5 MARKER-TRAITS ASSOCIATION OF AGRONOMIC TRAITS RELATED TO STAGNANT FLOODING
TOLERANCE IN RICE
Abstract
The objective of this study was to identify SNP markers linked with agronomic traits related to stagnant flooding tolerance in rice. The characters
selected were plant height and number of productive tilers. The phenotypical data was collected from bi-parental population of IR 42 and IRRI 119. IR 42 was
sensitive parent, and IRRI 119 was donor parent. The genotyping was carried out using 384 SNPs Golden Gate Illumina assay. Association analysis between SNP
markers and phenotypical data was performed using General Linear Model in Tassel version 5.0 software. Based on GLM association analysis, significant
markers 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. Two of them, TBGI424383 and TBGI271066 were expected associated with family of protein kinase which play role in plant
stress signaling. Key words: association mapping, stagnant flooding stress, rice
5.1 Introduction
Rice has a unique ability to elongate its internode with increasing water depth. During submergence, rice promotes growth of shoots toward the air-water
surface which facilitates gas exchange with atmosphere Kende et al. 1998; Sauter 2000; Vriezen et al. 2003; Jackson et al. 2003. Stagnant flooding at depths greater
than 25 cm adversely affects the growth of rice in medium-deep areas 100 cm, even though the plants are not fully submerged. In particular, it reduces tillering
and increases lodging Singh et al. 2000; Tuong et al. 2000.
The grain yield-related rice traits are controlled by multiple genes which are significantly influenced by environment Huang et al. 2013; Ikeda et al. 2013. If
the amount and distribution of favorable genes are unknown, it is difficult to apply the rice germplasm in conventional breeding. There are interaction between
genesQTLs underlying yield-related traits and particular ecological environments. For instance, the efficiency of certain favorable genes can be enhanced by proper
environments, but countered by unsuitable environments Liu et al. 2006; Sreedhar et al. 2011. Thus, it is important and prerequisite to study favorable trait-related
genes in specific ecological environment for utilizing the rice germplasms.
Several studies have been conducted on the inheritance of early elongation ability Tripathi and Rao 1985, Suge 1987, Eiguchi et al. 1993. The results differed
from cross to cross, apparently because rice is traitized by a variety of phylogenetic backgrounds Glaszamann 1987. Two quantitave trait loci QTLs that control the
early elongation ability on chromosomes 3 and 12 in F2 population. Another study
performed a diallel analysis of early elongation ability showed that some additional recessive alleles might confer the earlier elongation Nemoto et al. 2004.
Single nucleotide polymorphisms SNPs can be converted into genetic markers that are scored in mapping populations using various high-throughput
SNP-typing technologies Gabriel and Ziaugra 2004; Gunderson et al. 2005; Hui et al. 2008. High-throughput SNP discovery Marth et al. 1999;Weckx et al. 2005;
Zhang et al. 2005; Barbazuk et al. 2007; Li et al. 2008; Li et al. 2009 and genotyping technologies have simplified the generation of genetic maps and the
analysis of recombinants Shifman et al. 2006. Dense maps in economically important crops will be invaluable for marker-assisted selection programs Prigge
et al. 2009, analyzing linkage disequilibrium Kruglyak 2008; Wang et al. 2008, detection of intra-species cis-regulatory variation Stupar and Springer 2006, and
other quantitative genetic studies Cookson et al. 2009.
The elongation of internode, as well as the degree of elongation during submergence is regulated by environmental and hormonal factors viz ethylene, GA,
and ABA Vriezen et al. 2003. Further, Hattori et al. 2009 reported that this traits is controlled by two major SK genes driven by the rice actin promoter. There are
few reports available that specify the pattern of internode elongation under deep- water. However, very scanty report are available on response of physiological leaf
area, photosynthesis, and non-structural carbohydrates and morphological shoot length, tillers, culm thickness, spikelet fertility, shoot biomass, and grain yield
traits under deep-water rice Amante 1968; Sakagami et al. 2009, 2013; Vergara et al. 2014.
The objective of the study was aims to identified SNPs marker associated with number of productive tillers and plant height related to stagnant flooding
tolerance in rice.
