Screening of Blast Resistance Genes from South Tapanuli Rice Cultivars, North Sumatra, Indonesia

Corresponding Author: Saleha Hannum Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia Email: saleha@usu.ac.id Abstract: Rice blast disease caused by a phytopathogenic fungus, Magnaporthe oryzae, has been considered as a major threat to global rice productivity, including Indonesia. South Tapanuli is a regency in North Sumatra, known for its intensive and significant rice production activities. A collection of 13 rice cultivars originating from the region, is claimed for its resistances against the rice blast disease based on the practices by the local farmers. Molecular screening of blast resistance (R) genes was initiated to the 13 accessions using the Polymerase Chain Reaction (PCR) targeting five blast resistance (R) genes, Pup1, Pi-37, Pi-d2, Pi-ta, Pib and Pi-kh. Out of the 13 accessions, the Pi-d2, Pi-ta and Pib were detected in all cultivars, showing 100% of R gene frequency. The second dominant, Pup1 and Pi-37 gene frequencies were 76.9 and 61.5%, respectively while the least dominant, Pi-kh gene frequency was 15.4%. The number of R genes detected among cultivars was ranged between 3 to 6 genes. The genetic associations among thirteen germplasms were determined using unweighted pair group method with arithmetic mean (UPGMA) analysis. Cluster analysis revealed that the least blast-resistant cultivars: Putri Kembar, Silatian and Siganteng, were grouped into cluster 1, showing polymorphism for Pi-d2, Pi-ta and Pi-b. The high-resistant cultivars, Martabe-Sicondong and Sayuti, Sitolas, Pulo Raja, Pulo Pandan, Siporang and Pulo Manggis, were grouped into cluster 2 and 3, respectively. The medium blast-resistant cultivars, IR 64 and Sitampan were grouped into cluster 4, showing polymorphism for Pup1, Pi-d2, Pi-ta and Pi-b. These results indicated that the utilization of blast-resistant cultivars in North Tapanuli was supported by the presence of R genes.


Introduction
Rice (Oryza sativa L.) is one of the essential agricultural commodities in the world and becoming a staple food to almost all populations in the tropical region. The increasing demands upon rice agricultural practices are related to the increasing population in Asia, Africa and Latin America (Wang and Li, 2005).
In Indonesia, intensive research and development have been efforted to yield a higher rice productivity annually as well as developing a stable or higher production in a dynamic environment modified by the presence of abiotic and biotic stressors (Panuju et al., 2013). Indonesia's rice production is constantly threatened by several biotic stressors, from important pathogenic diseases to disruptive physical damages by insect pests. Rice blast disease is one of the most defective conditions with a fatality of plant deaths, caused by Pyricularia grisea (Cooke) Sayc., (cyn.: P. oryzae Cavara), or the teleomorph Magnaporthe oryzae (Hebert) Barr. (Rossman et al., 1990;Couch and Kohn, 2002;Choi et al., 2013).
The substantial loss of global rice productivity impacted by the diseases, may reach 30% or equivalent to a food supply for 60 million people (Nalley et al., 2016). Moreover, the presence of rice blast disease directly decreased the rice yields along with increased production costs, which become one of the largest challenge in increasing rice production (Skamnioti and Gurr, 2009).
Control and management of blast diseases in rice fields incorporated the use of chemical pesticides, fungicides and fertilizers which are considered highly cost and detrimental to the environment, involved in the presence of residual toxins and pollution (Sha et al., 2016). Furthermore, resistant populations of the pathogen may also emerged due to extensive use of antibiotics or fungicides (Chen et al., 2019). In the brink of inconsistencies, the utilization of blastresistant varieties is considered as the most economical, efficient, effective and environmentalfriendly method to prevent the rice blast disease incidence (Ghazanfar et al., 2009;Idowu et al., 2013;Mau et al., 2018).
Blast-resistant rice cultivars are genetically structured by numerous R genes and QTLs responsible for blast disease resistances. The genes are priorly identified through molecular screening and marker-assisted selections to produce superior hybrids for field application (Fukuoka et al., 2014;Yan et al., 2017). The functional traits may be studied thoroughly by preliminary screening of R genes and associated genes within the wild species and local cultivars.
Indonesia is rich in local cultivars and varieties, produced independently by local farmers, breeders and researchers (Vasudevan et al., 2014). According to the International Rice Genebank Collection Information System, approximately 10,205 rice accessions are currently reported in database which are originating from Indonesia (IRGCIS, 2020). The prospect upon finding potential blast-resistant cultivars is progressively conducted due to the richness of local species which has not been fully revealed across provinces and regions, especially in North Sumatra.
Rice farming in North Sumatra province was ranked third in the Sumatra region, with the yield of 2,078,901.59 tons in 2019 (ISA, 2019). In 2016, South Tapanuli was placed 7 th among regencies, producing 173,444 tons of rice with a trend of increasing productivity year to year (ISA, 2017). The local farmers utilized the highlands as planting areas for 13 indigenous rice cultivars, including the oldest IR 64 cultivar, which were claimed to be considerably resistant toward blast rice disease. There are still limited informations on the genetic basis of blast resistance among the collection of 13 rice germplasms originating from this region. This study then aimed to analyze the genetic relationship of blast resistance genes (R) in the local rice cultivars.

