Volume 6 Special Issue 1 2012
Including select papers from the 6th International Rice Genetics Symposium

ISBN 978-4-903313-88-7

How to reference: Sié M, Sanni K, Futakuchi K, Manneh B, Mandé S, Vodouhé R, Dogbe S, Dramé K-N, Ogunbayo A, Ndjiondjop M-N, Traoré K (2012) Towards a Rational Use of African Rice (Oryza glaberrima Steud.) for Breeding in Sub-Saharan Africa. In: Ismail A (Ed). Genes, Genomes and Genomics 6 (Special Issue 1), 1-7

Guest Editor

Abdelbagi M. Ismail

International Rice Research Institute (IRRI), Los Baños, The Philippines

www.irri.org

CONTENTS AND ABSTRACTS

Moussa Sié, Kayode Sanni, Koichi Futakuchi (Benin), Baboucarr Manneh (Senegal), Semon Mandé (NIgeria), Raymond Vodouhé, Selome Dogbe, Khady-Nani Dramé, Ayoni Ogunbayo, Marie-Noelle Ndjiondjop (Benin), Karim Traoré (Senegal) Towards a Rational Use of African Rice (Oryza glaberrima Steud.) for Breeding in Sub-Saharan Africa (pp 1-7)

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Invited Review: Africa Rice Center (AfricaRice) plays an important role in germplasm enhancement for their adaptation to different ecologies of upland, lowland, irrigated, mangrove and deep water. The genebank of AfricaRice contains over 20,000 rice accessions, notably two cultivated species (Oryza sativa and O. glaberrima) and wild species (O. longistaminata, O. barthii and O. stapfii), which are being used in breeding programs. In recent years, NERICA rice suitable for upland, lowland and irrigated ecologies were developed from interspecific crosses between O. sativa and O. glaberrima. For the better utilization of O. glaberrima, characterization studies of genetic diversity have been madeusing both molecular and conventional tools. The screening of this material for biotic and abiotic stresses has allowed us to identify firstly, genes for resistance to major rice diseases such as rice yellow mottle virus, rice blast disease and insect pests, and secondly, new sources of tolerance to drought, salinity and iron toxicity. A thorough exploitation of these resistance/tolerance genes will lead to rice varieties of better performance, under the heterogeneous production conditions in Africa. AfricaRice therefore planned to better exploit O. glaberrima and wild species conserved in its genebank through the use of biotechnology tools. Emphasis shall also be on the improvement of grain quality, nutritional values and post harvest techniques; this will greatly enhance the achievement of the objective of producing better quality rice in Africa. The objective of this paper is to propose several ways to better exploit O. glaberrima as breeding materials than the current interspecific breeding program through the critical review of published data and new additional data on the performance of NERICA lines.

Charles P. Chen (Japan), Michael Frei (Germany), Juan Pariasca Tanaka, Yoshihisa Kohno, Matthias Wissuwa (Japan) Tropospheric Ozone Poses a Rising Threat to Yield Stability in Rice: Tolerance Mechanisms and Underlying Genetic Factors (pp 8-15)

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Invited Mini-Review: Rice (Oryza sativa L.) is the major staple food for most of the developing countries in Asia, whose populations are predicted to steadily increase in this century. However, global change will put increasing pressure on crop production, as it is associated with a range of abiotic stress factors that limit crop yields. One such stress factor is tropospheric ozone (O₃), whose rising background concentrations are a consequence of rising emissions of precursor gases such as nitrogen oxides and volatile organic compounds. Surface O₃ levels that exceed the critical threshold beyond which yield declines are expected are now common in many rice growing areas of South and East Asia and climate models predict further increases for the next three decades. It is therefore imperative to assess the potential to develop new breeding lines with improved tolerance to ozone exposure. This review summarizes the current state of research on the effect of ozone on rice, focusing on recent efforts in the breeding for ozone tolerance. Moreover, novel data are presented that characterize physiological and agronomic features of previously reported quantitative trait loci (QTL) for ozone tolerance, including two QTLs influencing leaf bronzing (OzT3 and OzT9) and one QTL influencing dry weight (OzT8). In addition, the first ozone response data of ‘IR64’, the most widely-grown tropical rice cultivar in South and South-east Asia, is presented, including data on photosynthetic performance and yield loss under season-long ozone exposure.