5.2 Materials and Methods
Analysis was conducted in Indonesian Centre for Rice Research for DNA extraction and Molecular Biology Laboratory of Indonesian Centre for
Agricultural Biotechnology and Genetic Resource for genotyping assay on February to June 2016. Genetic materials used are population of P1 IR 42, P2
IRRI 119, and F2.
Markers used were 384 SNPs linked with morphological and agronomical traits. All markers used were validated by Rice Genome Division of Indonesian
Center for Agricultural Biotechnology and Genetic Resource BB Biogen 2015. Phenotypic data associated with the genotypic data were plant height and number
of tillers. Plant height and number of tillers are characters related to stagnant flooding tolerance based on Chapter 2 supported by Amante 1986, Singh et al.
2011, Collard et al. 2013, Kato et al. 2014. Background selection method was used in this experiment. There are two steps for genotyping that are DNA extraction
and Genotyping Assay: 1. DNA extraction
DNA extraction for rice was using a modified version of a method developed by Murray and Thompson 1980. It is a simple effective method
utilizing cetyl tri-methyl-ammonium bromide CTAB.
Materials used were 2-3 inches leaves of 3-week old plant one leaf per sample, scissors, sterile 2 ml micro tubes, steel balls, liquid nitrogen,
micropipettes, tips and tubes, water bath, centrifuge with rotor, -20 C freezer, 800
µl 2x CTAB buffer warmed to 65 C, 0.8 ml chloroform-isoamyl alcohol 24:1,
600 µl isopropanol, 70 ethanol, 200 µl TE buffer, 2 µl RNAse 10 mgml, 20 µl 110 volume solution sodium acetate, 400 µl 2 volumes absolute ethanol.
Procedure: 1. 2-3 inches of young leaves were cut into pieces and put inside of 2 ml
autoclaved tubes. Then it is placed in two steel balls per tube. The tubes then are closed and arranged in the rack provided. Racks then are placed onto
cryogenic container. 500 µl 2x CTAB buffer was poured over the tubes and then immediately ground using the Genogrinder Grinding may take minutes or more
until the samples are powdered-repetition may be needed
2. The mixture then is incubated at 65 C for 30 minutes to 1 hour
3. The mixture then is cooled briefly and added 0.8 ml chloroform-isoamyl alcohol 24:1, shaken at room temperature for 20 minutes, and spun at 12,000 rpm for
10 minutes 4. The aqueous phase then is transferred into a new tube
5. Into the tube is added with 500 µl isopropanol 1 volume and incubated at -20 C for 1 hour
6. Then the tube of mixture is spun at 12,000 rpm for 3 minutes, then decanted the isopropanol and pellet is washed with 70 ethanol and then drained to dry
7. Pellet is dissolved in 200 TE, then added with 2 µl RNAse 10 mgml and incubated at 37
C for 30 minutes 8. In the mixture then is added with sodium acetate 20 µl 110 volume solution
and 2 volumes of absolute ethanol 400 µl, and incubated at -20 C for 1 hour or
overnight 9. The tubes are spin at 12,000 rpm for 5 minutes, drained. The pellet is rinsed
with 70 ethanol, dried, and dissolved in 100-200 µl TE 10. Absorbance of the resulted pellets A at 260 nm and 280 nm and background
absorbance at 320 nm was measured using Nanodrop, for determining the yield and purity of the DNA.
DNA was bulked based on phenotypic clustering using plant height and number of tillers in F2 population. Clustering analysis resulted 53 groups of
genotypes. DNA was bulked based on the groups. Technique of sampling which will be used is bulked segregant analysis. Bulked segregant analysis BSA has been
proposed as an efficient strategy for identifying DNA markers linked to the genes or genomic regions of interest Brauer et al. 2006. BSA has been successfully used
in rice for identifying large effect QTLs linked to rice yield under severe drought stress Quarrie et al. 1999; Venuprasad et al. 2009.
Dilution will be done to obtain the required concentration of DNA in Genotyping Assay. DNA diluted by ddH2O up to DNA concentration is suitable
with its concentration for Genotyping Assay. Dilution formula is V1 x V2 x M1 = M2, with V1 is DNA volume before dilution, M1 is DNA concentration before
dilution, V2 is final volume dilution, and M2 is concentration DNA for Genotyping Assay, that is 25 ngmL.
