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PCR Analysis for Genetic Distances of Two Charybdis Crab Populations

Abstract Genomic DNA (gDNA) set apart from two populations of Korean Charybdis crab (Charybdis japonica) was augmented by PCR experiments. The five oligonucleotides primers (ONT-primers) were spent to yield the number of unique loci shared to each crab population (ULSECP) and number of loci shared by the two crab populations (LSTCP). 305 fragments (FRAGs) were identified in the Charybdis crab population A (CCPA), and 344 in the Charybdis crab population B (CCPB): 44 number of ULSECP (14.43%) in the CCPA and 110 (31.98%) in the CCPB. 44 number of LSTCP, with an average of 8.8 per primer, were detected in the two crab populations. The bandsharing (BS) value between entity’s no. 01 and no. 10 was the lowest (0.371) between the two CCPs. The average bandsharing (ABS) values of individuals in the CCPA (0.575±0.014) were lesser than in those originated from the CCPB (0.705±0.011) (p < 0.05). The polar hierarchical dendrogram (PHD) achieved by the five ONT-primers denotes three genetic clusters (GCs): cluster I (CHARYBCRAB 01, 04, 05, 06, and 08), cluster II (CHARYBCRAB 02, 03, 07, 09, 10, and 11) and cluster III (CHARYBCRAB 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). The shortest genetic distance (GD) displaying significant molecular difference (MD) was between individuals CHARYBCRAB no. 18 and CHARYBCRAB no. 17 (0.055).


INTRODUCTION
Korean C. japonica is one of ecologically significant portunid crab species in the Yellow Sea, belonging to the family Portunidae, and the order Decapoda. In the environmental ecosystem, Charybdis crab is broadly inhabited in the seawater areas of the Korean peninsula, besides in some regions in the East China Sea, Taiwan, Malaysia and Hawaii. Apparently, the dorsal surface of shell is habitually smooth. The crab has very large and long both claws and the body color of this crab is light grey form. Since crabs are night-time organisms, they nestle under the grits, sandy soil and rocks of the shallow sea. Many explores have displayed that the change of water temperature by earth warmth, population density, tide embankment, thoughtless overhunting and environmental pollution are serious in the early larval development of this portunid crab (Yeon et al., 2011). Charybdis crab is one of commercial portunid crab in Korea all the year round.

