QUANTITATIVE TRAIT LOCUS FOR SUBMERGENCE TOLERANCE IN THE RECOMBINANT POPULATION FROM THE CROSS BETWEEN SUSCEPTIBLE RICE

VARIETY IR42 AND TOLERANT VARIETY MA-ZHAN (RED): EXTENSIVE MAPPING AND EVALUATION

BY

REUBEN JACOB LABIOS College of Arts and Sciences - Bachelor of Science in Biology

Submitted in partial fulfillment of the Requirements of

ENG10 Writing of Scientific Papers, T-1R. Second Semester, 2011-12 UP Los Baos March 5, 6, 2012

BIOGRAPHICAL NOTE The author was born on November 24, 1992 in Bay, Laguna. He is the fourth of five children of Romeo Labios and Jocelyn Labios.

He finished his secondary education from Morning Star Montessori School Incorporated in 2009. He passed the University of the Philippines College Admission Test and entered the Bachelor of Science in Biology program. Upon ending his first semester of his second year, 2010-2011, he became the member of the UPLB Biological Society or Symbiosis. He earned the position of Membership Committee Head 2010-2012 on his second semester as a member and recently had been elected as the Vice President for Academic Affairs 2012-2013.

In the summer of 2011, he got the chance to work as Professional Service in Plant Breeding, Genetics and Biotechnology Laboratory under Dr. Endang Septiningsih and Mr. Tobias Kretzschmar in their project involving the search for the molecular response of rice varieties undergoing anaerobic germination.

Jacob, known to his friends and colleagues, first dreamt of being a cardiothoracic surgeon working in a prestigious hospital. His change of mind and pursue of the same academic path as his parents as scientists and researchers lead him to believe that a coveted position like a surgeon will not be enough to help him feed his unending curiosity as a member of the scientific community.

REUBEN JACOB LABIOS

I. INTRODUCTION A. Significance of the Study Rice (Oryza sativa L.) is the most important staple food for a large part of the worlds human population, especially in Asia, sub-Saharan Africa and the Caribbean region. With regards to its benefits to human nutrition (i.e. energy and carbohydrate component) and calorie intake, rice is the most produced grain in the world according to the Department of Agriculture in United States of America (2012). Countries and regions with high rainfall as well as low labor expense are the preferred environment for cultivating rice. Rice can be virtually grown anywhere but it requires intensive labor care and water (Pandey, 2002). However, challenges arise when cultivating rice and one of the prominent challenges is flooding. Flooding due mainly of typhoons regularly afflicts some 12 million hectares in South Asia; and as much as one-third of the rainfed lowland areas in sub-Saharan Africa are thought to be affected by submergence (IRRI, 2011). According to the October 2011 assessment of Food and Agriculture Organization of the United Nations, typhoon Nesat (PAGASA name: Pedring) and the subsequent localized floods had a critical impact on the paddy production of the main 2011 wet season, approximating to 55 percent of the national rice output. Preliminary official reports indicate substantial damage to 420, 337 hectares with losses estimated at about 728, 379 tons of paddy or 16 percent of the national production. Overall, latest estimates indicate that about 4 million people have been affected and at least 485 000 hectares of standing crops, including rice and other high value commercial crops (HVCC) have been damaged or lost to the floods. The affected cropped area covers 6 percent of the total national cropped area.

Since modern rice varieties like Nipponbare and Indica varieties are not adapted to flooding conditions, International Rice Research Institute in Laguna, Philippines determined rice varieties that are tolerant to submergence of two weeks. Included in the list is the China rice variety Ma Zhan (Red) (Das, 2009). Through molecular biology and biotechnology techniques, mechanisms associated with tolerance to flooding during germination and early seedling growth in rice were determined (Ismail, 2008). Using Marker Assisted Selection (MAS) technique and Quantitative Trait Locus (QTL) analysis, Ignacio et al. (2011) determined the chromosome and region in that chromosome the location of the gene responsible for the tolerance to submergence of offspring from the cross fertilization between Ma Zhan (Red) and IR42, a common rice varieties in South East Asian countries that is susceptible to submergence. While the locus of the submergence tolerance gene has been identified, further refinement of the region with the unique sequence of the submergence tolerant gene from Ma Zhan (Red) is still aimed. This could be done by further implementing backcrossing and MAS succeeded by drawing recombination maps for the heterozygote allele that indicates the presence of submergence tolerance gene in the offspring population. In this study, backcrossing complemented with MAS technique will be implemented as well as phenotypic and genotypic evaluations of the offspring population are to be studied. Knowing the recombination map of all offspring that passed the phenotypic and genotypic evaluation will help in the transformation of flood-susceptible varieties, commonly planted in the Philippines and other countries, to tolerant ones. This will minimize crop damages done by flooding, which is a commonplace in the agricultural routine of rice farmers.

