Marker-assisted Breeding and F₃ Progenies Characterization for Improving Local Rice Variety “Tinggong”

Tinggong is a rice variety cultivated by farmers in the southwest region of Aceh, Indonesia. The variety is adaptive to environmental conditions and has excellent cooking quality. Despite its advantages, characteristics of the plant need to be improved as its height reaches 143-180 cm, harvesting age 5-6 months and susceptibility to BLB disease caused by Xanthomonas oryzae pv. oryzae (Xoo) (Zakaria et al., 2021). Rice plants with a high stem architecture are not capable of supporting the weight of the grains, potentially causing the plants to fall and eventually yield losses. This circumstance is made worse by the occurrence of infectious diseases such as BLB that occurs in all major rice-growing areas in the world, especially in Asia, Northern Australia, Africa and the United States of America (Adhikari et al., 1995; Gnanamanickam et al., 1999; Sere et al., 2005). The disease causes yield losses of up to 70% and is a serious threat to agriculture and food security worldwide (Ke et al., 2019; Singh et al., 2009; Verdier et al., 2012).

Crossing indigenous rice with introduced types that bestow valuable genes might allow for the enhancement of rice varieties. IRBB27 is an introduced isogenic line that carries both Xa-27 and sd-1 genes. Xa-27 is an important R gene, effective against BLB disease while the sd-1 gene improves plant architecture (Luo et al., 2014; Makino et al., 2006; Radhamani et al., 2015). The utilization of the resistance gene (R) for rice improvement is the most efficient method for controlling BLB disease (Ikeda et al., 1990; Pradhan et al., 2015). In addition, the Xa-27 locus provided high resistance to 27 Xoo strains (Dossa et al., 2015). There are six R-genes includingXa-1, Xa-5, Xa-13, Xa-21, Xa-26 and Xa-27 that have been obtained through genetic map-based cloning (Luo et al., 2012). In Indonesia, there are 11 strains of Xoo bacteria with different virulence levels. Two Xoo strains that have a high virulence level are strain IV which has a high virulence level in the generative phase and strain VIII which is virulent in the vegetative phase and is dominant in several endemic locations (Suparyono et al., 2016).

Pathotype IV and pathotype V were two representative strains of Xoo pathotypes prevalent in South China (Zeng et al., 2002). Specifically, pathotype V and IV can overcome the resistance conferred by Xa4 and Xa21 (Zeng et al., 2002; Zhang, 2009), respectively. Moreover, two strains of Xoo from Indonesia were virulent to cultivars containing the bacterial blight resistance gene Xa5. In addition, the varieties of containing Xa21 and Xa5 genes were resistant to almost Asian Xoo strains. Indonesian rice cultivar Pandan Wangi” was susceptible to Xoo strain III and VIII and very susceptible to strain IV (Hadiwiyono et al., 2021).

The use of the sd-1 gene results in a short plant phenotype and improves plant architecture, increasing plants’ ability to assimilate substances into seeds (Vikram et al., 2016). Luo et al., (2014) employed the sd-1 gene to improve other Aceh local rice, called Siputeh, to shorten the harvesting age from 5.5 months to 3.5 months.

We have studied crossbreed between Tinggong and IRBB27 (Tg/IRBB27) in F2 and learned that 73.91% of plants with 115 cm height, 27.27% inherited resistance to Xoo strain IV. From these progenies, 21.73% of plants inherited both sd-1 and Xa-27 genes. Only 20% of these progenies have the potential to be developed as they can be harvested between 110-122 days after sowing and seed weight per hill up to 32 g or an estimated yield of 8 tons ha^-1.

The purpose of this study was to analyse the variation in genotypic and phenotypic levels of the characters F3 progenies from cross-breeding between Tinggong and IRBB27. Through this research, it is hoped that prospective progenies can be obtained, those that inherit both the Xa-27 gene and the sd-1 gene and have a phenotypic character resistant to BLB and better agronomic characteristics compared to their parent ‘Tinggong’.

Research implementation and genetical resources
 
The research was carried out in the experimental farm and Plant Breeding Laboratory, Faculty of Agriculture, Universitas Syiah Kuala, Darussalam Banda Aceh from March to July 2018. This study utilized 48 plants of F₃ progeny of the cross between Tg/IRBB27 as planting material. The plants were selected from F₂ Tg/IRBB27 that inherited sd-1 and Xa-27 as well as showed good agronomic characters (harvesting time 110-120 DAS, plant heigh 125-135 cm with production per hill 43.55 g (estimation yield 8 ton ha^-1).
 