Khin Thanda Win, Takahiko Kubo, Yuta Miyazaki, Kazuyuki Doi, Hideshi Yasui, Yoshiyuki Yamagata, Atsushi Yoshimura (Japan) Molecular Mapping of Two Loci Conferring F₁ Pollen Sterility in Inter- and Intraspecific Crosses of Rice (pp 16-21)

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Original Research Paper: Hybrid sterility, a major form of post-zygotic reproductive barriers, often appears in crosses between relatively distant species as well as between closely related subspecies. To unravel the genetic mechanism and cytological features of F₁ pollen sterility, we identified and characterized the loci causing F₁ pollen sterility using introgression lines of the donor parent O. nivara in the genetic background of O. sativa ssp. japonica cv. ‘Taichung 65’ and chromosome segment substitution lines carrying O. sativa ssp. indica cv. ‘IR24’ segments in the genetic background of ssp. japonica cv. ‘Asominori’. In this study, we report two F₁ pollen sterility loci, designated as S36 and S25, found in inter- and intraspecific crosses, respectively. Genetic analyses revealed that allelic interaction at the heterozygous locus caused the sterility of male gametes carrying the japonica alleles in both cases. Both loci are located on the distal end of the short arm of rice chromosome 12 and comparison of the map positions of S36 and S25 indicated that these two loci might be the same locus. Cytological investigation demonstrated that abnormality of sterile pollen grains caused by S36 occurred mainly at the late bicellular stage after initiation of starch accumulation. The present study provides a better understanding on the genetic nature and the cytological aspect of F₁ pollen sterility and the evolutionary dynamics of post-zygotic reproductive isolation in rice, and consequently, could help to overcome the reproductive barriers in inter- and intraspecific hybridization for the improvement of cultivated rice.

Muthurajan Raveendran (India/The Philippines), Krishna S. V. Jagadish (The Philippines/UK), Peter Q. Craufurd (UK/India), John Bennett (The Philippines) Proteomic Response of Rice Floral Organs and Lemma-palea to High Temperature Stress (pp 22-25)

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Original Research Paper: In rice, a brief period of 6 h of high temperature at flowering had a significant effect on reproductive processes. High temperature stress had a significant effect on the number of spikelets opening per day and spikelet fertility (P<0.001). High temperature stress inhibited normal anther dehiscence and pollination, thereby reducing spikelet fertility. 2D-PAGE analysis of control and high temperature-stressed floral organs of rice spikelets, i.e., anthers (male), pistil (female reproductive organ), and lemma-palea, revealed that anthers had more proteins with significant changes in expression, followed by the lemma-palea. The pistil, on the other hand, had no significant changes in protein expression. This study provides evidence at the molecular level that the anther is the most responsive and sensitive floral organ to high temperatures, facilitating tissue-specific physiological and molecular studies to induce high temperature tolerance in rice varieties.

Sushma M. Awaji, Madhavi D. Naidu, Prashantkumar S. Hanjagi, Sashidhar V. Ramakrishna, Rohini Sreevathsa (India) Possibility of Improved Salt Tolerance in Rice Transgenics Overexpressing pgNHX1 (pp 26-33)

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Original Research Paper: In the present study, transformants in rice were developed with PgNHX1 gene following a tissue culture-independent in planta transformation protocol. Analysis of T₁ plants by a stringent salt screening test at seedling and plant level identified putative transformants. Integration of the transgenes in T₁ generation plants was confirmed at the molecular level by PCR, genomic Southern and RT-PCR analysis. Physiological studies such as chlorophyll estimation and membrane permeability tests revealed that some of the T₁ transformants showed lower percent reduction in chlorophyll content and less membrane leakage compared to wild type under salt stress. These results clearly demonstrate that transgenic rice plants overexpressing PgNHX1, a vacuolar antiporter have better salt-tolerance. The stable integration and inheritance of the transgene in subsequent T₂ generation was also confirmed by seed germination assay and PCR analysis.

