Philippine Journal of Science 150 (S1): 17-28, Special Issue on Biodiversity ISSN 0031 - 7683 Date Received:27 Jul 2020 Genetic Diversity and Structure of Oryza rufipogon Griff. Populations in the Philippines Sandy Jan E. Labarosa1*, Neah Rosandra Sevilla2, Dindo Agustin A. Tabanao2, Nenita B. Baldo1, Helen L.V. Ebuña1, and Joy M. Jamago1* 1Department of Agronomy and Plant Breeding, College of Agriculture Central Mindanao University, Musuan, Maramag, Bukidnon 8710 Philippines 2Plant Breeding and Biotechnology Division, Philippine Rice Research Institute Science City of Muñoz, Nueva Ecija 3119 Philippines Oryza rufipogon Griff., or “Rufi” in the Philippines, was previously known to be found only in Lake Apo, Bukidnon. However, a new population was identified in Lake Napalit in the same province. A better understanding of the genetic diversity of both Rufi populations using molecular methods may be beneficial to further ascertain its usefulness in rice breeding and in the development of effective conservation strategies. Population genetic analysis was conducted to estimate the degree of genetic diversity and population structure of the two Philippine Rufi populations using 98 genome-wide simple sequence repeat (SSR) markers. Four Oryza sativa indica and three O. sativa japonica cultivars were added for comparison. Results indicate that both Rufi populations exhibit low genetic diversity but with significant population structure and differentiation. Low genetic diversity suggests that both populations might be in a genetic bottleneck, perhaps due to observed unsustainable farming practices near their habitat and lack of awareness among locals of their importance. Also, geographical isolation that prevented gene flow between the two populations, as well as the unique climatic conditions between the two lakes might have contributed to the significant population structuring and differentiation. Thus, in situ and ex situ conservation should be observed for both Rufi populations in the Philippines. Keywords: conservation genetics, crop wild relatives, diversity analysis, Oryza sativa, simple sequence repeats INTRODUCTION 2012), rice tungro disease (Shibata et al. 2007), and other biotic stresses (Shibata et al. 2007). It also has tolerance Oryza rufipogon Griff. (nicknamed “Rufi”, AA genome, mechanisms to several environmental stresses like acid 2n = 2x = 24) is the ancestor of the cultivated Asian rice sulfate soils (Bui and Nguyen 2017) and chilling stress (Oryza sativa L.) (Song et al. 2005). For breeding, Rufi (Cen et al. 2018). and 20 other wild rice species offer a reservoir of genetic diversity as essential sources of resistance and tolerance to Bon and Borromeo (2003) reported that the only natural some biotic and abiotic stresses, among others (Jacquemin populations of Rufi in the Philippines were in Lake Apo, et al. 2013). Rufi has resistance mechanisms to various Barangay Guinoyoran, Valencia City, Bukidnon. However, pests and diseases, such as bacterial leaf streak (He et al. in a class excursion by Jamago and her students in 2012, they recorded the existence of new natural populations *Corresponding Authors: [email protected] along Lake Napalit, Barangay Pigtauranan, Pangantucan, [email protected] 17 Philippine Journal of Science Labarosa et al. Genetic Diversity and Structure of Oryza Vol. 150 No. S1, Special Issue on Biodiversity rufipogon Griff. Populations in the Philippines Bukidnon. This new population might harbor novel alleles (PCR) was conducted per 6-µl reaction solution composed useful for current and future rice breeding programs. of 1.55 µl sdH20, 1.5 µl 10x PCR buffer, 0.6 µl 25 mM Balos and Jamago (2013) compared some morpho- magnesium chloride, 0.35 µl 5mM deoxynucleoside ecological parameters of Rufi populations in both lakes triphosphates, 0.5 µl forward primer, 0.5 µl reverse primer, in situ. Their findings showed that Lake Apo populations 1.0 µl 1:100 (concentrated suspension: storage buffer) Taq (Apo) had longer leaves, culms, and awns than the Lake DNA polymerase, and 1.5 µl diluted DNA. Napalit populations (Napalit). Also, they reported that Lake Napalit had higher average rainfall (73.50 mm) and Each locus was amplified using a PTC-100® Thermal was relatively colder (23.5 °C) than Lake Apo (average Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) rainfall: 26.33 mm; average temperature: 25.3 °C). These for 2 h and 30 min, following the PCR profile: 5 min variations in morphological and ecological parameters initial denaturation at 94 °C, followed by 35 cycles of between the two populations offer an opportunity for 1 min denaturation at 94°C, 1 min annealing at 58 °C, researchers and breeders to explore for their possible use and 2 min extension at 72 °C; then a final extension at in rice breeding programs. 