Plants cultivation technique
 
The seeds were soaked for 24 hours for the imbibition, then germinated on straw paper for 48 hours. The germinated seeds were then sown on the sowing media consisting of soil and manure mixed with a ratio of 2:1. The seeds were transplanted after 15 days to a 5 kg pot containing soil and manure in a ratio of 3:1 (v/v). As much as 2.6 g pot^-1 of basic fertilizer, consisting of nitrogen, phosphorus and potassium (15-15-15) and 0.6 g pot^-1 urea were applied one day before planting. At the age of 15, 30 and 45 days after planting, urea was applied as top dressing at a dose of 0.6 g pot^-1.
 
Bacterial inoculation and disease scoring
 
The bacterial suspension of Xoo used in this study is Pathotype IV obtained from the Muara Rice Research Centre, Bogor, Indonesia. Reisolation was carried out at the Plant Diseases Laboratory, Faculty of Agriculture, Universitas Syiah Kuala. The bacterial suspension was grown on Natrium Agar (NA) media at 28°C for two days. The composition of the media was peptone 10 g.L^-1, sucrose 10 g.L^-1, glutamic acid 1 g.L^-1, bakto-agar 16 g.L^-1  and pH 7.0. Bacterial cells were mixed in distilled water at an optical density of 0.5 (OD 600) (Luo et al., 2012). Inoculation of Xoo was carried out 14 days after planting (DAP) by applying the leaf-clipping method (Kauffman, 1973). The tip of the leaves was cut (starting from the 3^rd leaf in each clump of 5 leaves) with scissors that have been dipped in Xoo suspension. Inoculation was carried out in the afternoon to avoid scorching heat and high evaporation.

The length of the lesion was observed 3 weeks after inoculation. The length of the lesion was measured from the tip of the cut leaf to the base of the leaf indicating Xoo infection. Disease scoring was measured as described by Gu et al., (2004).
 
Molecular analysis of sd-1 and Xa-27 gene presence in F₃ progenies
 
DNA was extracted from 0.2 g young leaves of each sample of rice plants at the age of 14 DAP. The leaves were cut into small pieces and placed into a 2 ml microtube containing one stainless steel bead. The samples were set in a microtube box and stored at -86°C for 3-5 hours. After the sample was crushed by shaking the tube until the leaf sample was crushed, each micro centrifugation tube contained the sample was filled with 300 µl of TPE buffer. The samples were then incubated in a water bath at a temperature of 65°C for 20 minutes. After incubation, the samples were centrifuged at 13,000 gravity for 10 minutes. 100 µl of supernatant DNA was taken and transferred into a fresh 1.5 ml microtube. The PCR template was constructed by mixing 1% DNA supernatant with distilled water that has been prepared in a 200 µl microtube (Koeda and Fujiwara, 2019).
 
PCR-based molecular markers and PCR conditions
 
The molecular marker for gene Xa-27 is the codominant SSR marker M964, located at 0.964 kb (F: 5-TGT GCA ATG CAG GAT TTC AGT TACT-3; R: 5-TTT CAC CTG CAT AAT GCA AAA GCT AA-3) (Gu et al., 2004). The molecular marker for the sd-1 locus is a co-dominant STS (sequence-tagged site) marker derived from the sd-1 gene. (F: 5-CAC GCA CGG GTT CTT CCA GGT G-3; R: 5-AGG AGA ATA GGA GAT GGT TTA CC-3) (Spielmeyer et al., 2002; Srivastava et al., 2019).

PCR amplification (My CyclerTm thermal cycler) for the Xa-27 gene was performed according to (Gu et al., 2004) with initial denaturation of DNA at 94°C for 2 minutes. The next denaturation was 35 cycles for PCR amplification, consisting of 94°C for 1 minute, 37.0°C for 45 seconds and 72°C for 1 minute and 30 seconds. Final extension at 72°C for 5 minutes. Whereas PCR amplification for the sd-1 gene was conducted with initial denaturation of DNA at 94°C for 5 minutes. The subsequent denaturation was 35 cycles for PCR amplification, consisting of 94°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes. Final extension at 72°C for 7 minutes (Spielmeyer et al., 2002; Srivastava et al., 2019). The amplified product was resolved on 1.5% agarose in 0.5xTAE buffer. For identification of amplification of Xa-27 gene, initially 5 µl of PCR product was used for gel electrophoresis. Electrophoresis was performed for 35 minutes for primary Xa-27 and 30 minutes for primary sd-1. The gel was visualized under ultraviolet light using UV Transilluminator.
 
