | TGAAAGACAACAAACGAG | TTACACCCACCTAAATAAGA |
| mPgCIR13 | CCTTTTTCCCGACCATTACA | TCGCACTGAGATTTTGTGCT |
| mPgCIR16 | AATACCAGCAACACCAA | CATCCGTCTCTAAACCTC |
| mPgCIR19 | AAAATCCTGAAGACGAAC | TATCAGAGGCTTGCATTA |
| mPgCIR22 | CATAAGGACATTTGAGGAA | AATAAGAAAGCGAGCAGA |
| mPgCIR23 | GTCTATACCTAATGCTCTGG | CCCAGGAAAATCTATCAC |
| Reagents | Stock concentration* | Individual concentration | Volume x one tube (μl) |
|---|---|---|---|
| DNA | 25 ng/ μL | 5 ng/ Μl | 5 |
| Enzyme | 5U/ μL | 0.05 U/ Μl | 0.25 |
| Primer Forward | 10 μM | 0.5 Μm | 1.25 |
| Primer Reverse | 10 μM | 0.5 Μm | 1.25 |
| dNTPs | 10 mM** | 0.2 Μm | 0.5 |
| Buffer | 10 X | 1 X | 2.5 |
| Water | ----------- | ---------- | 14.25 |
| Total | ----------- | ---------- | 25 |
| Temperature (°C) | Time | Number of cycles |
|---|---|---|
| 95° | 3 min | 1 |
| 61°-55° | 30 seg | 10 |
| 56° | 30 seg | 23 |
| 72° | 1 min | |
| 72° | 5 min | 1 |
| Locus | N° Allele | Allele | Patio Bonito | Maracayo | Santa Lucia |
|---|---|---|---|---|---|
| 161 | 0.000 | 0.143 | 0.000 | ||
| 163 | 0.000 | 0.143 | 0.000 | ||
| 166 | 0.167 | 0.143 | 0.000 | ||
| mPgCIR9 | 9 | 168 | 0.417 | 0.143 | 0.125 |
| 170 | 0.083 | 0.429 | 0.625 | ||
| 173 | 0.000 | 0.000 | 0.250 | ||
| 181 | 0.083 | 0.000 | 0.000 | ||
| 186 | 0.083 | 0.000 | 0.000 | ||
| 189 | 0.167 | 0.000 | 0.000 | ||
| 275 | 0.000 | 0.000 | 0.500 | ||
| 277 | 0.000 | 0.429 | 0.250 | ||
| mPgCIR11 | 6 | 280 | 0.250 | 0.143 | 0.000 |
| 283 | 0.250 | 0.429 | 0.250 | ||
| 286 | 0.250 | 0.000 | 0.000 | ||
| 295 | 0.250 | 0.000 | 0.000 | ||
| 224 | 0.000 | 0.250 | 0.000 | ||
| 227 | 0.083 | 0.063 | 0.000 | ||
| 229 | 0.167 | 0.188 | 0.000 | ||
| 232 | 0.500 | 0.375 | 0.167 | ||
| mPgCIR13 | 9 | 235 | 0.000 | 0.000 | 0.111 |
| 236 | 0.167 | 0.000 | 0.444 | ||
| 239 | 0.083 | 0.063 | 0.000 | ||
| 241 | 0.000 | 0.063 | 0.222 | ||
| 244 | 0.000 | 0.000 | 0.056 | ||
| 240 | 0.375 | 0.000 | 0.000 | ||
| 246 | 0.000 | 0.857 | 0.000 | ||
| 254 | 0.000 | 0.000 | 0.800 | ||
| mPgCIR16 | 7 | 256 | 0.125 | 0.000 | 0.000 |
| 270 | 0.500 | 0.000 | 0.000 | ||
| 274 | 0.000 | 0.000 | 0.200 | ||
| 281 | 0.000 | 0.143 | 0.000 | ||
| 237 | 0.000 | 0.000 | 1.000 | ||
| 250 | 0.000 | 0.500 | 0.000 | ||
