AGRICULTURE AND FISHERIES

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AF-01

AMPLIFICATION AND SEQUENCE ANALYSIS OF dhaS, ONE COMPONENT OF THE INDOLE-3- PYRUVIC ACID SYNTHETIC PATHWAY OF THE PHYTOHORMONE INDOLE-3-ACETIC ACID

Krizzia Mae R. Lumangaya, Joan Christine O. Adajar, Mannix S. Pedro and Karen B. Alviar
ABSTRACT

​Several species of Bacillus are plant growth-promoting rhizobacteria that can produce the phytohormone Indole-3-acetic acid (IAA) which regulates plant growth and development, and in some species, protects the host plant from pathogen invasion. Previous studies reveal several pathways for IAA biosynthesis in various bacterial species including genes and enzymes that take part in the biosynthetic pathway. In this study, we aim to amplify dhaS, one of the component genes from the indole-3-pyruvic acid pathway (IPyA) for IAA synthesis, and to conduct 16S rRNA sequence analysis from two Bacillus spp. isolated from Fermented Plant Juice biofertilizer. Genomic DNA extraction was performed in isolate 1 (B. amyloliquefaciens) using CTAB method followed by dhaS gene amplification through polymerase chain reaction using designed gene specific primers. Sequenced DNA was then analyzed and BLASTn results showed 98% identity with B. velezensis strain FJAT-45028 and a translated BLAST hit with an aldehyde dehydrogenase protein, having percent identity of 96%. Secondary structure of the protein coded by dhaS gene in isolate 1 was also examined using Phyre2 and Protein Data Bank (PDB) displayed models of proline dehydrogenase and aldehyde dehydrogenase.  Furthermore, 16s rRNA sequences from two Bacillus isolates were analyzed. 16S rRNA from Isolate 1 showed a top BLAST hit with Bacillus sp. strain 1CY1 (99.67%) while Isolate 2 showed a top BLAST hit with B.subtilis strain GX S-11 (95.65%). Phylogenetic trees were generated using Maximum likelihood method to reveal the relationship of the two isolates to their top five BLAST hits. To further understand the potential roles of dhaS in the IPyA pathway for IAA synthesis, transcriptional responses to l-tryptophan and functional genomic studies must be done. These will help us further understand the physiological bases of biofertilizers towards sustainable agriculture in reducing problems associated with the use of chemicals fertilizers. 
AF-02

MEMBRANE LIPID UNSATURATION CONFERS COLD GERMINATION ABILITY IN FATTY ACID MUTANTS OF UPLAND COTTON (GOSSYPIUM HIRSUTUM)

Lakhvir K. Dhaliwal, Junghyun Shim, Rosalyn B. Angeles- Shim
ABSTRACT

​Seed germination is a highly regulated process that begins with the imbibition of a mature, dry seed and ends with the protrusion of the radicle from the seed coat. During imbibition, cell membranes reorganize from a hexagonal to a lamellar phase to restore the normal metabolic functions of the seed. Perturbations in the cell membrane during this transition result in cytoplasmic leakage. Under cold stress, leakage of solutes out of the cell is exacerbated, leading to membrane damage and eventually, to embryo death. In this study, we determined the effects of fatty acid composition on the germination under cold stress (12°C and 15°C) of upland cotton mutants with higher unsaturated:saturated fatty acid (FA) ratios. Results of the study showed a more uniform and higher germination (60-80%) of the FA mutants compared to the wild type (0-20%) under the critically low temperature of 12°C and cardinal temperature of 15°C. Consistent with the reported benefits of hydropriming on seed germination under cold stress, the treatment significantly improved the germination rate, mean germination time, mean daily germination, peak value and germination index of all the experimental materials even under low temperatures. Biochemical assays showed that lipid peroxidation, an established measure of oxidative stress, was generally higher in the FA mutants after imbibition at 12°C and 15°C for 8 hours compared to the wild types. Conversely, electrolyte leakage was higher in the wild types than in the mutants after imbibition at 12°C and 15°C for 8 hours. Results suggest that the higher proportions of unsaturated fatty acids in the mutants enhanced the fluidity of the cell membrane during reorganization, facilitating the rapid restoration of cellular functions and allowing the faster and higher germination of the FA mutant seeds even under cold stress.
AF-03