B. Review of Related Literature

Germination stresses caused by climate change continuously threaten the production of valuable grains, one of which is rice. As it is consumed by about 3 billion of the worlds population, rice requires a number of ecosystems where there is constant irrigation and rain to sustain food security in many countries (Fig. 1). However, threats including variation of temperatures and rainfall season are already being experienced by countries dependent on optimal rice harvesting conditions to continuously provide the already strained needs for food consumption of the worlds population. Agricultural systems must adapt to the unpredictability of the climate not only for short-term conditions but also for long-term ones. Facets of climate change include high temperature and humidity, drought, salinity and flash floods (Wassman et al. 2009). Philippines, located west of the Pacific Ocean, experiences yearly typhoons and storms by the dozen, and this number still increases due to climate change. One of the after effects of continuous typhoons and storms is flooding.

Flooding is one of the major causes of crop yield losses estimating to US $1B every year in 10-15 million hectares of rice fields in South and South East Asia, according to Dey and Upadhyaya (1996). These numbers still rises as seawater level increases and gradual worsening of weather events continues to occur. There are two common practices being adapted by farmers today. Conventional transplanting involves grown seedling being planted in a paddy field filled with water levels of about one centimeter (IRRI, 2011). Another method, being introduced to famers is direct seeding wherein pre-germinated or primed rice seeds are directly planted on soil. This reduces labor costs (Singh et al. 2010). However, both methods are prone to crop damages

as either pre-grown rice plant or seed is sensitive to partial to stagnant flood water or anaerobic conditions (Bailey-Serres et al. 2010). Death of seedlings due to submergence is mainly caused by anoxia or little or no oxygen present in the environment of the rice plant, and light penetration is limited. Limitations to these requirements will inhibit mitochondrial oxidative pathway and photosynthesis which are important for plant survivability (Bates, 2008). Reduction in diffusion of gases in water relative to air limits the exchange of carbon dioxide and oxygen necessary for photosynthesis and respiration and increases the cellular concentration of the gaseous hormone ethylene that catalyzes the death of chlorophyll (Bailey-Serres et al. 2010).

As early as 1970s, tolerant rice varieties, that can survive in complete submergence for about 14 days and have the ability to recover after water receded, were identified. Through molecular assisted selection (MAS) and genetics techniques, the chromosomal region that mainly confers the tolerance of FR13A, a rice variety from India, to submergence was identified and mapped in the locus named Submergence 1 located at chromosome 9 (Mackill, 1996).

Figure 1. Irrigated and rain fed rice in East, South and Southeast Asia (Wassmann et al. 2009)

Anaerobic Germination Rice plants do not immediately die while submerged. There are two mechanisms rice plants adapt to survive in submergence conditions. The first one is called escape strategy wherein rice plants adapt by elongating a white coleoptile tissue that allows rice seedling in waterlogged soils to reach an aerobic zone from which the oxygen can be transferred to the seed, with subsequent normal growth of the radicle and leaves (Angaji et al. 2009). In the case of slow rising flood, investment of energy into elongation growth is a successful survival strategy. Imbalance, however, happens if floods are either too deep to allow renewed contact with the air or when floods are transient to outgrow the flood water, exhausting energy reserves and causing death to the plant (Perata and Voesenek, 2007). Another problem with this strategy is that when the flood waters recede, the elongation growth will leave a spindly plant that easily falls down due to weight and carbohydrate exhaustion (Bailey-Serres et al. 2010).

Another strategy that can be adopted by rice plant under submergence is called the quiescence strategy characterized by slow growth and thus, conservation of energy and carbohydrates. Species exploiting this strategy have higher survival rates during long-term submergence than those that use elongation strategy (Perata and Voesenek, 2007) as they maintain growth rate similar to plants in air (Angaji et al. 2009). When the flood is deep and prolonged, the protection of energy reserves and growth meristems provides an advantage (Bailey-Serres et al. 2010). Identification of the ability of rice plants with this adaptation through molecular techniques leads to the locus or region of the chromosome 9 in flood resistant 13A (FR13A) rice variety which is named Submergence 1 or Sub1. Reception of FR13A variety was low because of characteristics like photoperiod sensitive, tall and provides low yield of poor

quality grain (Singh et al. 2010). Experiments were employed to introgress the Sub1 locus without the undesirable qualities to mega-varieties or varieties widely planted as these varieties have high yield and good performance under normal conditions.