| mPgCIR19 | 5 | 254 | 0.333 | 0.333 | 0.000 |
| 257 | 0.667 | 0.111 | 0.000 | ||
| 269 | 0.000 | 0.056 | 0.000 | ||
| 229 | 0.000 | 0.000 | 0.500 | ||
| 233 | 0.000 | 0.000 | 0.375 | ||
| 238 | 0.250 | 0.125 | 0.000 | ||
| mPgCIR22 | 7 | 241 | 0.000 | 0.625 | 0.000 |
| 252 | 0.000 | 0.000 | 0.125 | ||
| 254 | 0.250 | 0.000 | 0.000 | ||
| 259 | 0.500 | 0.250 | 0.000 | ||
| 203 | 1.000 | 0.250 | 0.143 | ||
| mPgCIR23 | 3 | 206 | 0.000 | 0.750 | 0.714 |
| 210 | 0.000 | 0.000 | 0.143 | ||
| Pop | Locus | Allele | Frecuencia |
|---|---|---|---|
| Patio Bonito | mPgCIR9 | 181 | 0.083 |
| Patio Bonito | mPgCIR9 | 186 | 0.083 |
| Patio Bonito | mPgCIR9 | 189 | 0.167 |
| Patio Bonito | mPgCIR11 | 286 | 0.250 |
| Patio Bonito | mPgCIR11 | 295 | 0.250 |
| Patio Bonito | mPgCIR16 | 240 | 0.375 |
| Patio Bonito | mPgCIR16 | 256 | 0.125 |
| Patio Bonito | mPgCIR16 | 270 | 0.500 |
| Patio Bonito | mPgCIR22 | 254 | 0.250 |
| Maracayo | mPgCIR9 | 161 | 0.143 |
| Maracayo | mPgCIR9 | 163 | 0.143 |
| Maracayo | mPgCIR13 | 224 | 0.250 |
| Maracayo | mPgCIR16 | 246 | 0.857 |
| Maracayo | mPgCIR16 | 281 | 0.143 |
| Maracayo | mPgCIR19 | 250 | 0.500 |
| Maracayo | mPgCIR19 | 269 | 0.056 |
| Maracayo | mPgCIR22 | 241 | 0.625 |
| Santa Lucia | mPgCIR9 | 173 | 0.250 |
| Santa Lucia | mPgCIR11 | 275 | 0.500 |
| Santa Lucia | mPgCIR13 | 235 | 0.111 |
| Santa Lucia | mPgCIR13 | 244 | 0.056 |
| Santa Lucia | mPgCIR16 | 254 | 0.800 |
| Santa Lucia | mPgCIR16 | 274 | 0.200 |
| Santa Lucia | mPgCIR19 | 237 | 1.000 |
| Santa Lucia | mPgCIR22 | 229 | 0.500 |
| Santa Lucia | mPgCIR22 | 233 | 0.375 |
| Santa Lucia | mPgCIR22 | 252 | 0.125 |
| Santa Lucia | mPgCIR23 | 210 | 0.143 |
| Population | Locus | Na | Ne | Ho | uHe | F |
|---|---|---|---|---|---|---|
| mPgCIR9 | 6.000 | 4.000 | 0.333 | 0.818 | 0.556ns | |
| mPgCIR11 | 4.000 | 4.000 | 0.000 | 0.857 | 1.000ns | |
| Patio | mPgCIR13 | 5.000 | 3.130 | 0.167 | 0.742 | 0.755** |
| Bonito | mPgCIR16 | 3.000 | 2.462 | 0.250 | 0.679 | 0.579ns |
| mPgCIR19 | 2.000 | 1.800 | 0.000 | 0.533 | 1.000ns | |
| mPgCIR22 | 3.000 | 2.667 | 0.000 | 0.714 | 1.000* | |
| mPgCIR23 | 1.000 | 1.000 | 0.000 | 0.000 | ----- | |
| Average | 3.429 | 2.723 | 0.107 | 0.621 | 0.815 | |
| mPgCIR9 | 5.000 | 3.769 | 0.000 | 0.791 | 1.000** | |