EXTRACTION OF HIGH MOLECULAR WEIGHT DNA SUITABLE FOR NEXT-GENERATION SEQUENCING FROM THE FIBER CROP ABACA

Cris Francis C. Barbosa, Rhosener Bhea L. Koh, Vermando M. Aquino and Leny C. Galvez
ABSTRACT

​The abaca (Musa textilis Née), globally known as Manila hemp, is considered to be the strongest of all the natural fibers. DNA extraction in abaca is difficult due to its fibrous nature, high cellulose content and polyphenol compounds. Thus an optimized DNA extraction method is required if abaca DNA will be used for next-generation sequencing applications. This study therefore aimed to determine the most appropriate DNA extraction method for obtaining high quality DNA from abaca leaf sample to be used for NGS applications. We compared five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional CTAB method (Protocol 1), CTAB with PVP method (Protocol 2), CTAB with 0.3% β-mercaptoethanol method (Protocol 3), SDS-method (Protocol 4), and CTAB with Triton X-100 and PVP method (Protocol 5). The use of a high throughput homogenizer (TissueLyserII) was also tested in tandem with select extraction protocols for applications in high-throughput DNA extraction. DNA from Abuab and ‘Ihalas’ abaca varieties were extracted via Protocol 3 and were submitted for sequencing via Illumina HiSeq platform after passing the quality control parameters for library preparation. Protocol 3 was found to be the simplest and most consistent method for extracting NGS-quality DNA, while Protocol 4 was determined to have the shortest processing time. Together with TissueLyserII-facilitated homogenization, Protocol 4 is the most appropriate combination for high-throughput extraction of abaca samples, and will be useful for genotyping-by-sequencing (GBS) strategies as a molecular tool for plant breeding.

Keywords: DNA extraction, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Musa textilis, next-generation sequencing (NGS)
AF-04

GENOTYPING-BY-SEQUENCING BASED QTL MAPPING FOR YIELD, GRAIN ZINC AND IRON CONCENTRATION OF RICE (Oryza sativa L.)

Mark Ian C. Calayugan, Gwen Iris Descalsota-Empleo, Chau Thanh Nha, Alvin D. Palanog, Amery Amparado,
​Mary Ann Inabangan-Asilo, Teresita H. Borromeo, Jose E. Hernandez, and B.P. Mallikarjuna Swamy
ABSTRACT

​Rice (Oryza sativa L.) is one of the most important food crops and serves as the principal source of calories for more than half of the world's population. Development of high yielding rice varieties with improve grain Zn and Fe concentration is essential to help solve micronutrient malnutrition. This study was conducted at the ZES, Strategic Innovation Platform of the International Rice Research Institute (IRRI), Los Baños, Laguna, Philippines. Genotyping-by-sequencing (GBS) based high-density linkage map of rice was developed using two recombinant inbred line (RILs) mapping populations namely IR95040/Kaliboro (2018 SNPs covering 1232.22 cM) and IR95097/Kaliboro (2213 SNPs covering 1071.53 cM) with average marker density of 0.61 cM and 0.48 cM, respectively. The parental lines IR95040 and IR95097 are irrigated lowland breeding lines while Kaliboro is an Aus-type rice variety known to have high grain Zn and Fe concentrations. RILs were grown in an alpha lattice design with two replications each during the 2017 dry and wet seasons, and 2018 dry season. In total, five QTL for YLD were identified on chromosomes 4, 6, and 7, with phenotypic variance explained (PVE) ranged from 7.07-14.86%. For Fe, three QTL (qFe4.1, qFe6.1, and qFe6.2) were identified on chromosomes 4 and 6. The favorable donor allele Kaliboro has contributed for the high Fe concentration in qFe4.1 and qFe6.1. A total of 12 QTL associated with Zn were identified on chromosomes 3, 4, 5, 6, 7, 8, 10, and 12, which accounted for 5.17-18.33% of PVE. Four QTL were consistently detected on chromosomes 3, 7, and 10 in IR95040/Kaliboro and on chromosome 4 in IR95040/Kaliboro viz. qZn3.1, qZn7.3, qZn10.1, and qZn4.1. Validation of the genomic regions reported in this study will help accelerate the development of SNP chip for breeding healthier rice varieties and will be beneficial in rapid development and deployment of biofortified rice varieties. 

