Identifying Phl citrus varieties using molecular markers

Citrus is the most important fruit in the world in terms of production, processing and trade. In the Philippines, calamansi is the most popular type of citrus due to its abundance and popularity of use as an ingredient in our local cuisine. Aside from the popular calamansi, there are many other types of citrus grown in the country like sweet orange, orange, ponkan, pummelo, and mandarin.

Experts mentioned how the origin and identification of citrus is a complicated, confusing, and controversial matter due to the intra- and interspecific hybridization, high frequency of mutations, long history of cultivation, and dispersal. In fact, even the most widely-accepted classifications of citrus have differences in their concepts of classifying it.

According to Karen Tonogbanua of the Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños (UPLB), “a well-established identity is the key in protecting and preserving any plant genetic resource. Researchers and breeders can properly utilize plant genetic resources in their researches if the materials are well characterized and identified to exploit their potentials as breeding materials.” She also mentioned that, “in other countries, researchers geared their studies toward profiling their materials.” Therefore, she together with Dr. Rene Rafael C. Espino, UPLB professor, took it upon them to develop an identification key for citrus varieties and species in the Philippines using molecular markers.

The research project titled, “Identification of Variety-Specific Alleles and Loci in Philippine Citrus Collection Using Simple Sequence Repeat (SSR) Markers,” was recently published at the Philippine Agricultural Scientist Journal in September 2017. Dr. Espino acknowledged the Bureau of Agricultural Research for funding their research, saying that “it serves as a contribution of the Bureau to furthering science in the country.”

DNA profile of citrus collection in the Philippines

For the research project, they utilized 44 citrus varieties belonging to nine citrus species obtained from Baguio National Crop Research and Development Center, Bureau of Plant Industry, Baguio City. These include 13 mandarins (Citrus reticulata and C. unshiu Marc.), 11 pummelos (C. grandis), 10 sweet oranges (C. sinesis), three lemons (C. limon and C. volkameriana), three limes (C. aurantifolia), three trifoliates (P. trifoliata) and calamansi (x Microfortunella microcarpa).

Over the past 20 years, SSRs are used for genotyping plants because they are highly informative, codominant, multi-allele genetic markers that are experimentally reproducible and transferable among related species, according to Mason (as cited in Vieira et. al., 2016). For the research project, 44 SSR markers were used to construct the DNA profiles of the 44 citrus varieties from nine species. These SSR markers that were screened from previous works provided sufficient information to identify unique alleles and/or loci that were used to establish an identification key for citrus.

“A haplotype map was constructed where alleles are presented accordingly with the loci that were observed. A total of 268 alleles were observed for 44 polymorphic primers used in this study, “according to Tonogbanua. More so, the identification of markers for each agronomic type (mandarins, pummelos, sweet oranges, lemons, limes, trifoliates, and calamansi) was done manually by using the banding patterns on the haplotype map. Tonogbanua also mentioned that, “the use of markers in determining varieties for the study is arbitrary; no strict order of the markers was followed.” ### (Rena S. Hermoso)

2018-02-23 lab

Photo Caption

Karen A. Tonogbanua, research assistant, preparing the polymerase chain reaction (PCR) cocktail. PCR is the technique used to create multiple copies of a particular section of DNA.

Photo Courtesy of Karen A. Tonogbanua