Figure 2. Sub1 region gene composition and submergence induced mRNA accumulation in rice (Xu et al. 2006)

Figure 3. Introgression of the FR13A Sub1 haplotype into an intolerant variety by MAS confers submergence tolerance (Xu et al. 2006)

Figure 4. Ethylene responsive factor (ERF) gene composition (haplotype) at the Sub1 locus of submergence tolerant and intolerant rice (Bailey-Serres et al. 2010)

Submergence 1 Sub1 locus mechanism is to confer tolerance through the suppression of plant elongation under submergence (Singh et al. 2010). O. sativa ssp. indica cultivar FR13A are highly tolerant and survive up to two weeks of complete submergence owing to the Sub1 locus. Two qualities submergence-tolerant rice has are that it can survive 10-14 days of complete submergence, and renew growth when the flood water subsides (Xu et al. 2006) (Fig. 3). This quality is correlated with better maintenance of total soluble carbohydrates and limited elongation growth, lower aldehyde contents, less chlorophyll degradation, and less oxidative damage upon re-oxygenation, all of which are characteristics of the quiescence strategy (Bailey-Serres et al. 2010). With these qualities, Sub1 locus needs molecular markers system that can efficiently facilitate selection of this trait which is low in heritability (Angaji et al. 2009). This marker system, which will be used in MAS technique to breed rice, will limit only to the region size capable of submergence

tolerance and exclude genes involved in the poor agronomic performances exhibited by tolerant varieties like FR13A (Septiningsih et al. 2009).

The approximately 200 kilobase-long Sub1 locus involves three ethylene-response (ERFs) transcription genes designated as Sub1A, Sub1B and Sub1C (Xu et al. 2006, Singh et al. 2010) (Fig. 2). Plant proteins that contain ERF domains are known regulators of abiotic and biotic stress response. While the Sub1A is present in all tolerant rice varieties, Sub1B and Sub1C are invariably present in the Sub1 region of all rice varieties (Xu et al. 2006). In five out of seventeen indica varieties and all four japonica varieties including mega-variety Nipponbare, Sub1A gene is either absent or present in the intolerant allelic variant Sub1A-2. However, genotypes that possess the Sub1 haplotype or combination of Sub1A-1/Sub1C-1 are tolerant rice varieties. There was no Sub1B allele identified as being specific to submergence tolerance (Xu et al. 2006) (Fig. 4). With this discovery, molecular markers specific for Sub1A and Sub1C were employed to study the correlation of the two ERF genes in submergence tolerance (Singh et al. 2010).

Submergence tolerance is strongly correlated with the presence of Sub1A-1 allele through rapid, prolonged and pronounced transcript accumulation for tolerance in plant tissues (leaf collar, node, internode, shoot elongation zone) (Singh et al. 2010), of 14 to 28 days old plants in response to submergence, whereas intolerance to submergence is associated with Sub1A-2 allele through the promotion of lower levels of transcript induction by the stress, or with the complete absence of Sub1A gene (Bailey-Serres et al. 2010). This correlation was demonstrated by Xu et al. (2006) through the transformation of a submergence-intolerant

japonica variety with Sub1A-1 conferred submergence tolerance to transgenic plants (Perata and Voesenek, 2007). Sub1A-1 and Sub1A-2 encode identical proteins, with the exception of a specific single nucleotide polymorphism (SNP) within the Sub1A coding region that causes an amino acid substitution (intolerant: CCG=proline; tolerant: TCG=serine) (Septiningsih, et.al. 2009).