Agronomic character analysis
 
The variables used for agronomic character analysis consist of harvesting time, plant height at harvesting time, the weight of 1,000 grains, the weight of grain per hill and production estimation per ha based on the population of 250,000 hills ha^-1.

Molecular analysis of sd-1 and Xa-27 genes presence in F3 progenies of Tg/IRBB27
 
The performance of F3 individual progenies based on the presence of Xa-27 and sd-1 genes by molecular analysis for 48 individual plants is shown in Fig 1.

Molecular analysis showed that 48 plants of F3 progenies analysed for the presence of the Xa-27 gene, 30 plants (62.50%) inherited the Xa-27 gene through DNA band formation at 0.96 kb. Meanwhile, based on the analysis of the presence of the sd-1 gene based on the co-dominant sd-1 STS marker, it showed that of the 48 plants analysed only 32 plants (66.67%) inherited the sd-1 gene through the formation of DNA bands at 0.85 kb. Fig 1 also shows that 48 individual plants derived from Tg/IRBB27, there were only 28 (58.33%) plants that inherited both the Xa-27 gene and the sd-1 gene.


 
The resistance of Tinggong/IRBB27 to X. oryzae and their agronomic characters
 
The symptom of BLB indicated by lesion length on the leaves of 48 F₃ progenies is shown in Table 1. The table showed that among all of the progenies 14 plants showed resistant reactions (29.17%) including #T5-8, T13-16, T18, T27, T29-30, T43 and T46. The table also showed that 19 plants (39.58%) showed moderately resistant reaction (MR), 4 plants (8.33%) showed moderately susceptible reaction (MS) and 11 plant (22.91%) showed susceptible reaction (S) at the third week after inoculation. The resistance of the F3 progenies genotype tested with Xoo strains IV after inoculation shows a positive response when viewed from the level of lesion length caused by Xoo disease inoculation (Fig 2). Plant resistance to BLB symptoms was evaluated quantitatively, such as disease index, lesion area, or lesion length (Bhattacharjee et al., 2020; Jerish et al., 2022; Ogawa, 1993; Sharma et al., 2020).



BLB intensity is highly dependent on the virulence level of X. oryzae, environmental conditions and climatic conditions in the area. Environmental conditions with lots of weeds and high humidity can stimulate the development of virulent Xanthomonas. High humidity conditions are usually often found in plants with close spacing with continuous watering conditions (Chen et al., 2020; Jiang et al., 2020). The mechanism of virulent bacteria in plants is that bacterial cells enter plant tissues through pores or leaf stomata. After entering the plant tissue, the bacteria then multiply or grow, then attack the vascular system of the plant. The liquid containing bacteria eventually comes out onto the leaf surface, which then forms a lesion.

Moreover, Table 1 also showed that the whole F₃ progenies had harvesting time from 104-129 DAS with plants height ranging from 90-135 cm or their harvesting time and plant height were 36.07% faster and 20.70% shorter compared to their female parents Tinggong respectively. The table also showed that the whole progenies had productive panicles ranging from 5-12, with panicle lengths ranging from 16.90-28.00 cm. The 1,000 grains weight and the weight of filled grains per hill of the progenies ranged from 1.81-38.78 and 26.24-34.12 respectively.



The age of the rice plant is the main factor for farmers in the selection of rice varieties to be cultivated. Rice plant age criteria based on Committee Standards (1980) early 110-125 DAS, moderate 126-145 and deep >145 DAS. In addition to improving plant architecture, the sd-1 gene can indirectly shorten the lifespan of rice plants. The sd-1 gene has been used by (Devi et al., 2019; Luo et al., 2014) to improve the architecture of rice plants and the lifespan of TS4 with its harvesting time and plant height shorter compare to its parent Siputeh.

Research results showed that 62.50% of F₃ progenies inherited the Xa-27 gene, 66.67% inherited the sd-1 gene and 58.33% inherited both the Xa-27 and sd-1 genes. Based on the existing both of these genes, resistance to strain IV of BLB diseases and agronomic performance, there were four F₃ progenies consisting #T5, T6, T29 and T30 plants were prospective to be developed further based on the criteria for early maturing age ranging from 116-120 DAS, short plant height of 109-128 cm and estimated yield per ha of 6.56-8.53 tons ha^-1.