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Assessment of the Genetic Diversity of Philippine Arabica Coffee (Coffea Arabica L.) Using SSR Markers

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Assessment of the Genetic Diversity of Philippine Arabica Coffee (Coffea Arabica L.) Using SSR Markers Philippine Journal of Science 149 (3-a): 993-1003, October 2020 ISSN 0031 - 7683 Date Received: 18 May 2020 Assessment of the Genetic Diversity of Philippine Arabica Coffee (Coffea arabica L.) Using SSR Markers Miriam D. Baltazar1,2* and Jermaine Marie Ann O. Fabella2 1Department of Biological Sciences 2National Coffee Research, Development and Extension Center Cavite State University, Indang, Cavite 4122 Philippines Arabica coffee (Coffea arabica L.) plays a significant contribution to the Philippine coffee industry. Many important genes are continuously lost due to an increase in population, urbanization, and the promotion of registered and popular varieties in the country. This study was conducted to assess the genetic diversity of 27 Philippine C. arabica accessions currently maintained at the Cavite State University – National Coffee Research, Development and Extension Center (CvSU-NCRDEC) field genebank using 19 simple sequence repeat (SSR) markers. Around 80% of the markers used showed polymorphism. A total of 56 alleles were detected, 47 of which were polymorphic. The average number of alleles per locus (3.7) and the polymorphism information content (PIC) (~ 0.40) found in this study were higher than those reported in the literature. Duplicate accessions were identified despite their striking morphological differences, and differentiation of synonymous accessions was also noted. The average genetic similarity was 0.83 and ranged from 0.64–1.0. Overall, the genetic diversity of Philippine C. arabica collection was low. Nonetheless, the higher number of alleles and PIC obtained provide more information in the selection of materials that can be used as parents in hybridization works. Cluster analysis showed three major clusters: Cluster I consisted of most of the accessions from Benguet State University (BSU), while Cluster II consisted of all accessions from the Bureau of Plant Industry (BPI) except Yellow Bourbon. MCA and Yellow Bourbon banded in Cluster III. The cluster analysis offers valuable information in the selection of parents for the development of vigorous F1 hybrids. Further, the results obtained in this study can be utilized in developing strategies to widen the low genetic variability of C. arabica through proper management of the country’s coffee genetic resources, development of effective breeding and selection programs, and varietal registration and identification. Keywords: Arabica coffee, coffee, Coffea arabica, genetic diversity, molecular markers, SSR markers INTRODUCTION Woehl et al. 2020). The worldwide demand for coffee is continuously increasing. The Philippines consumes 3.0 Coffee is a highly traded commodity that provides M bags of coffee every year, and there has been a steady livelihood for around 12.5 million households per annum increase in per capita coffee consumption from 0.7 kg per around the world (Browning 2018). The coffee industry person in 2008 to 1.5 kg and 1.7 kg in 2014 and 2017, is estimated to generate some USD 74 bn (Pruvot- respectively. Net imports are 2.8 M bags of coffee or 93% *Corresponding Author: [email protected] of total consumption (ICO 2017). Commercial production 993 Philippine Journal of Science Baltazar and Fabella: Genetic Diversity of Vol. 149 No. 3-a, October 2020 Philippine Arabica Coffee is mainly attributed to Coffea arabica L. (Arabica coffee) Assessment of the genetic diversity of coffee is important. and C. canephora (known as Conillon when produced in This can be done by using morphological characters; Brazil and Robusta in other parts of the world). However, however, this could lead to inconsistent data as they other less popular ones are also cultivated: C. liberica var. are highly affected by the environment. Morphological liberica (Liberica coffee) and C. liberica var. dewevrei, characterization is time-consuming and requires also known as Excelsa (IPNI 2020). expertise. Many of these morphological keys may also be effective only for a particular life stage; for example, The center of origin of C. arabica is the South Western many distinct characteristics can be observed only when forests of Ethiopia and the Boma Plateau of South Sudan. the coffee trees are already bearing flowers and fruits. th From Ethiopia, seeds were introduced in Yemen in the 15 This necessitates a more effective and reliable tool in century. From Yemen, coffee was introduced to i) Bourbon germplasm characterization and identification. Island (today French Reunion Island), giving the Bourbon- based varieties; and ii) to India and from India to Indonesia, The use of molecular markers is one efficient way to giving the Typica-based varieties in the late 17th and early genetically assess the collection. This method is more precise 18th centuries (Pruvot-Woehl et al. 2020). Instrumental to and reliable. Many DNA-based markers were widely used the introduction of the first coffee tree to the Philippines such as restriction fragment length polymorphism, random