Keywords: rice, zinc, GBS, Quantitative Trait Loci
AF-05

VALIDATION OF CHALCONE SYNTHASE GENE SINGLE NUCLEOTIDE POLYMORPHISM FOR RED COLOR IN MANGO (Mangifera indica L.)

Aira Janella L. Elec, John Albert P. Lachica, and Eureka Teresa M. Ocampo
ABSTRACT

​The Carabao mango is the Philippines’ vital and premium export variety because of its nutritive value and its distinctive sweet taste compare to other varieties. Carabao variety is one of the top fruit exports that has been essential in the growth of the Philippine economy. According to PSA (2015), mango ranks third in terms of production volume of 885,000 metric tons. Its export potential is however, hampered by industry problems and a growing preference for mangoes with red blush. Red blush trait correlates to plant resistance against cold and pathogens and has a potential benefits to human health. Ten newly designed primers based on SNP of chalcone synthase gene derived from the GBS data of 341 mango genotypes were used to amplify chalcone synthase from sixteen mango genotypes with red and yellow skin color. This was an attempt to develop SNP markers for red blush that can be utilized for mango breeding programs. Only four out of ten primers were successfully optimized for the amplification of chalcone synthase namely FLVS, Chs2, DRPRGA3, and At4g. The optimum annealing temperatures for each of the markers were 51.6°C, 49°C, 47.9°C, and 46°C, respectively, resulting in amplification of desired DNA bands after PCR. The primers were able to differentiate mangoes with red peel from those with yellow peels. After sequencing of selected PCR products followed by sequence analysis, it was found that red and yellow varieties exhibited highly conserved sequences for the chalcone synthase. However, different insertions and deletions within the conserved region were also observed. The phylogenetic tree based on Chs2 sequence of sixteen mango genotypes showed that the same colored mangoes tended to cluster together in one clade, showing the usefulness of chalcone synthase SNPs for identifying red skin color in mango. 

Keywords: Carabao variety, SNP marker, chalcone synthase, red blush
AF-06

IDENTIFICATION OF LINAMARASE- PRODUCING LACTIC ACID BACTERIA AND YEASTS FOR CASSAVA (Manihot esculenta Crantz) SOURDOUGH FERMENTATION

Francisco B. Elegado, Margarita A. Mercado, Hazel Alena D. Tan, Johanna A. Bangoy, Ralph Ryan M. Gibas
ABSTRACT

​Cassava (Manihot esculenta Crantz) is a tropical rootcrop considered as staple food to millions of people worldwide. However, one of the drawbacks of this rootcrop is the presence of toxic cyanogenic compound used by the plant as defense mechanism against pests.  Fortunately, the toxic compound can be degraded during fermentation, facilitated by linamarase which is a hydrolytic enzyme for cyanide degradation and can be produced by lactic acid bacteria (LAB) and yeasts. Thus, this project aimed to screen LAB and yeasts for their ability to produce linamarase which can then be utilized for cassava sourdough fermentation. Previously, we have screened a total of 166 LAB from existing collection of cultures and from cassava sourdough, and 113 yeast isolates from forest canopy for their ability to degrade cyanide. To ensure that the isolates are safe to be used for the fermentation of cassava sourdough, the isolates were identified through 16S and 18S rRNA sequencing. The high linamarase-producing LAB isolates were that from papaya flower, identified as Enterococcus faecalis, and that from cassava sourdough, identified as Leuconostoc mesenteroides. Moreover, the three yeast isolates with high linamarase activity were identified as Cyberlindnera mrakii. The HCN degrading abilities of these isolates were characterized during sourdough and bread preparation. 