Another main difference between Sub1A-1 and Sub1A-2 is the specific presence of mitogen-activated kinases (MAPK) site in the Sub1A-1 allele which affects the post-translational regulation of proteins and also the regulation of downstream genes, including Sub1C. Xu et al. (2006) wrote in his article that overexpression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica cultivar during submergence conferred enhanced submergence-tolerance to the plants, down regulation of Sub1C and up regulation of Alcohol Dehydrogenase (Adh1), (Fig. 5). According to the reports made by Fukao et al.(2006) and Perata and Voesenek (2007), suppression of Sub1C in intolerant plants is important to prevent the Sub1C-mediated up regulation of the alpha amylase gene Ramy3D involved in starch degradation, therefore preserving carbohydrates reserves needed for recovery after de-submergence. At the same time, Sub1A-1 is important for the down regulation of Expansin A and the sucrose synthase Sus3, proteins that promote cell elongation and carbohydrate catabolism, respectively. (Septiningsih et al. 2009; Singh et al.2010) Also, an increase in levels of hormone ethylene inside submerged plant tissues drives the accumulation of Sub1A proteins that increases the transcripts responsible for the fermentation of ethylene-derived ethanol (e.g. Alcohol dehydrogenase and Pyruvate decarboxylase). This will prevent the senescence of chlorophyll in leaves (Perata and Voesenek, 2007).

Figure 5. RT-PCR analyses for tolerant hybrids BR11-Sub1, IR64-Sub1 and their original intolerant parents. *S=Submerged; C=Non-submerged Controls (Septiningsih et al. 2009)

According to the study by Septiningsih et al. (2009), there seemed to be a correlation between the amount of Sub1A expression in the original parents and the level of tolerance of their corresponding improved varieties. Heterozygous plants, or offsprings where one parent carries the tolerant allele and the other the intolerant allele, were significantly less tolerant than the homozygous plants, or both parents carries the tolerant allele, for tolerance. This was proven by the evidence that the expression of the Sub1A allele in the heterozygotes was less than in homozygotes (Fig. 6). This is important to reach the critical threshold of expression needed for tolerance.

Submergence 1 Introgressed Conventional breeding approaches have since been used to develop submergence-tolerant varieties improved with agronomic performance (Singh et al. 2010). Introgression, or the entry or introduction of a gene from one gene complex into another, of Sub1 into flood-intolerant rice

cultivars improves submergence tolerance significantly and has no negative side effects in terms of yield, harvest index, and grain quality when grown under control or non-submerged conditions (Perata and Voesenek, 2007). Using marker-assisted backcrossing technique, a small genomic region containing Sub1A has been introgressed into modern high-yielding varieties, such as Swarna, Samba Mahsuri, IR64, Thadokham (TDK1), CR1009 and BR11 (Bailey-Serres et al. 2010; Mackill et al. 2012) (Table 1). Swarna-Sub1, the first example of a submergencetolerant mega-variety, is currently being evaluated in submergence-prone areas of India and Bangladesh. Swarna-Sub1 showed a two-fold or higher yield advantage over Swarna after submergence for 10 days or more during the vegetative stage (Septiningsih et al. 2009) (Fig. 3 & 7).

Table 1. Submergence tolerance of the Sub1 mega varieties measured in different experiments at IRRI, Philippines (Septiningsih et al. 2009)

Figure 6. RT-PCR of Sub1A with Rubisco as control with their corresponding phenotypes (S=Susceptible, M=Moderately tolerant, T=Tolerant) (Septiningsih et al. 2009)

To establish an efficient MAS system to incorporate tolerance of flooding during germination, it is necessary to find additional and more diagnostic DNA markers that are tightly linked with the quantitative trait locus of interest, which will primarily come from fine-mapping of the identified QTLs. The isolation of genes underlying the QTLs will help design more precise-gene specific diagnostic markers for MAS and also advance our understanding of the genetic and physiological mechanisms of tolerance (Angaji et al. 2009). This will be important in regions, including marginal fields, which need varieties with tolerance to submergence during germination. Some advancement has been made in the breeding of advance lines conferring tolerance to submergence during germination using tolerant donors such as Ma-Zhan (Red) (Table 2), in combination with other beneficial traits, such as high-yielding and better adaptation including varieties with Sub1 gene. (Septiningsih et al. 2009)

Table 2. Rice accessions tolerant of flooding during germination selected during initial screening, together with 3 sensitive checks, IR42, IR64 and FR13A (Angaji et al. 2009)

Figure 7. Field plot test of submergence tolerance of Sub1 and non-Sub1 varieties. IR49830 is a FR13A-derived tolerant rice variety. (Bailey-Serres et al.2010)