was a Spanish Franciscan monk in 1740 in Lipa, Batangas, amplified polymorphic DNA, amplified fragment length from which it eventually spread to the whole province and polymorphism, SSR or microsatellites, etc. Molecular nearby areas and the whole country (Juan and Francisco markers are very useful especially in identification as they 2007). This school of thought that is believed by many must directly represent the genotype and are not affected by the be treated with caution due to the paucity of research and environment. They are also diverse and highly distributed lack of refereed scholarly works (Castro 2003). in the genome. Numerous studies on coffee demonstrated the success of the use of molecular markers for species Coffee belongs to the genus Coffea of Rubiaceae family. and varietal identification as well as analysis of genetic It consists of 124 species (Davis 2011) with chromosome diversity of collections in various countries (Anthony et number 2n = 22 except for C. arabica (2n = 4x = 44). al. 2002; Aga et al. 2003; Ruas et al. 2003; Cubry et al. C. arabica is the only self-fertile among the other 2008; Teressa et al. 2010; Mishra et al. 2011; Geleta et al. commercially cultivated species (Lashermes et al. 1999). C. 2012; Razafinarivo et al. 2013). SSR markers are the most arabica is prized over C. canephora and C. liberica due to desired molecular markers for genetic diversity analysis its superior quality. In 2018, it accounted for ~ 23% of the because of their high-information content, co-dominant Philippines’ total production (www.psa.gov.ph). Despite its nature, sensitivity, and ease to analyze with minimal high cup quality, production is limited in high elevations. quantities of test samples. It is also increasingly being used They are mostly planted in the highlands of Sultan Kudarat, for linkage analysis and molecular breeding (Baruah et al. Davao del Sur, Sulu, South Cotabato, Iloilo, Benguet, and 2003). Despite its widespread use in coffee, there is little Mountain Province (www.psa.gov.ph). information on its use in evaluating the genetic diversity As the wild coffee population is generally under threat due of coffee varieties in the Philippines. Hence, the study was to its natural habitat disturbance mainly by deforestation conducted to assess the genetic diversity of C. arabica and land-use change (Poncet et al. 2004; Teressa et al. of the country. This is vital in coffee improvement and 2010), the effect is also evident in the Philippines. There selection, screening of important traits such as reaction was a continuous decline in the area of production of to pests and diseases, tolerance/resistance to biotic and C. arabica since 2001 with an average reduction of ~ abiotic stresses, high cup quality, and other traits. The 270 ha/yr (www.psa.gov.ph). This situation, along with information generated in this study could lead to numerous continuous urbanization, could lead to the genetic erosion possibilities in coffee breeding and selection, in ensuring of C. arabica in the country. Because of its high market the authenticity of coffee varieties, DNA fingerprinting, potential, the use of the National Seed Industry Council and many more. (NSIC)-registered C. arabica varieties is promoted. This – together with encroachment by agricultural activities, population pressures, and economic hardships – also contribute to this threat. The CvSU identified this gap of MATERIALS AND METHODS the coffee industry, limiting coffee genetic diversity that will greatly impact the sustainability of coffee production. Plant Materials Thus, a formal exploration and collection of coffee genetic A total of 68 accessions of coffee representing all resources throughout the country were initiated in 2013. commercially cultivated species that were collected from The collection is conserved in a field gene bank inside all over the Philippines were established in a field with the main campus. a land area of 1.3 ha located inside CvSU (14˚12.407’N, 994 Philippine Journal of Science Baltazar and Fabella: Genetic Diversity of Vol. 149 No. 3-a, October 2020 Philippine Arabica Coffee 120˚52.803E; approximately 266 masl). Of these, the 27 stain (Biotin GelRedTM Nucleic Acid; 10,000x in water), 1x accessions of C. arabica were used as samples (Table Tris-acetate-EDTA (TAE) buffer. Only samples with intact 1). Additionally, one representative accession of the DNA and had sufficient quantities were used. Thirty-seven other Coffea was included in this study for reference and (37) SSR primers were screened based on their reported comparison of SSR profiles. polymorphism across Coffea spp. Of these, 29 gave clear, distinct, consistent, and scorable bands. Nineteen (19) primers were found polymorphic across Coffea spp., 15 of DNA Extraction and Primer Selection which were polymorphic in C. arabica (Table 2). Fresh young leaves of a representative tree of each accession were ground with liquid nitrogen in a small mortar and pestle. Two DNA extraction protocols were Polymerase Chain Reaction (PCR) Amplification used and followed in the experiment: modified cetyl The primers were tested on the genomic DNA of the trimethylammonium bromide method [modified from coffee accessions.
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