Plant Materials
Thirteen rice germplasms including the local cultivars and standard cultivar, IR 64 originating from various sites in South Tapanuli regency were selected, based on the claims upon their medium to high resistance to blast disease ( Table 1). The seeds were collected and germinated in the dark on moistened filter papers for 2 days at 30C in an incubator. The germinating seeds were then planted in plastic pots at maintained temperature, 27-30C with daily sunlight exposure for 12 h. The seedlings were grown for 3 weeks prior genomic DNA isolation.
The supernatants were collected and mixed with 700 µL cold isopropanol and incubated in a freezer for 2 h. Solutions were centrifuged at 12,000 rpm for 10 min at 4°C. Pellets were collected and suspended in 500 µL 70% ethanol with further centrifugation at 12,000 rpm for 10 min at 4°C. Pellets were air-dried and suspended into 50 µL ddH2O and incubated at 37°C for 1 h.

DNA Marker Analysis
The rice germplasms or accessions were genetically screened for the presence of six major blast resistance (R) genes, Pup1, Pi-37, Pi-d2, Pi-ta 2 , Pib and Pi-kh using PCR markers with details of information listed in Table 2. All of the synthesized primers were obtained from Macrogen, Inc. South Korea. The PCR analyses were conducted in a thermal cycler (SensoQuest GmbH, Germany) with PCR reaction set as follows: 5 µL GoTaq® Green Master Mix solution, 3 µL nuclease-free water, 0.5 µL forward primer, 0.5 µL reverse primer, 1 µL DNA template.  PCR amplification was performed with following specification: 94°C for 1 min, annealling at different temperature (Table 2) for 45 s, 72°C for 2 min and 72°C for 5 min. All PCR reactions for each sample were repeated three times. PCR products were separated by electrophoresis on 1.2% agarose gels in 1× TAE buffer at 70 V for 60 min. The gels were stained with ethydium bromide solution and visualized under UV transilluminator (Fire-Reader XS-D56, England). The DNA amplicons were recorded as absence (0) or presence (1) accordingly.

Data Analysis
All data sets were arranged using Microsoft Excel. Cluster analysis was performed using the Multivariate Statistical Package (MVSP) ver. 3.1A to produce a dendrogram of relationship among rice cultivars based on the binary matrix or Sørensen-Dice coefficient and unweighted pair group method with arithmetic mean (UPGMA) analysis (Kovach, 2007).

Results
Six R primers were used to amplify and detect the presence or absence of blast resistance genes and associated R genes from the thirteen South Tapanuli germplasms. All the 13 accessions possessed at least three to six R genes as revealed by the bands for different markers in agarose gel electrophoresis. The 'Sicondong' and 'Martabe' cultivars showed DNA bands in all rice blast resistance genes, Pup1, Pi-37, Pi-d2, Pi-ta 2 , Pi-b and Pi-kh. Five others were positive for five markers. Three cultivars possessed four markers, while two cultivars showed positive bands for three markers (Fig. 1).