Quantitative Trait Locus Identification and Mapping of Submergence Tolerance in Rice Derived From Tolerant Variety Ma-Zhan (Red) Ignacio et al. (2010) started the identification and mapping of the QTL responsible for the submergence tolerance in rice derived from tolerance donor Ma-Zhan (Red). In the study, IR42, an intolerant variety, was crossed with Ma-Zhan (Red). Through phenotypic evaluations, survival rates of the seedlings after submergence were collected. The surviving offsprings were evaluated by genotyping. Eighty-three (83) SSRs were used and distributed across the rice genome and four markers identified a QTL responsible for tolerance to flooding on chromosome 7. Fine-mapping implements MAS for different recombination along the QTL region. The authors concluded that the major QTL represents a valuable target for MAS to rapidly transfer tolerance to flooding into improved varieties.

C. Objectives of the Study The general objective of the study is to further fine-map the major QTL of submergence tolerance in chromosome 7 of the recombinant populations (Ma-Zhan (Red) IR42 cross). Specifically. This study will attempt: 1. to produce a recombination or genetic map of the major QTL for the presentation of the recombination process of the backcross between BC2F1 from the experiment done by Ignacio, et.al. (2011) and IR42 (BC3F1 generation), and 2. to determine the effect and presence of the major QTL of submergence tolerance in the recombinant population BC3F1 generation through phenotypic and genotypic evaluation, respectively.

D. Date and Place of Study The experiment will be conducted in the Molecular Biotechlogy Laboratory of the International Rice Research Institute under the supervision of Dr. Endang Septinighsih from April to August 2012.

II. MATERIALS AND METHODS The experiment will be conducted in the Molecular Biotechlogy Laboratory of the International Rice Research Institute under the supervision of Dr. Endang Septinighsih from April to August 2012. It will be arranged in completely randomized design with three to four replications.

A. Materials
Pre-germinated rice seedlings of BC2F1 (Ignacio et al. 2011) and IR42 (incubated in a petri plates lined with moistened coarse filter paper at 30oC for 3 days in the dark) Greenhouse concrete tanks Garden soil CTAB (Cetyl trimethylammonium bromide) dissolved in sterile ddH2O NaCl 1M Tris pH 8.0 0.5M EDTA (Ethylenediaminetetraacetic acid) pH 8.1 Autoclave, Centrifuge b-mercapthoethanol SDS (Sodium dodecyl sulfate) chloroform-isoamyl alcohol (24:1) isopropanol 70% ethanol TE (Tris-EDTA) buffer RNAse sodium acetate absolute ethanol 10x TB buffer 1 mM dNTP Taq DNA polymerase MJ Research single or dual 96-well thermal cycler bromophenol blue gel loading dye 8% polyacrylamide gel 0.5 mg/ml ethidium bromide DNA ladder Micropipette tips, microtubes, thermal cycler tubes, chemical glass wares Alpha Imager 1220 20 cm x 15cm x 10 cm trays

B. Methods 1. Plant materials and crossing scheme (Neearaja et al. 2007; Mackill and Ismail, 2011) a) BC2F1 (Backcross population # 2, 1st progeny generation) pre-germinated rice seeds from the research study by Ignacio et al. (2011) will be obtained. This will be used as the donor of the major QTL of submergence tolerance.

b) Soil will be placed into the concrete beds then the entries will be labeled
according to the layout. c) Well-germinated twenty rice seeds from BC2F1 will be selected and will be sown not more than 1 cm below the soil surface. Replicated trials will be made. The number of seedlings per entry will be recorded. d) After sowing one row, the seeds will be covered with sieved garden soil. e) The seedlings will be grown for 14 to 21 days after germination. The plants will be watered and weeded regularly. Plant height (14-21d plants) usually measure around 25 to 35 cm at the start of submergence. The seedlings will not be fertilized. f) BC2F1 will be backcrossed with IR42, a susceptible variety that is the recipient of the major QTL for submergence tolerance, to obtain BC3F1 seeds. g) The BC3F1 seeds will be self-bred to increase the number of the BC3F1 population (BC3F2 seeds)