Keywords: Cassava, Lactic Acid Bacteria, Yeast, Linamarase, sourdoug
AF-07

SEQUENCING AND IDENTIFICATION OF WRKY TRANSCRIPTION FACTORS IN ABACA (Musa textilis Neé)

Richard I. Encarnacion and Vermando M. Aquino
ABSTRACT

​The abaca plant (Musa textilis Neé) is a fiber crop indigenous to the Philippines and is a close relative of banana.  Diseases due to viral pathogens such as the abaca bunchy top virus (ABTV), banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV) can be detrimental to the quality and yield of fiber produced. Though of great economic importance to the country, there has been only a few published works that delve into the molecular genetic mechanisms that underly processes such as fiber production and disease resistance. This study is focused on the WRKY genes, which are a family of transcription factors ubiquitous to green plants that are identified by the conserved WRKYGQK motif and a C2H2 or C2HC zinc-finger binding motif. These transcription factors have been implicated in a variety of biological functions dealing with plant development and stress responses, among others.  Total DNA was extracted from M. textilis var ‘Abuab’ and used as a template to amplify WRKY genes with PCR using degenerate primers targeting the WRKY motif. Amplified fragments of 250 bp, 300 bp, 800 bp, and 1000 bp were purified, cloned, and sent for sequencing. Comparison of the DNA and predicted translated protein sequences obtained to online databases with BLAST revealed 27 sequences contained WRKYGQK and partial C2HX domains that were closely related to predicted Musa acuminata and Musa balbisiana WRKY transcripts. Mapping and phylogenetic analysis revealed that the sequences clustered with either Group II or Group III WRKY domain types.  This is the first reported study on sequence identification of WRKY transcription factors in abaca and is a first step to identify genes for targeted genetic engineering for increased disease and stress tolerance in abaca.

Keywords: abaca, PCR, WRKY, transcription factor
AF-08

ISOLATION AND CHARACTERIZATION OF BIOTIN SYNTHASE GENE IN COCONUT (Cocos nucifera L.)

Rafrel E. Caisip and Roberta N. Garcia
ABSTRACT

​Coconut, Cocos nucifera L., is an important oil crop in the Philippines.  Recently, there had been growing interests in increasing its oil content through metabolic engineering.  The first committed step in the biosynthesis of lipids is catalyzed by the tightly regulated enzyme, Acetyl-CoA carboxylase (ACCase) which significantly controls the carbon flux in this pathway (Ali and Tyagi, 2016).  Biotin acts as a cofactor for ACCAse and its synthesis involves four significant enzymes. One of which, biotin synthase, is considered as the rate-limiting step in the biotin synthesis pathway. Increasing the concentration of biotin on its maximum level as the co-factor of ACCAse would also increase the rate of lipid biosynthesis. This study aimed to isolate and characterize the biotin synthase gene from coconut towards understanding its utility in increasing oil production through biotechnology.   The isolated 749-base pair nucleotide sequence revealed significant homology with the biotin synthase of various plant species such as Elaeis guineensis, Phoenix dactylifera, Musa acuminata, Asparagus officinalis, Ananas comosus, Sorghum bicolor and Zea mays with 76- 85% identity. The deduced amino acid sequence consisted of 244 residues and shared 59-73% homology with the Radical S-adenosylmethionine (SAM) region and the Biotin and Thiamin Synthesis (BATS) associated domain of other oil crops.Phylogenetic analysis showed that the coconut biotin synthase gene was closely similar to those of the oil palm and the date palm.Sequence analysis of the isolated gene fragment from coconut ascertained its identity to published biotin synthase gene sequences.  This characterized partial coconut biotin synthase gene sequence could be used to further isolate the full-length gene.

Keywords: Coconut, lipid biosynthesis, biotin, biotin synthas
AF-09

DEVELOPING MORPHOLOGICAL AND SIMPLE SEQUENCE REPEATS MARKERS FOR PUTATIVE DROUGHT TOLERANT PAPAYAS*

Pablito M. Magdalita and Alangelico O. San Pascual
ABSTRACT

​Drought as a result of climate change has adverse effects on plant production. It is known to negatively impacted food production. One important crop in the Philippines which is affected by drought is papaya. Papaya ringspot virus (PRSV) and bacterial crown rot (BCR) are two other pressing problems of this crop. Under drought conditions in the field, papaya showed decrease productivity as it produces smaller and fewer fruits but had sweeter flesh.  Ten papaya genotypes were selected as putative drought tolerant.  The basis for drought tolerance are their relative growth rate in terms of plant height, stem diameter, the chlorophyll content of leaves, and number of closed stomata during imposition of drought.  Compared to drought susceptible genotypes, putative drought tolerant papaya genotypes have more closed stomata, higher chlorophyll content, bigger stem and crown diameter and have more number of leaves. To mark the putative drought tolerant selections, Screening of 21 simple sequence repeats (SSR) primers to reveal polymorphism among 15 genotypes was done.  Polymorphic bands were consistently revealed by 5 SSR primers across 15 genotypes.  From the 21, 2 SSR primers have been selected to mark putative drought tolerant genotypes.