72 °C for 5 min. PCR products were subjected to 6% polyacrylamide gel electrophoresis in 1x TBE buffer at 90 This study estimated the magnitude of genetic diversity V for 1–2 h depending on the size of PCR products. Gels and determined the population structure of in situ were stained using an ethidium bromide solution for 10–15 populations of O. rufipogon Griff. in the Philippines using m. DNA bands were visualized using the AlphaImager® SSRs. Further, allelic patterns and degree of population HP system (ProteinSimple, San Jose, CA, USA). Detected differentiation across the populations were also calculated. allelic bands were scored from the fastest to the slowest Information on Rufi’s genetic diversity and population migrating alleles by letters a, b, c, and so on. structure would provide more clarity on its potential for utilization and even insights for its conservation. Data Analysis A total of 128 SSR markers were initially selected to assess the genetic diversity and population structure of 41 Rufi samples along with seven rice cultivars for comparison, MATERIALS AND METHODS which represented the two O. sativa subspecies, i.e. japonica and indica. SSR markers with > 20% missing Leaf Sampling and Genotyping band across all samples were excluded and only 98 Twenty-seven and 14 Rufi strains from Lake Napalit (~ markers were retained for further analysis (Appendix I). 1,041 masl) and Lake Apo (~ 640 masl) populations, Rufi samples per lake were examined for genetic diversity respectively, were sampled in situ in 2014 spaced at per population. Genetic diversity indices per population least 30 m to minimize the collection of leaves from were assessed by calculating the mean number of alleles, possible clonal plants. Leaf sampling was done by cutting observed heterozygosity (Ho), expected heterozygosity 4–5 in from tips of flag leaves. Leaf cuttings per strain (He), Shannon information index (I), and fixation index were secured in individual 200 ml centrifuge tubes that (F). Calculations were performed using GenAlEx ver. 6.5 were placed immediately in an icebox before storing at (Peakall and Smouse 2012). In addition, allelic richness –20 °C until DNA extraction. Total genomic DNA was (AR) and private allelic richness (PAR) were calculated extracted from lyophilized leaf tissues following the using the rarefaction method implemented in HP-Rare modified cetyltrimethylammonium bromide method v1.1 (Kalinowski 2005). This accounts for the differences of Perez and colleagues (2012), then dissolved in tris- in sample size between the pre-defined population groups ethylenediaminetetraacetic acid buffer. DNA samples of Rufi and O. sativa. were quality-checked through electrophoresis on 1% agarose gel. DNA stocks of four O. sativa indica (i.e. Moreover, the population structure of Rufi and rice NSIC Rc192, PSB Rc14, PSB Rc18, and PSB Rc82) and populations were evaluated by phylogenetic analysis. three O. sativa japonica cultivars (i.e. Azucena, Li-Jiang- The pairwise genetic distance matrix for all 48 genotypes Xin-Tuan-Hei-Gu, and Nipponbare) from the Philippine was constructed using Nei’s genetic distance (Nei 1972). Rice Research Institute were added for comparison. Neighbor-joining (NJ) tree was used for cluster analysis. A boot function from “poppr” package (Kamvar et al. 2015) One-hundred twenty-eight polymorphic genome-wide was used to build and visualize the unrooted dendrogram SSR markers for O. sativa were initially selected based on following 1000 bootstraps. The analysis was done using a 3-mb bin system to allow uniform marker distribution in R (R Core Team 2020). Two models were implemented the chromosomes. These were used to evaluate the genetic to determine the patterns of the genetic structure of variation of 41 Rufi samples, including seven O. sativa populations. Model 1 only analyzed the Apo and Napalit cultivars, for comparison. Polymerase chain reaction Rufi populations, whereas Model 2 included the japonica 18 Philippine Journal of Science Labarosa et al. Genetic Diversity and Structure of Oryza Vol. 150 No. S1, Special Issue on Biodiversity rufipogon Griff. Populations in the Philippines and indica rice cultivars. A systemic Bayesian clustering in both the japonica and indica group (Table 1). This approach applying Markov Chain Monte Carlo (MCMC) higher diversity observed among O. sativa compared estimation was performed using STRUCTURE ver. 2.3.2 to Rufi might be due to its diverse countries of origin, (Pritchard et al. 2000). The analysis was set using the and which were also bred for different ecotypes and admixture ancestry model, with the number of assumed came from different genetic background. The former is populations (K) set from 1–10, and each was run 10x. different from the natural populations of Rufi – which Each run started with 500,000 burn-in periods followed were geographically isolated, with restricted gene flow, by 500,000 MCMC iterations. MCMC was ran using a and might have been predominantly inbred – resulting in correlated allele frequency model based on the default heterozygote deficiency. frequency model information under the advanced option of STRUCTURE. Results from STRUCTURE were collated Population Structure and Differentiation and imported to the web-based application Structure Population genetic structure was analyzed using the NJ Harvester (Earl and von Holdt 2012) to calculate for tree based on Nei’s genetic distance.