Materials and Methods Studi Genetik Toleransi Genangan Pada Padi

18-28 . Meanwhile for grain yield, 8 SNPs markers were significant with probability 0.05 and R 2 was ranged 11-18 . R 2 showed magnitude of QTLs effect controlling the phenotype. Although it was detected with less statistical power, according to Suwarno et al. 2015, MAF0.05 but 0.02 is still appropriate to help in identification of smaller set of candidates genes using bi-parental population. Based on General Linear Model GLM association analysis, significant genomic regions for plant height and number of tiller were identified based on P value 0.05. The significant markers for plant height were TBGI275345, TBGI275367, dan TBGI424383. TBGI275367 was expected on same position with LOC Os06g09630 which probably related with 3-oxoacyl-synthase gene. LOC Os06g09630 were functionally annotated as chloroplast precursor putatively expressed genes on the basis of the homology with the previous annotated genes. Chloroplasts have been observed to be involved in light perception and interact with photoperiodic development acting as major light harvesting organs Lepistö et al., 2009. TBGI424383 is located in chromosome 11 and was mapped near from probably gene controlling serine protease. Serine protease have been shown to be involved in diverse processes regulating plant development and defense response to abiotic and biotic stress Nakagami et al. 2005; Schaller 2004; Adam et al. 2001; Palma 2002. The significant SNPs for number of tiller were TBGI000722, TBGI258600, TBGI270843, TBGI271066, TBGI271076, TBGI272122, TBGI272241, and TBGI327790. The SNPs marker TBGI271066 is associated with OsWAK61 gene. The Wall-Associated Kinase WAK is a subfamily of receptor-like kinase RLK, with some members identified as associated to cell wall, suggesting these genes are strong candidates to act as sensors directly linking the extracellular environment to the cytoplasm and triggering intracellular signals. TBGI327790 was associated with Glucan endo-1,3-beta glucosidase. Glucan endo-1,3-beta glucosidase protein that were up regulated under stress Chen et al. 2016. Proteins involved in multiple pathways showed significant changes in expression in response to stress environment, including oxidative stress response proteins, beta-glucanases, protein; kinases, glycine, and photosynthesis proteins. Signal transduction is an important molecular response which plays a major role in protecting the plants by modulating the stress-responsive gene expression. These signals may be transmitted to neighboring cellsplants by emitting volatile organic compounds Bruinsma et al. 2008. TBGI270843 was allegedly in same position with LOC_Os06g04810.1 which is associated with leucine rich repeat protein gene. Table 5.2 SNP marker significantly associated with plant height, no of tiller, weight of 100 grain, panicle length, and grain yield of rice genotypes under stagnant flooding stress Traitmarker Chr Position P value R 2 Nearby gene candidate Plant Height TBGI275345 6 4869776 0.036 0.129 - TBGI275367 6 4897700 0.038 0.130 3-oxoacyl-synthase, chloroplast precursor TBGI424383 11 6199852 0.037 0.128 Serine protease-like proteins No. of tiller TBGI000722 1 421982 0.031 0.113 Expressed protein TBGI258600 5 25247484 0.039 0.106 Glucose 1 dehydrogenase GlcDH TBGI270843 6 2096700 0.038 0.107 Leucine rich repeat protein, putative, expressed TBGI271066 6 2229688 0.034 0.110 OsWAK61 - OsWAK receptor-like protein kinase TBGI271076 6 2234192 0.018 0.129 OsEF3 TBGI272122 6 2829380 0.044 0.067 - TBGI272241 6 2877413 0.01 0.152 - TBGI327790 7 21148555 0.034 0.110 Glucan endo-1,3-beta glucosidase precursor The most significant marker for the characters was described with QQ plot Fig 5.1. QQ plot is a visualization of Table 5.1. Significant marker was showed by highest Log 10P-value. The SNP marker associated with number of tiller, TBGI272241, which located in chromosome 6 at 2877413bp. TBGI2241 is not yet uncharacterized http:oryzasnp.plantbiology.msu.edu so that its gene has not been identified yet. This provides an opportunity to identify a new gene associated with number of tiller in relationship with stress water tolerance. Figure 5.2 Quartile-quartile plot QQ plot determines marker-traits association compare to the expected result of rice genotypes

5.4 Conclusion

Based on General Linear Model GLM association analysis, significant marker for plant height with P value 0.05 were TBGI275345, TBGI275367, and TBGI424383. Significant markers for number of tiller were TBGI000722, TBGI258600, TBGI270843, TBGI271066, TBGI271076, TBGI272122, TBGI272241, and TBGI327790. TBGI424383 and TBGI271066 markers probably associated with family of protein kinase which play role in plant stress signaling. TBGI272241 6 GENERAL DISCUSSION

6.1 Agro-morphological Traits Associated with Stagnant Flooding Tolerance in Rice

The experiment was less favourable because of leaking ponds and technical obstacles on irrigation. When the water reached 40 cm of depth, the water receded to 20 cm for 2-3 days at the end of the vegetative stage. This condition caused the plant recovered so that it carried out metabolic activity and saving assimilates. Thus, when the stressed water was occurred, the plant used the reserve of assimilate to survive. This may explain the why the decreasing of grain yield were not extreme. Plants were also attacked by stem borer at the low to moderate intensity. Water regime based on Vergara et al. 2014, which was gradual flooding with maximum of the depth of 50-60 cm. This condition was less effective because it is difficult to distinguish between tolerant and susceptible genotypes. As shown by grain yield of IRRI 119 tolerant check was not significantly different with IR 42 check-sensitive. The effectiveness of screening methods depends on water level and light intensity. The flood stress level besides depends on water level, also depends on light intensity, temperature, turbidity of water, soil fertility, and age of crops Setter et al. 1997; Das et al. 2009. For artificial selection, the water level should be increased because the floods could reach at 100 cm of depth in swampy area. A flooded environment can also suffer from low light- thus reducing photosynthesis, and from high concentration of toxic soil compound Bailey-Serres and Voesenek 2008. Artificial screening should be conducted in wet season to get similar condition in swampy area, where rice is only grown in wet season. Genotypes tested have narrow genetic diversity, as shown by several genotypes which having the closer genetic background. IR64, Ciherang, INPARA 4 IR 64 SUB1, and INPARI 30 Ciherang SUB1 had the same parent Source: Official documents of released plant varieties. The closer relationship caused similar response of stress on grain yield, as shown by genotype x water regimes interaction were not significantly different on traits number of productive tiller, number of filled grain, 100-grain weight, grain yield, stem length, stem diameter, and length of leaf blade. This means that the tolerant genotypes based STI classified as intermediate tolerant. Previous study reported that the performance of Swarna SUB1 INPARA 5 under stagnant flooding was uncertain, as the first generation of variety introgressed with SUB1 were relatively short, and if water depth remains at above the canopy level for longer than two weeks, the plants may not elongate sufficiency. The SUB1 mediated suppression of elongation could limit the growth of the variety and render it more sensitive to stagnant floods, particularly if the floods persist after a period of complete submergence Vergara et al. 2006; Kotera et al. 2007; Septiningsih et al. 2009; Barley-Serres et al. 2010. However, in our study performed that percentage of grain yield decreasing of varieties with SUB1 gene INPARA 4, INPARA 5, INPARI 29, INPARI 30 were relatively low. It may because screening method in our study was different. In our study, the complete submergence was never occur so that the SUB1 varieties were still could elongated their stem. Therefore they still could produce grain yield relatively higher. The genotypes with good performance based on the STI were irrigation varieties, namely IR 64, Ciherang, and INPARI 30. The genotypes have high yield