2. Molecular markers analysis (Neearaja et al. 2007) a) DNA will be extracted from young leaves, selected at random, of 2-week old plants of the BC3F2 population b) Polymerase chain reaction (PCR) will be performed in 10 l reactions containing 5-25 ng of DNA template, 1 l 10x TB buffer, 1 l of 1 mM dNTP, 0.50 l each of 5 M forward and reverse primers* that flanks the molecular marker and 0.25 l of Taq DNA polymerase using an MJ Research single or dual 96-well thermal cycler. c) After PCR, the PCR products will be mixed with bromophenol blue gel loading dye. d) The solution in the previous step will be analyzed by electrophoresis on 8% polyacrylamide gel using mini vertical polyacrylamide gels. e) The 8% polyacrylamide gels will be produced and stained in 0.5 mg/ml ethidium bromide. f) The gels will be analyzed using Alpha Imager 1220. g) Microsatellites or simple sequence repeats (SSR) markers that are tightly linked, flanking, and unlinked** with the Sub1 locus will be used for selection. *Forward and Reverse primers will be designed, with the major QTL sequence for submergence tolerance identified by Ignacio et al. (2011) as the basis, using Primer3. (http://genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi)

** Molecular markers produced will be compared with reported BAC clones that correspond to the Sub1-linked, Sub1-flanked and Sub1unlinked SSR markers identified by Ignacio et al. in 2011, Xu et al. in 2006 and by IRGSP in 2005.

3. Genomic DNA extraction from rice leaves (Modified CTAB Method) (McCouch et al. 2007) a) Sample young leaf tissues will be grind. About 50ul ground tissues will be placed into 2ml microtubes. 500 l of 2x CTAB buffer warmed to 65C and containing 40ul/20ml b-mercapthoethanol will be added (for older leaves) or optional or will be replaced with SDS. The solution will be mixed thoroughly. The solution will be incubated at 65C for 30 minutes to 1 hour. b) The solution will be cooled briefly and then 500-ul chloroform-isoamyl alcohol (24:1) will be added. The solution will be shook at room temperature for 20 minutes, and will be spun at 5000 rpm for 15 minutes c) The aqueous phase (top phase) will be decanted into a new 2ml tubes. d) A total of 500 l isopropanol (0.5 ml, 1 volume) will be added to the tubes and the tubes will be incubated at -20C for 30 minutes. e) The tubes will be spun at 5000 rpm for 10 minutes (12,000 rpm for 5 min). The isopropanol will be decanted and the pellet will be washed with 500 l 70% ethanol and then will be drained dry.

f) The pellet will be dissolved in (100 l) TE buffer. (1.0 l) RNAse (10mg/ml) will be added and the pellet will be incubated at 37C for 30 minutes. g) One-tenth volume of sodium acetate (10l) and 2 volumes of absolute ethanol (200 l) will be added. The solution will be incubated at -20C for 1 hour or overnight. h) Afterwards, the plate will be spun at 5000 rpm for 15 min, and after, the pellet will be drained and rinsed with 70% ethanol. Air dry. The pellet will be dissolved in 100-200 l TE. i) The deep well plate will be spun for 5 minutes at 5000 rpm to pellet the debris. Dilute as necessary, and the stock solution will be stored in 20C and the working solution at 4C.

4. Marker-assisted backcross selection(Neearaja et al. 2007) a) The individual plants from the BC3F2 population that is heterozygous, or with molecular markers tightly linked for Sub1 locus, will be identified and selected (foreground selection). b) From these heterozygous for Sub1, those that were homozygous for the recipient allele at one marker distally flanking the Sub1 locus will be identified (recombinant selection). c) From these plants, individuals with the fewest number of markers from the donor genome will be selected (background selection).

All selection will be done through the same procedure as with molecular marker analysis and results will be compared for presence of same molecular markers with the results of the first molecular marker analysis.

5. Screening for submergence tolerance (Neearaja et al. 2007; Mackill and Ismail, 2011) a) Seeds of selected plants of BC2F1, BC2F2, and BC3F2 along with parents and susceptible checks IR42 will be germinated in rows of 20 cm x 15cm x 10 cm trays. b) Fourteen- to Twenty one-day-old seedlings will be transplanted into the pond field at 20 20 cm2 spacing, with 2 seedlings per hill. c) Five to ten extra rows of susceptible local variety IR42 will be transplanted on one side of the deep ponds to observe the extent of damage of the sensitive check to be used as a guide to determine when to end the submergence treatment. d) 100% plant population will be ensured before submergence by replanting missing hills. e) Transplanted seedlings will be allowed to grow normally in the field for another 1421 days. The number of plants before submergence will be counted. The plant height will be measured (from soil base to tip of longest leaf) before and after submergence. f) For the submergence treatment, ponds will be filled with water until a depth of 1 to 1.25 m. Flooding can be started at noon to give plants