Keywords: Carica papaya L. chlorophyll, drought, simple sequence repeats, stomata
AF-10

PERTURBATION OF RICE (Oryza sativa L.) LEAF ARCHITECTURE THROUGH ECTOPIC OVEREXPRESSION OF POLYADENYLATE BINDING PROTEIN (PABP).

Robert A. Nepomuceno, Jolly Chatterjee, Robert Coe, Jacqueline Dionora, and William Paul Quick
ABSTRACT

​Functional genomics through the use of gain of function approach such as full-length cDNA overexpression is often utilized to potentially identify gene function. Gain-of-function approach offers an advantage over loss-of-function mutants since most species has a large proportion of redundant genes, and the function of such genes are impossible to identify using a gene-knockout approach because the redundant copy just compensates for the loss of function created. In this study, full-length cDNA was generated from RNAs derived from rice seedling root tissues. A rice overexpression mutant manifesting debilitating dwarfism was generated, however, closer examination of the fl-cDNA revealed it to be polyadenylate binding protein of Schizosaccharomyces cerevisiae origin. The fl-cDNA is 2,254 bp with the longest predicted ORF of 2,013 bp, representing 89.31% of the total length. The predicted polypeptide is 670 amino acid residues. Analysis of the protein sequence revealed a dominant RNA recognition motif (RRM) at amino acid positions 134-204, 227-297, and 330-400. RNA recognition motif (RRM) is characterized by 90 amino acid residues with two conserved sequences of eight and six amino acids, called RNP-1 and RNP-2, respectively, in the protein primary structure. Proteins with RNA recognition motif have multitudes of functions which include the heterogeneous nuclear ribonucleoproteins (hnRNPs) that function in the processing of hnRNAs into a mature mRNA as well as a trans regulatory factor in gene expression proteins involved in the regulation of alternative splicing, RNA stability and translation and protein components of small nuclear ribonucleoprotein.

Keywords: full-length cDNA; RNA recognition motif; Schizosaccromyces cerevisiae; heterologous overexpression
AF-11

IMPROVEMENT OF ASIAN RICE CULTIVARS THROUGH MARKER-ASSISTED INTROGRESSION OF YIELD QTLS GRAIN NUMBER 1A (GN1A) AND WEALTHY FARMER'S PANICLE (WFP)

Vincent P Reyes, Rosalyn B. Angeles-Shim, Ruby S. Lapis, Jung-Hyun Shim, Hidehiko Sunohara,
​Kshirod K Jena, Motoyuki Ashikari, Kazuyuki Doi
ABSTRACT

​Food insecurity is one of the major problems of our growing population. With the threats of climate change and an exponential decrease in arable land, crop productivity improvement is needed more than ever. The advent of molecular markers has given the field of plant breeding the ability to fast track plant selection with target genes for selected traits in shorter generations. Introgression of abiotic and biotic related QTLs has been a common practice for crop improvement; however, utilizing yield-related QTLs has remained limited. In this study, major QTLs Grain number 1a (Gn1a), for an increase in spikelet number, and Wealthy Farmer's Panicle (WFP), for an increase in primary branching, were introgressed in four Asian rice cultivars (Kinandang patong, IRAT 109, Silewah, Basmati) from the donor parents ST12 and ST6. The rice microsatellite (RM) markers RM3360, RM 5493, and RM3452 were used to confirm the introgression. Introgressed lines at BC3F5 and BC3F6 generation from each genetic background were evaluated for phenotypic traits, focusing on primary branching per panicle (PBPP) and total spikelet per panicle (TSPP). Improvements in TSPP and PBPP were observed in selected lines representing four genetic backgrounds. Improved lines with introgressed Gn1a or WFP were comparatively similar to its recurrent parent in yield-related traits such as heading date (HD), plant height (PH), hundred-grain weight (HGW).  This study demonstrates the success of utilizing yield QTLs for improving the yield performance of our existing rice cultivars. 

