Low Association of Bph17 Allele in Landraces and Improved Varieties of Rice Resistant to Brown Planthopper

Resistance traits to brown planthopper on rice varieties are controlled by dominant and recessive genes called Bph/bph. Bph17 is one of dominant genes that control rice resistance to brown planthopper. Marker of Bph17 allele can be used as a tool of marker assisted selection (MAS) in breeding activity. Association of Bph17 allele and resistance to brown planthopper in Indonesian landraces and new-improved varieties of rice is not clearly known. The study aimed to determine the association of Bph17 allele in landraces and new-improved varieties of rice resistant to brown planthopper. Twenty-one rice genotypes were used in the study, consisting of 13 landraces, 5 improved varieties, 3 popular varieties and a check variety Rathu Heenati. Two simple sequence repeat markers linked to Bph17 allele were used, i.e. RM8213 and RM5953. The results showed that association of Bph17 allele in landraces and new-improved varieties of rice resistant to brown planthopper resistance was very low (r = -0.019 and -0.023, respectively). The presence of Bph17 allele did not constantly express resistance to brown planthopper. The study suggests that Bph17 allele cannot be used as a tool of MAS for evaluating resistance of landraces and new-improved varieties of rice to brown planthopper. Further research is needed to obtain a specific gene marker that can be used as a tool of MAS and applicable for Indonesian differential rice varieties.


INTRODUCTION
Brown planthopper (BPH; Nilaparvata lugens) is a major pest of rice crop around the world, including Indonesia. The pest is cosmopolitan, potentially reducing rice production even causing crop failure (Watanabe et al. 2009;Direktorat PTP 2016). Rice plants stricken by BPH show symptoms of leaf yellowing and dry, stunted growth, and eventually die (Baehaki 2012). The 100-140-day old rice plant had higher number of BPHs per hill compared to 80-90-day old crop (Prashant et al. 2012). Current technology that effectively controls BPH is a resistant variety (Baehaki and Mejaya 2014).
Resistance traits to BPH on rice varieties are controlled by major and minor genes called Bph/bph ). The genes were mapped on chromosomes 2, 3, 4, 6, 7 and 9 (Liu et al. 2001;Liu et al, 2009). Bph17 is one of dominant genes that control resistance trait to BPH ). The gene is located on chromosome 4 (Rahman et al. 2009) and derived from BPH donor resistance gene of Rathu Heenati (Sun et al. 2005).
Bph17 gene has been used as a donor in breeding program of BPH-resistant rice varieties (Iswanto et al. 2015). Sun et al. (2005) and Jena et al. (2006) revealed that RM8213 and RM5953 DNA markers are closely linked to Bph17 gene that controls the expression of resistance trait. These markers can be used as a tool of marker assisted selection (MAS) with marker alleles at 177 bp on RM8213 PCR products and 140 bp on RM5953 PCR products (Sun et al. 2005;Jena et al. 2006). DNA products of RM8213 follow the Mendelian inheritance pattern of 1:2:1 (susceptible: heterozygous segregation: resistant) (Pertiwi et al. 2014;Carsono et al. 2016).
The success of MAS such as Bph17 marker depends on several factors, including genetic base of trait, degree of association between molecular marker and target gene, number of individuals analyzed, and genetic background of the target gene to be transferred (Francia et al. 2005;Wang et al. 2008). DNA markers such as Bph17 need to be evaluated for identifying BPH biotypes. The use of MAS to suspect and avail the selection of simply inherited traits is increasingly important in breeding programs, allowing an acceleration of breeding process, and is not affected by the environment or growing conditions (Guimarães et al. 2007;Bahagiawati 2012). Many of MAS are used as a marker assisted breeding (MAB) for selection of segregated population, but sometimes cannot be used for selection of non-breeding populations as well as landraces or local rice (depending on the marker trait). Carsono et al. (2016) found 63 selected lines from F2 progenies of resistant parent based on the linked marker of Bph17 allele of Rathu Heenati as the check variety.
Landrace is a germplasm containing resistance genes to pests and diseases (Sitaresmi et al. 2013). Some landraces and new-improved varieties of rice have been identified for resistance to BPH (Yunani et al. 2014;Jamil et al. 2015). However, the presence of resistance genes, especially Bph17 in Indonesian landraces and some new-improved varieties has not been intensively studied. To support breeding program based on landrace populations, it is necessary to study the presence of these BPH resistance genes in Indonesian landraces and several new-improved varieties of rice and find out the association of Bph17 allele position on Rathu Heenati.
The study aimed to determine the association between Bph17 allele in landraces and improved varieties of rice and resistance to BPH.