enough time to photosynthesize in the morning. The water depth will be maintained by adding additional water regularly to completely submerge plants. Daily floodwater conditions will be monitored in the submergence tanks in the morning (0700-0800) and in the afternoon (1300-1400) and at various depths during submergence. If equipment is available, water temperature, light, dissolved O2, etc., at the water surface (within 2.5 cm) and at 40- and 80-cm water depth will be measured. g) Ten plants of the susceptible check IR42 will be randomly uprooted after 7 days of submergence daily from the extra rows (step c) to observe the extent of damage and to decide on the proper time to terminate the submergence treatment (at approximately 1015 days). This can be determined by examining the base of the stem, which becomes soft when the growing point (shoot-root junction) is damaged. h) The floodwater will be released when 4050% damage is observed. Desubmergence will be started after noon. i) Algal growth will be minimized by partially removing algae from the water surface daily using a small fish net. Extra care should be done when scooping the algae to minimize disturbance of floodwater. Hand weeding, snail control, and plant protection measures will be adopted when needed. j) For survival rating, number of hills before submergence/flooding and after submergence/ flooding of 57 days when the plants start to develop new leaves will be recorded. Number of plants that survive will be divided by the total number of hills before flooding/submergence and will be

multiplied by 100. This will be the submergence tolerance/intolerance data collected. k) The field will be allowed to remain without water for 34 days. Afterward, the field will be filled with not more than 12 cm of water until another 1521 days go by, and then increase the water to a normal 57 cm. l) DNA from twelve surviving recombinant plants from BC3F2 population will be extracted and will undergo PCR using the forward and reverse primers from the molecular markers analysis section to confirm genotypically the presence of the Sub1 locus in the recombinant plants. m) DNA analysis will be implemented to form the recombination or genetic map of the major QTL for the presentation of the recombination process of the backcross between BC2F1 from the experiment done by Ignacio, et.al. (2011) and IR42 (BC3F1 generation).

6. Data analysis a) As written in step j of Screening for submergence tolerance, number of hills before submergence/flooding and after submergence/ flooding of 57 days when the plants start to develop new leaves will be recorded and also, the number of plants that survive will be divided by the total number of hills before flooding/submergence and will be multiplied by 100 (survival percentage). b) The percent survival of BC2F1, BC2F2, and BC3F2 along with parents and susceptible checks IR42 populations will be compared using a one-way

ANOVA (Minitab for Windows, Version 14) and 95% Confidence Intervals to determine if they are statistically different.

C. Detailed schedule of activities Date April 2, 2012 April 16-23, 2012 Activity Preparation of Concrete Beds and Planting of Seeds Plant height will be measured BC2F1 will be backcrossed with IR42 Place Greenhouse Greenhouse Molecular Biotechlogy Laboratory Greenhouse Molecular Biotechlogy Laboratory Molecular Biotechlogy Laboratory Greenhouse

April 30, 2012 May 7, 2012 May 11, 2012 May 14, 2012

BC3F1 plants will be self-bred Molecular marker analysis of the leaves of the plants Marker-assisted backcross selection for Sub1 of BC2F3 plants Seeds of selected plants from BC2F1, BC2F2 and BC3F2 along with parents and susceptible checks IR42 will be germinated Transplantation of seedlings Seedlings for submergence treatment will be submerged with water Termination of Submergence conditions; Survival Rates will be measured Fill the field with 1-2cm of water Increase water to 5-7 cm; Survival Rates will be measured DNA extraction and analysis of Recombinant Plants