Keywords: Rice, Molecular Breeding, Marker-assisted breeding, Gn1a, WFP, QTL, Crop Improvement
AF-12

THE GENETIC DIVERSITY OF BROWN AND RED RICE SAMPLES AND THEIR ANTIOXIDANT ACTIVITIES, ANTICANCER, AND LOW GLYCEMIC PROPERTIES

Yariv Brotman, Cindy Llorente, Saurabh Badoni, Glenn Oyong, Gopal Misra, Roslen Anacleto, Sabiha Parween, Erstelle Pasion, Rhowell N. Tiozon Jr. , Joanne J. Anonuevo,Maria K. deGuzman, Edwige G.N. Mbanjo, Lesley A. Boyd, Alisdair R. Fernie, Nese Sreenivasulu
ABSTRACT

​The double burden nutrition problem (i.e. lack of essential nutrients and calorie rich food) is a
growing epidemic in Asia and around the globe, where cancer and diabetes are part of the major causes of death. Hence, bringing diet-based nutritional intervention by identifying rice varieties with grains rich in nutrient density is becoming increasingly significant. In this research, metabolome-wide association study (mWAS) was conducted by linking the metabolites with ultra-dense genotyping data of 1.7 million single nucleotide polymorphisms (SNPs). It revealed novel genetic regions and/or candidate genes influencing the levels of various amino acids and catechin as pivotal molecules in determining the nutritional quality of brown and red rice. Importantly, the novel genetic variants identified through mWAS which was found to affect catechin accumulation on chromosome 7 (6.06-6.43 Mb region), co-located with GWAS peaks of red colour and amylopectin composition and thereby the glycemic index. Contrasting lines resulted from mWAs analysis were used to extract the free and bound phenolic components. Red rice extract has higher phenolic content thereby, effective antioxidant activities than brown rice extract in concordance with its potent antiproliferative property against different cancer cell lines. The extract of red rice exhibited strong anti-proliferative activity with mean IC50 values of 8.25, 17.13, and 24.47 mg/mL while the brown rice extract with mean IC50 values of 1580.57, 23007.56, and 55400.58 mg/mL against HT-29, MCF-7, and HepG2, respectively. Further, in vitro GI analysis has demonstrated a lower glycemic index in milled red rice with high resistant starch. The selected rice samples may be used as a functional food to address diabetes and certain forms of cancer.   
AF-13

TGW6 KNOCKDOWN CAUSES PLEIOTROPIC EFFECTS IN ELITE RICE VARIETIES INCREASING YIELD

Lawrence Yves Uy, Yvonne Ludwig, Merlyn Mendioro, Ma Carmina Manuel, Jorge Gil Angeles, Inez Slamet-Loedin
ABSTRACT

​An estimated 4 billion people depend on rice as their staple food and this number increases rapidly. As such, increasing its production is a major challenge. A suggested solution for this is gene editing yield-related regions of interest in elite indica rice varieties such as IR64 and Samba Mahsuri (SM) since these varieties are already widely accepted in the market. One promising region of interest is the THOUSAND GRAIN WEIGHT 6 (TWG6) because it has showed promising influence in grain weight when knocked out in japonica varieties. In this study, TGW6 was knocked-down in IR64 and SM rice varieties using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cpf1 system. A CRISPR Cpf1 vector construct (IRS-1466) was assembled to target TGW6 in both IR64 and SM. The resulting knockout IR64 and SM were analyzed through PCR screening for the presence of CRISPR gene while cutting efficiency and mutational changes in the region of interest were analyzed using T7 assay and sequencing, respectively. Knockout SM had 81.7% cutting efficiency while knockout IR64 had 94.34% cutting efficiency.  Sequence analysis revealed minimal single nucleotide insertion-deletions (indels) in the region of interest. Phenotypic data showed significant increase in total grain weight of both knockout SM (increased up to 33.5%) and IR64 (105.11%) compared to the wild-type controls. In addition, increase in grains per panicle   were also observed in both SM and IR64 knockouts. These results reveal that targeted knockdown via CRISPR-Cpf1 of TGW6 increases grain weight thus, increasing rice grain yield which has great potential as a possible solution in the foreseeable rice production challenges ahead.  

Keywords: CRISPR-Cpf1, Gene editing, Rice, and Yield.