MATERIALS AND METHODS
The study was conducted in 2015 in DNA Laboratory of Plant Breeding Division, Indonesian Center for Rice Research (ICCR) at Subang, West Java.

Plant Materials
The study used 21 rice genotypes, consisted of 13 landraces from various provinces in Indonesia, one positive check variety (Rathu Heenati), five new improved varieties and three popular varieties ( Table  1). The rice genotypes belonged to ICRR. Ten to twenty seeds of each accession were germinated in the planting medium then put into the germinator cabinet. The 21 day-old rice seedings were transplanted into polybags containing a mixture of soil and sand growth medium (50:50). The plants were kept in the greenhouse of Plant Breeding Division, ICRR, and maintained according to protocol of The Crop Manager version 1.0 by IRRI (http://webapps.irri.org).

Molecular Analysis
Molecular analysis was done using simple sequence repeat (SSR). The analysis consisted of five major activities, namely DNA isolation, DNA quantity and quality test, polymerase chain reaction (PCR) amplification, electrophoresis of PCR products, and visualization of electrophoresis products.

DNA Isolation and DNA Quantity and Quality Test
Five young leaves of ten-day old rice seedlings of each accession were taken and used for DNA isolation. The DNA was extracted following the method of Murray and Thompson (1980) by small modification on leaf crushing. The leaves were crushed in a mortar without liquid nitrogen and homogenized with 800 µl CTAB buffer. DNA quality and quantity were measured using NanoDrop 2000/UV-Vis Spectrophotometer at 260 and 280 nm.

PCR Amplification
DNA of Bph17 allele in the leaf samples was amplified using SSR markers, i.e. RM8213 and RM5953 (Sun et al. 2005) (Table 2). Extracted DNA was amplified using PCR machine (BIO-RAD T100 TM Thermal Cycler) applying the ICRR DNA Laboratory procedure. PCR cocktail was made consisting of 50 ng DNA sample, 0.25 μM forward and reverse primers, 100 μM dNTPs, 1x PCR buffer (consisting of 20 mM Tris pH 8.3, 50 mM KCl, 1.5 mM M g Cl 2 , and 0.01% gelatin), and 0.5 units Taq DNA polymerase. PCR amplification was performed under the following conditions: denaturation at 95ºC for 5 min, 35   (2015) cycles of 1 min denaturation at 94ºC, 1 min annealing at 55ºC, 1 min extension at 72ºC, and a final extension at 72ºC for 5 min. The amplification was verified by continuous polyacrylamide gel electrophoresis (8%) to ascertain the presence of amplifiable DNA under 100 volts for 60 minutes in 1 x TBE buffer.

Data Analysis
The amplification data were analyzed based on the presence (1) or absence (0) of DNA bands. DNA polymorphism of Rathu Heenati was used as a reference of the Bph17 allele. The presence of Bph17 allele on each accession was ascertained at a distance of 177 bp and 129 bp based on RM5953 and RM8213 markers existing on the check variety Rathu Heenati. Association of the presence of Bph17 allele bands and plant resistance to BPH was analyzed using correlation analysis by Minitab version 13 software.