Discussion
Utilization of medium to high blast-resistant rice cultivars in agricultural practices needs to consider the genetic stability of the germplasms. In addition, rice fields planted with only a monoculture of even highresistant cultivar may lead to the evolution of a more virulent strain of Magnaporthe oryzae (Vasudevan et al., 2016). Therefore, screening of new blast resistance (R) genes from local cultivars and wild relatives are still relevant until now to maintain the availability of blastresistant germplasms.
Contribution of finding new blast resistance genes involved in Pathogen Associated Molecular Pattern (PAMP), is to introduce new traits to hybrid cultivars with superior resistance against blast disease through genetic engineering or introgression (Bao-hua et al., 2017). Moreover, the genetic diversity of the most rice accessions has not been fully investigated despite the abundant source of germplasms, especially across regions in Indonesia (McCouch et al., 2012).
In this study, we performed genotyping to the 13 South Tapanuli rice cultivars, by detecting the presence of six R genes, Pup1,  and Pi-kh with the genetic frequencies ranged from 15.4 to 100.0% or at least three positive bands out of the genes. Our results were similar to the study by Kim et al. (2010) in 84 aromatic rice accessions from Korea, possessing at least three positive bands out of eight R genes (37.5%). Imam et al. (2014) reported the lowest presence of one positive bands out of nine R genes (11.1 %) in 32 rice accessions from North East and Eastern India. Singh et al. (2015) reported the R genetic frequencies from 19.79 to 54.69% in 192 rice accessions from India with at least one positive bands detected out of 10 R genes (10.0%).
In a more recent study, Yan et al. (2017) reported the genetic frequencies of 11 R genes, ranging from 9.4 to 100.0% in 32 Chinese rice germplasms while Yadav et al. (2019) reported the genetic frequencies ranged between 4.96 and 100.0% in 161 Indian rice landraces. Prior to this study, Hannum et al. (2018) have screened 15 rice accessions originating from other districts in North Sumatra and reported higher genetic frequencies from 47.0 to 100.0%. Despite the genetic variability of the studied rice germplasms across regions, selection of blast resistance genes will produce variable results in genetic frequencies yet to be confirmed in the field tests.
The 13 local rice cultivars have been claimed for its blast resistance based on the field trials by the local farmers. The results can then be regarded as a bottom-up evaluation to prove the scientific basis of the resistance. The existence of R genes in our study may indicate a possibility of finding other local germplasms with a larger number of blast resistance genes from other regions in North Sumatra (Vasudevan et al., 2014).
The 13 accessions possessed at least three to six blast resistance genes in which Pi-d2, Pi-ta 2 and Pi-b were detected in all cultivars. Pi-d2 is located in chromosome 6, which encodes for a receptor-like kinase involved in PAMP-Triggered Immunity (PTI) responses (Chen et al., 2006). Pi-ta or Pi-ta 2 are two tightly-associated blast resistance genes located in chromosome 12 (Rybka et al., 1997). Recently, a novel broad spectrum R protein was reported to be encoded by Pi-ta 2 although the varieties studied by International Rice Research Institute (IRRI) were relatively low in frequency despite its high potential for future breeding of blast resistant varieties (Meng et al., 2020). Pi-b is located in chromosome 2 and a member of the Nucleotide Binding Site -Leucine-Rich Repeats (NBS-LRR) class of blast resistance gene (Wang et al., 1999). However, previous study reported that the detection of Pi-b produced none effect or even a susceptibility to M. oryzae based on the field study (Imam et al., 2014).
Pup1 and Pi-37 were detected as second dominant R genes in our study. Pup1 or phosphorus uptake 1 locus is located in chromosome 12, which encodes major Quantitative Trait Locus (QTL) for phosphorus (P) tolerance (Heuer et al., 2009). Chithrameenal et al. (2018) stated that majority of the modern rice cultivars lacked Pup1 loci, yet highly susceptibility to P starvation. Therefore, genetic introgression of both P tolerant gene and blast resistance gene into new cultivars is a strategy to yield a higher rice productivity in the future. Meanwhile, Pi-37 is a member of NBS-LRR which is also responsible for the blast resistance (Lin et al., 2007). In addition, Yan et al. (2017) reported that both Pi-36 and Pi-37 were less likely detected from rice accessions.
Pi-kh was the least detected R gene with only two cultivars in our study. Pi-kh was a former name for Pi54, cloned from rice line Tetep which only induced in response to pathogen infections (Sharma et al., 2010). The over-expression of Pi-kh with a strong promoter CaMV 35S, has proven to improve rice tolerance to M. oryzae blast disease (Azizi et al., 2016).
Based on the dendrogram, it can be seen that there was no spatial effect to the detection of R genes in different districts. The result suggested that tolerance of the cultivars against pathogenic fungus were not based on environmental condition of rice fields. Moreover, pathogenicity assay is needed to confirm the interaction between associated rice R genes with disease incidences in field trials since less frequent detected R genes may be related to the increased susceptibility of plants to M. oryzae (Imam et al., 2014). In the future, a large scale analysis of detecting multiple R genes may be investigated as well as identifying other rice cultivars originating from North Sumatra, as a hidden source of blast-resistant cultivars in the future.

Conclusion
Thirteen rice cultivars from South Tapanuli, North Sumatra previously claimed to be moderately or highly resistant to blast disease, were proven to possess at least three R genes, with the most frequent genes, Pi-d2, Pita 2 and Pi-b (100.0%), followed with Pi-37 (61.5%), Pup1 (76.9%) and Pi-kh (15.4%) detected from all cultivars. Although our study may be considered as the first to reveal the genetic basis of the indigenous rice cultivars, more efforts are needed to confirm the contribution of single or multiple R genes involved in resistance on the field or through pathogenicity assay.