| mPgCIR11 | 3.000 | 2.579 | 0.000 | 0.659 | 1.000** | |
| mPgCIR13 | 6.000 | 4.000 | 0.250 | 0.800 | 0.667** | |
| Maracayo | mPgCIR16 | 2.000 | 1.324 | 0.000 | 0.264 | 1.000** |
| mPgCIR19 | 4.000 | 2.656 | 0.111 | 0.660 | 0.822** | |
| mPgCIR22 | 3.000 | 2.133 | 0.000 | 0.567 | 1.000** | |
| mPgCIR23 | 2.000 | 1.600 | 0.000 | 0.429 | 1.000* | |
| Average | 3.571 | 2.580 | 0.052 | 0.596 | 0.927 | |
| mPgCIR9 | 3.000 | 2.133 | 0.000 | 0.567 | 1.000** | |
| mPgCIR11 | 3.000 | 2.667 | 0.000 | 0.714 | 1.000* | |
| Santa | mPgCIR13 | 5.000 | 3.447 | 0.111 | 0.752 | 0.843** |
| Lucia | mPgCIR16 | 2.000 | 1.471 | 0.000 | 0.356 | 1.000* |
| mPgCIR19 | 1.000 | 1.000 | 0.000 | 0.000 | ----- | |
| mPgCIR22 | 3.000 | 2.462 | 0.000 | 0.633 | 1.000** | |
| mPgCIR23 | 3.000 | 1.815 | 0.000 | 0.484 | 1.000** | |
| Average | 2.857 | 2.142 | 0.016 | 0.501 | 0.974 | |
| Total | 3.286 | 2.482 | 0.058 | 0.572 | 0.906 | |
| Na: Number of alleles; Ne: Effective number of alleles; Ho: Observed heterozygosity; uHe: Unbiased expected heterozygosity; F: Fixation index; HWE: Hardy-Weinberg Equilibrium (ns: not significant *: P-Value < 0.05 **: P-Value < 0.01). | ||||||
| Locus | PIC |
|---|---|
| mPgCIR9 | 0.738 |
| mPgCIR11 | 0.740 |
| mPgCIR13 | 0.787 |
| mPgCIR16 | 0.732 |
| mPgCIR19 | 0.713 |
| mPgCIR22 | 0.795 |
| mPgCIR23 | 0.443 |
| Locus | FIS | FIT | FST | Nm |
|---|---|---|---|---|
| mPgCIR9 | 0.835 | 0.857 | 0.136 | 1.591 |
| mPgCIR11 | 1.000 | 1.000 | 0.166 | 1.256 |
| mPgCIR13 | 0.753 | 0.781 | 0.110 | 2.024 |
| mPgCIR16 | 0.784 | 0.895 | 0.514 | 0.236 |
| mPgCIR19 | 0.896 | 0.950 | 0.522 | 0.229 |
| mPgCIR22 | 1.000 | 1.000 | 0.294 | 0.600 |
| mPgCIR23 | 1.000 | 1.000 | 0.495 | 0.255 |
| Media | 0.895 | 0.926 | 0.320 | 0.885 |
| FIS: individual fixation index with respect to subpopulations within a population; FIT: individual fixation index with respect to the total population.; FST: genetic differentiation coefficient among subpopulation; Nm: number of migrants per generation. | ||||
| A | B | C | |
|---|---|---|---|
| A | 0.000 | ||
| B | 1.066 | 0.000 | |
| C | 2.000 | 1.096 | 0.000 |
| A: Patio Bonito; B: Maracayo; C: Santa Lucia | |||
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