BPH Resistance
Resistance levels of rice genotypes to BPH biotype 3 varied (   36, Inpari 37 and Rojolele were susceptible to BPH. Data on resistance to BPH biotype 3 from ICRR Gene Bank showed that biotype 3 is the most virulent biotype. Therefore, the biotype was selected to investigate the existence of Bph17 allel. Brown planthopper has a high genetic plasticity, making it easier and faster in forming a new biotype (Baehaki and Widiarta 2009). Therefore, breeding of rice varieties having durable resistance to BPH is needed to compensate the development of BPH biotypes (Baehaki 2012;Baehaki and Mejaya 2014). Resistance trait to BPH has a narrow genetic variability. Nugaliyadde et al. (2016) reported that resistance to BPH was monogenic dominant based on the damage reaction at seedling stage of F1 and F2 generations from crosses between PTB33 and susceptible variety. On the other hand, Sai Harini et al. (2013) stated that resistance trait to BPH had a wide genetic variability based on molecular analysis results.
Bph17 allele could be used for marker assisted selection (Sun et al. 2005). For rapid identification requirements associated with resistance trait to BPH in Indonesian rice landraces, the role of this allele and its presence need to be investigated clearly. Specific markers for Bph17 were mapped at RM8213-177bp and RM-5953-129bp on Rathu Heenati. Table 3 shows that RM8213-177 bp was not only present in positive-check variety (Rathu Heenati), but also in nine landraces, three new improved varieties and Batanghari. Meanwhile, based on the results of RM5953 amplification at 129 bp, specific band was present on check variety and 10 landraces, but the band was absent in new improved varieties tested (Figure 1) (Table 3).

Identification of Bph17 Allele
One landrace (Si Awak) had Bph17 allele and medium resistance to BPH biotype 3. The contradictive results

Association Between Bph17 Allele and BPH Resistance
The presence of the bands that indicate Bph17 had a very low relationship with BPH resistance. The analysis result showed that the correlation between the presence of RM8213-177bp and RM5953-129bp and BPH resistance was very low and not significant (r = -0.019 and -0.023, respectively). This means that the presence of Bph17 allele had very low association with resistance traits to BPH in Indonesian landraces and new-improved varieties of rice. The presence of Bph17 allele mostly contradicted with the resistance information on landraces and new-improved varieties. The data revealed that some Indonesian landraces that had a Bph17 gene were not necessarily resistant to the most virulent BPH and the resistant varieties did not necessarily have a Bph17 gene. Some landraces that have a Bph17 allele (the same allele with Rathu Heenati, positive check variety) based on SSR analysis were susceptible to BPH. It is allegedly because although the size of DNA bands are the same, the DNA sequences are completely different.
The same result was observed by Damayanti (2014) on bph4 alelle. The allele was absent in resistant genotypes, but it was present in susceptible genotypes. The Bph1 allele also had a low association with resistance to BPH in promising lines. Rahmini et al. (2012) reported that IR42 has a bph2 allele, but the feeding activity of BPH of bitype 3 on this variety is very high. Sun et al. (2006) reported the great progress in MAS development in recent years, but relatively few varieties or lines successfully developed by this method due to low association of specific marker and resistance. The presence of one kind of Bph/bph allele was not enough information to guest plant resistance to BPH. Satoto et al. (2008) reported that pyramiding analysis is needed as resistance to BPH is controlled by many genes.  said that not all Bph/bph allele markers can be used as MAS. Bogadhi et al. (2015) found more than one BPH resistance genes in each resistant genotype.
Microarray analysis showed that BPH resistance in Rathu Heenati (donor of Bph17 gene) may be controlled by a series of resistance-related genes (Wang et al. 2012). Based on this research result, the presence of Bph17 allele in Indonesian landraces and new-improved varieties does not merely show plant resistance to BPH. Association of Bph17 alleles in landraces and new-improved varieties of rice does not constantly express resistance to BPH. Landraces and varieties resistant to BPH in this study had different resistance genes to those of Rathu Heenati.
So that, RM5953-129bp and RM8213-177bp cannot be used for analyzing rice varieties resistant to BPH of biotype 3. The presence of these markers had no correlation with resistance trait on new-improved rice varieties, i.e. Inpari 34, Inpari 35, Inpari 36, Inpari 37 and Inpari 38. Therefore, it is necessary to search for new genes that control the resistance trait to BPH in landraces or varieties originated from Indonesia.

CONCLUSION
The association of Bph17 alleles in landraces and newimproved varieties of rice resistant to brown planthopper was very low (r = -0.019 and -0.023, respectively). The presence of Bph17 allele does not constantly express resistance to brown planthopper. The study suggests that Bph17 allele cannot be used as a tool of MAS for evaluating resistance of Indonesian landraces and newimproved varieties of rice to BPH. Further research is needed to obtain a specific gene marker that can be used as MAS and applicable for differential rice varieties from Indonesia.