MATERIALS AND METHODS
Two assemblies of Charybdis crab (C. japonica) were taken from Seosan in the vicinity of the Yellow Sea of Korea. Two sample collections of crab muscle was gathered in disinfected cylinders, proximately positioned on cold ice, and retained at -79℃ until required. PCR inquiry was achieved the genomic DNAs from 22 individuals, consuming different five oligonucleotides primers (ONTprimers). The extraction/refinement of genomic DNA (gDNA) was accomplished under the requirements described previously (Yoon, 2018). Proteinase K solution was involved to the tubes and gradually pipetted for some minutes.
Similarity matrixs (SMs) were established based on the data of bandsharing (BS) rates constructed by molecular analysis. Comparing the two lanes, the BS rate was appraised as surveys: BS = 2 (Nab) / (Na + Nb), where Nab signifies the amount of FMs shared by examples b and a; Na points out the total quantity of FMs in a; and Nb signifies the total quantity of FMs in example b. The median within-group correspondence was rated via pairwise matching inquiry between the parties within a group. A polar hierarchical dendrogram (PHD) was generated renowned on SMs to take a cluster tree using Systat version 10 (SPSS, Chicago, IL, USA). some rounds by PCR experiments. The fragment (FRAG) amounts in each size interval have been computed from the integrated FRAGs obtained with every five ONT-primers. The higher FRAG extents (>1,900 bp) are perceived in the Charybdis crab population A (CCPA), as shown in Fig. 1. Seven ONT-primers produced a sum of 884 FRAGs in the black rockfish species and 632 in the Hwanghae rockfish species, with a DNA FRAGs amount stretching from 150 to 2,200 bp (Yoon et al., 2007). The extents of the DNA FRAGs also varied roughly, from 90 to 2,400 bp in Gracilaria vermiculophylla and G. chorda (Kim & Yoon, 2018). Yoon (2018) also described that the more FRAG amounts (>1,100 bp) are not identified in di-population of the razor shellfish (Solen corneus). The five ONT-primers created a whole of 367 FRAGs in the innate pufferfish population and 211 FRAGs in the farmed pufferfish population, with genetic FRAG extents fluctuating from 50 bp to 1,300 bp (Yoon, 2020). The mean quantity of separate FRAGs per primer within the innate and cultivated river pufferfish population was 14.68 and 8.44, individually (Yoon, 2020). The number of the FRAGs showed 354 and 390 DNA countable FRAGs, respectively, extending from 100 bp to 1,600 bp for the Yeosu and Jinhae populations of the arkshell species (Yoon, 2021a).
The BS value between individual's no. 01 and no. 10 was the lowest (0.371) between the two CCPs and the value between individual's no. 10 and no. 11 was the highest (0.818), as shown in Table 1. The five ONT-primers, OPA-16, OPB-10, OPB-15, OPD-10, and OPD-20 were spent to yield the number of ULSECP and number of LSTCP (Table 2). 305 FRAGs were identified in the CCPA, and 344 in the Charybdis crab population B (CCPB): 44 number of ULSECP (14.43%) in the CCPA and 110 (31.98%) in the CCPB. 44 number of LSTCP, with an average of 8.8 per primer, were detected in the two crab populations. The ONT-primer OPD-20 generated 44 identical DNA FRGMs, nearly 200 bp and 300 bp, in both the CCPA and CCPB. Assessed independently, the average molecular difference (MD) was higher in the CCPB than in the CCPA.
The ONT-primer OPC-07 created 44 unique loci shared to each pufferfish population (ULSEPP) of the refined river pufferfish (Yoon, 2020). The author contended that the ONTprimer OPC-05 discovered 44 loci shared by all samples of the two river pufferfish populations, as thick and/or thin FRAGs of 500 bp and 1,200 bp, separately. Also, the author stated that five ONT-polymers were expended making a full of 110 LUECP in first group and 132 in second group in two Scapharca subcrenata populations, separately, shifting in amount of DNA FRAGs from larger than nearly 50 to below 1,050 bp (Yoon, 2021b). As prepared reference of clams, shrimps and oysters, for PCR scrutiny, Yoon & Kim (2003a) 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21   that DNA FRAGs acquired by four ONT-primers ranged from 100 to 2,300 bp in the brittle star (Amphiura filiformis). It has been informed that 7 ONT-primers produced 317 FRAGs in a farmed shrimp population, and 385 in the untamed population, extending from 100 to 1,800 bp (Yoon & Kim, 2003b). The average bandsharing values (ABS values) was 0.457±0.007 between the CCPA and CCPB, as established in Table 3. The ABS values of individuals in the CCPA (0.575±0.014) were lesser than in those originated from the CCPB (0.705±0.011) (p < 0.05). To elucidate, reports have exposed that the median BS value attained expending five ONT-primers was 0.852 in the rainbow trout population, 0.704 in the masu salmon population (Yoon, 2020), and 0.282±0.008 between the two terrestrial oyster populations (Kim et al., 2004).
The PHD achieved by the five ONT-primers denotes three genetic clusters (GCs): cluster I (CHARYBCRAB 01, 04, 05, 06, and 08), cluster II (CHARYBCRAB 02, 03, 07, 09, 10, and 11) and cluster III (CHARYBCRAB 12,13,14,15,16,17,18,19,20,21,and 22), as shown in Fig. 2. The shortest GD displaying significant MD was between individuals CHARYBCRAB no. 18 and CHARYBCRAB no. 17 (0.055). The longest GD showing significant MDs between two Charybdis crab populations was observed in individuals CHARYBCRAB no. 06 and CHARYBCRAB no. individuals in the two Charybdis crab populations from cluster I (CHARYBCRAB 01, 04, 05, 06, and 08), cluster II (CHARYBCRAB 02, 03, 07, 09, 10, and 11) and cluster III (CHARYBCRAB 12,13,14,15,16,17,18,19,20,21,and 22)  In finfish, this grouping scrutiny acquired a form consistent with the one postulated by Nagarajan et al. (2006). Amid the three Channa punctatus collections gathered from a few waterways of south India, the maximum hereditary space (GD=0.9231) was acquired between two sample collections (Nagarajan et al., 2006). This analysis presented that great MDs could be achieved among individuals of terrestrial sample assembles. The rates of the pairwise appraisals of balanced GD between the populations of the tailfin anchovy (Coilia nasus) from the pooled records for the seven ONT-primers, ranged from 0.051 to 0.435 ( Jo & Yoon, 2021). The GD between individuals thus confirmed the reality of a neighboring affiliation in group II between two populations of tailfin anchovy. As mentioned formerly, the promise of genomic PCR to ascertain analytical markers for the recognition of the two CCPs has been validated. S. subcrenata bi-group can be obviously categorized by gDNA-grounded processes.