Development of Microsatellite Markers to Determine Genetic Diversity of Indonesian Betel Nut ( Areca catechu L.)

Areca nut ( Areca catechu ) belongs to the monocotyledonous and palm family of plants known as betel nut or supari plants. The Center of origin areca nut comes from India. Areca nut is a cross-pollinated plant with a heterozygous heterogeneous genetic constitution. Next-generation sequencing is widely used for plant breeding activities, including developing SSR markers with an in-silico approach. This research aims to develop an SSR marker to detect ge-netic diversity in local areca nut from Indonesia. The genomic data used to develop SSR markers were obtained from the Whole Genome Sequencing of Pinang Reyan Hainan Cultivars obtained from NCBI. Fragments containing SSR regions were isolated using MISA software, and primers were designed with Primer3web v.4.1.0 (https://bioinfo.ut.ee/primer3/). The average total size of the sequences tested was 168770731.8, with a total number of SSRs identified as many as 1149,813. SSR motifs are used as a step in genetic diversity research because these motifs will be used in primary attachments which in turn can affect genetic diversity. There were twenty-eight primers generated from four amplicons (100-150, 150-200, 200-250, and 250-300 bp) used for multiplex primers with motifs (AAT)37, (TA)53, (TA)54, (TC)52, (TC)52, (TG)53, and (AG)54.


Introduction
Areca nut (Areca catechu) belongs to the monocotyledonous and palm family of plants known as betel nut or supari plants.Center of Origin areca nut comes from India (Rajesh & Ananda, 2019).Areca nut has 32 chromosomes (2n=2x=32).Center of Diversity areca nut from Bangladesh, Madagascar, Arabian Peninsula, Vietnam, Indonesia, Fiji Islands, Malaysia, Papua New Guinea, Solomon, and Laos (George et al., 2006).Center of betel nut diversity in Alamgan, Madagascar, Anatahan, Pagar, Africa, and the Marshall Islands (Raghavan & Baruah, 1958).Indonesia has betel nut germplasm scattered in various areas such as in Gorontalo, Manado, Papua, North Kalimantan, West Kalimantan, and Jambi.The morphological diversity of areca nut between various regions is very high because areca nut is a cross-pollinated plant.Areca nut with a heterozygous genetic constitution so that in the selection of parents, it takes a long time to carry out morphological characterization.
The potential for areca nut production, which can be used as a medicinal plant, is one of the technological innovations in the development of palm plant commodities.The germplasm of areca nut spread throughout Indonesia can be used as genetic material.Differences in the performance of areca nut are not only caused by genetics, but differences in altitude and agro-climate make the diversity of areca nut even greater.Genetic diversity in a population between regions is caused by humans and nature over time (Zhou et al., 2020).The existence of genetic diversity is the main capital in the management of germplasm more effectively and efficiently.
Identification of areca germplasm can be made with a molecular approach.Confirming genetic diversity using a molecular marker approach aims to detect a variation between individuals caused by duplications, insertions, deletions, translocations, and point mutations at the DNA level (Ahmad et al., 2018).Molecular markers can be used to determine genetic diversity in areca nut, which has been done recently on RAPD markers (Bharath et al., 2015).The marker used in genetic diversity is SSR (Simple Sequence Repeats).Genomic SSR is very abundant and produces high polymorphism.The use of SSR markers in areca nut has not been carried out, so this study develops SSR markers from existing data.SSR has the advantages of codominant, multi allele, very informative, reproducible, and high abundance throughout the genome (Pan, 2010;Pan et al., 2006;Nadeem et al., 2018).The advantages of SSR over SNP markers are that genetic analysis activities can use SSR markers at cultivar, species, individual, and population levels (Ahmad et al., 2017).Research on SSR markers has been widely used carried out on plantation crop commodities such as sugarcane ((Ahmad et al., 2018), oil palm (Khomphet et al., 2017), coconut (Wu et al.,2018), and Cocoa (Everaert et al., 2020).
SSR markers can be sequenced according to genomic level or express sequence tag (EST).SSR is classified into three types namely nuclear (nuSSR), mitochondrial (mtSSR) (Soranzo et al., 1999), or chloroplast SSRs (cpSSR) based on genomic location (Weising & Gardner, 1999).Most SSRs are nuclear SSRs.EST led to significant advances in gene identification, transcriptional isoform biology, and genetic identification of species with complete genomes.Conventional construction and screening of genomic libraries are time-consuming and expensive, but it is cheaper and universally applicable (Vettore et al., 2003).So that a more efficient approach is used, namely sequencing in the next generation, searching for EST centers, and searching for genomic libraries that have been enriched to reduce SSR isolation time and increase the number of SSR loci (Cordeiro et al., 2000;Cordeiro et al, 2021).The genome-based NGS (Next-generation sequencing) approach is widely used in germplasm management.With NGS, an in-silico approach can be used to become an innovation in sequencing plant genomes that are cheap and fast in developing SSR markers.NGS helps increase the quality and quantity of plant conservation, so this study aims to develop markers SSR from the areca nut genome.

Results of microsatellite search
The data processed by Mass resulted in motifs of various types of nucleotides, namely dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides.The mononucleotide motif was not used in the primer design because it was not informative (Sari & Efendi, 2020).Types of nucleotides (dinucleotides and trinucleotides) are found in many living things.
The primer design was carried out with a specific primer design after checking the sequence containing SSR and the primer design with a length of 18-24 bp, complementary to the flanking area, then amplification and polymorphism were carried out.
The total size of the sequence examined is the number of nitrogenous bases in one chromosome.The average total size of the 16 areca nut chromosomes tested to determine the number of SSRs was 168770731.8.The average total number of SSRs identified was 1149,813.The average number of SSR in the compound formation is 47.75 bases.The three largest total sequence sizes examined were chromosome 2, chromosome 8, and chromosome 14.The total number of identified SSRs and the highest number of SSRs in compound formation on chromosome 2 were 1696 bases and 84 bases.While the number of SSRs identified was at least on chromosome 11, and the number of SSRs in compound formation was at least on chromosome 9, namely 27 bases (Table 1).

Multiplex primer designs
The primer design for this study was carried out by primary multiplexing with a product size range (amplicons) of 100-150, 150-200, 200-250, and 250-300.Multiplex PCR in this study was used to amplify some targets in one PCR experiment with several pairs of primers in one reaction mixture.This reduces energy and time but does not reduce the experiment's validity.Shen et al. (2010), Multiplex PCR was performed simultaneously from some DNA template regions or multiple DNA templates using more than one forward and reverse primer.Primary multiplexes are widely used in microbiological and environmental studies.However, these primer multiplexes have disadvantages, namely mispriming caused by primer dimerization, non-specific binding to non-target DNA templates, unable to purify and separate DNA amplicon with the same electrophoretic movement.The results of the primer design of the seven SSR motifs, namely (AAT)37, (TA)53, (TA)54, (TC)52, (TC)52, (TG)53, and (AG)54 (Table 4) obtained seven primers.Forward and reverse with four amplicons so that the total primer produced is 28 primers.

Table 1 .
Results of microsatellite search

Table 2 .
Distribution of different repeats of various sizes of nitrogenous bases in chromosome 1 to chromosome 8

Table 3 .
Distribution of different repeats of various sizes of nitrogenous bases in chromosome 9 to chromosome 16