May 28-June 4, 2012 June 18-25, 2012 July 5-10, 2012

Greenhouse Greenhouse Greenhouse

July 13-14, 2012 July 29-August 4, 2012 August 13, 2012

Greenhouse Greenhouse

Molecular Biotechlogy Laboratory

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Bailey-Serres J, FukaoT, Ronald P, Ismail A, Heuer S, and Mackill DJ. 2010. Submergence tolerant rice: SUB1s journey from landrace to modern cultivar. Rice. 3(2-3):138-147. Bates BC, Kundzewicz ZW, Wu S, and Palutikof JP. 2008. "Climate change and water," 2010. Technical Paper of the Intergovernmental Panel on Climate Change, IPCC Secretariat, Geneva. Retrieved from http://www.ipcc.ch/ipcreports/tp-climate-change-water.htm. Collard, B.C., D.J. Mackill. 2008. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos Trans R Soc Lond B Biol Sci. 2008 Feb 12; 363(1491):557-72. Das, K.K., D. Panda, R.K. Sarkar, J.N. Reddy, A.M. Ismail. 2009. Submergence tolerance in relation to variable floodwater conditions in rice. Environmental and Experimental Botany 66 (2009) 425434. Dey M, and Upadhyaya H. 1996. In "Rice research in Asia: Progress and priorities". R. Evenson, R. Herdy, and M. Hossain, Eds. 291-303. CAB International, Oxon, UK. Fenomenal. 2010. Important Facts about Rice. Retrieved from: http://fenomenal.ws/facts-aboutrice#2 Fenomenal. 2010. Important Facts about Rice in General. Retrieved from: http://fenomenal.ws/facts-about-rice#2 Ignacio, J.C., P.M. Sendon, D. Sanchez, A. Ismail, D. Mackill, and E. Septiningsih. 2011. Quantitative trait loci (QTLs) identification and fine mapping of a major QTL for submergence tolerance during germination in rice derived from a tolerant variety, Ma-Zhan (red). Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, Los Baos, 4030 Laguna, Philippines International Rice Research Institute. 2011. Methods of planting rice. 1-14. Retrieved from http://www.knowledgebank.irri.org/ericeproduction/PDF_&_Docs/PlantingRice.pdf Ismail, A.M., Ella, E.S., Vergara, G.V. and Mackill D.J. Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa). Annals of Botany 103: 197-209 Mackill DJ, Ismail AM, Singh US, Labios RV, and Paris TR. 2012. Development and rapid adoption of submergence-tolerant (Sub1) rice varieties. Adv. Agron.115:303-354.

Mackill, DJ, AM Ismail. 2011. Guide to rice genotypes screening for submergence tolerance. Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, Los Baos, 4030 Laguna, Philippines Manzanilla DO, Paris TR, Vergara GV, Ismail AM, Pandey S, Labios RV, Tatlonghari GT, Acda RD, Chi TTN, Duoangsila K, Siliphouthone I, Manikmas MOA, and Mackill DJ. 2011. Submergence risks and farmers' preferences: Implications for breeding Sub1 rice in Southeast Asia. Agric. Sys. 104(4):335-347. McCouch, S.R., N. Huang, Z. Li, S. Heuer, D.J. Mackill. 2007. Molecular Breeding (Genome Mapping) Laboratory Protocols (Compilation of Protocols from 1990-2007). Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, Los Baos, 4030 Laguna, Philippines Neeraja CN, Maghirang-Rodriguez R, Pamplona A, Heuer S, Collard BCY, Septiningsih EM, Vergara G, Sanchez D, and Xu K. 2007. A marker-assisted backcross approach for developing submergence-tolerant rice cultivars. Theor. Appl. Genet. 115:767-776. Pandey, S. 2002. Direct Seeding: Research Strategies and Opportunities. International Rice Research Institute. p. 152-154 Perata P and Voesenek LA. 2007. Submergence tolerance in rice requires Sub1A, an ethyleneresponse-factor-like gene. Trends Plant Sci. 12(2):43-6. Septiningsih, E.M., Pamplona, A.A., Sanchez, D.L., Neeraja, C.N., Vergara, G.V., Heuer, S., Ismail, A.M. and Mackill, D.J. 2009. Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond. Annals of Botany 103: 151-160. Singh N, Dang TT, Vergara GV, Pandey DM, Sanchez D, Neeraja CN, Septiningsih EM, Mendioro M, Tecson-Mendoza EM, Ismail AM, Mackill DJ, and Heuer S. 2010. Molecular marker survey and expression analyses of the rice submergence-tolerance gene Sub1A. Theor. Appl. Genet. 121(8):1441-53 Southeast Asian Flood Update. October 2011. Retrieved from: http://www.fao.org/giews/english/shortnews/seasia21102011.pdf Wassman R, Jagadish SVK, Heuer S, Ismail A, Redona E, Serraj R, Singh RK, Howell G, Pathak H, and. Sumfleth K. 2009. Climate change affecting rice production: The physiological and agronomic basis for possible adaptation strategies. Donald L. Sparks, Ed. Adv. Agron. 101:59-122. Xu K, Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Heuer S, Ismail AM, Bailey-Serres J, Ronald PC, and Mackill DJ. 2006. Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature. 442(7103):705-8.