Gautam Kumar, Hemant R. Kushwaha, Ram S. Purty, Sumita Kumari, Sneh L. Singla-Pareek, Ashwani Pareek (India) Cloning, Structural and Expression Analysis of OsSOS2 in Contrasting Cultivars of Rice under Salinity Stress (pp 34-41)

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Original Research Paper: Salinity is one of the major environmental factors limiting growth and productivity of crop plants in coastal areas and irrigated farmlands. Salinity tolerance is a very complex trait. Plants adapt to salinity stress by coordinated and orchestrated functioning of various complex mechanisms. In Arabidopsis thaliana, SOS (Salt Overly Sensitive) pathway has been established as a major player in ion homeostasis and salt tolerance. The SOS pathway has recently been shown to be conserved in rice as well. In the present study, we have isolated and characterized the OsSOS2 full-length cDNA from a salt sensitive Oryza sativa L. cv ‘IR64’, which encodes 50.65 KD protein. It was observed that OsSOS2 transcripts are induced by salinity and further showed differential accumulation at different time intervals at seedling stage in contrasting cultivars of rice i.e. ‘IR64’ (salt sensitive) and ‘Pokkali’ (naturally salt tolerant). We have also observed tissue specific expression for OsSOS2 in field grown mature plants of these contrasting cultivars. With the use of molecular modeling techniques, we have modeled OsSOS2 protein and present a comparative structural analysis with respect to its ortholog from model plant - Arabidopsis thaliana. Comparison of various orthologous sequences has shown high level of similarities between SOS2 members isolated from Arabidopsis thaliana and O. sativa. Experiments have established that the SOS3 protein senses Ca²⁺ and regulates SOS2 activity. Therefore, we have carried out the analysis of conserved binding site for SOS3 protein in SOS2 protein which can give an insight to the probable mechanism of the functioning of OsSOS2 protein. We propose that OsSOS2 is one of the important members of salinity stress response in rice functioning towards ion homeostasis.

M. M. Hossain, M. M. Islam, H. Hossain, M. S. Ali (Bangladesh), Jaime A. Teixeira da Silva, Atsushi Komamine (Japan), Shamsul H. Prodhan (Bangladesh) Genetic Diversity Analysis of Aromatic Landraces of Rice (Oryza sativa L.) by Microsatellite Markers (pp 42-47)

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Original Research Paper: The allelic diversity and relationship among 12 elite aromatic ricecultivars were determined through DNA fingerprinting using microsatellite markers. A total of 24 SSR markers were used to characterize and discriminate all rice genotypes, 10 of which were polymorphic for different chromosome numbers. The number of alleles per locus generated by each marker varied from 2 (RM256, RM510) to 7 (RM180), with an average of 3.8 alleles per locus. The polymorphic information content (PIC) values varied widely among loci and ranged from 0.239 to 0.765 with an average of 0.508. The highest PIC value (0.765) was obtained for RM180, followed by RM207 (0.746), RM224 (0.680), RM163 (0.593), and RM566 (0.505). The genetic distance-based results seen in the unrooted neighbor-joining tree and UPGMA clustering revealed five genetic groups in which closely related genotypes were clustered together. A moderate salt-tolerant variety, Rajasail, grown mainly in coastal areas, formed a distinct monogenic cluster. Another single cluster was formed by Tilockachari, which was 54.02% dissimilar with Rajasail; the remaining aromatic land races formed distinct clusters according to their relatedness. Pairwise genetic dissimilarity was also studied to determine the level of relatedness among the cultivars in which highest genetic dissimilarity was recognized between Gondhocosturi and Lalrodha dhan (81.03%), Gondhocosturi and Rajasail (81.03%), and other pairs. This information will provide maximum selection of diverse parents, background selection during backcross breeding programs and assist in broadening germplasm-based rice breeding programs in the near future.

Pallavi Pawar, Churkunnu Krishnan Suresh, Shailaja Hittalmani, Keshava Murthy B.C., Hanamareddy Biradar (India) DNA Marker-Assisted Analysis of Recombinant Inbred Lines Using Trait-Specific Markers and Candidate Genes in Rice (Oryza sativa L.) (pp 48-51)

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Original Research Paper: Rice (Oryza sativa L.) is the staple food for most people in Asia and is major caloric dietary source. As population and economy expand, the demand for consumption of rice will increase. To meet this growing demand it is required to increase the productivity levels of rice by combating biotic and abiotic stresses and other constraints. Early duration Recombinant Inbred Lines, 41 in total, developed using deep-rooted (Moroberekan) and high-yielding (IR50) parents were validated with 15 trait-specific SSR markers. These genotypes were validated by 4 candidate genes. Single marker analysis was done to study the association of markers with traits using the Student’s t-test. RM242 and RM282 were associated with plant height, RM302 with days to 50% flowering, RM242 with number of tillers per plant, while RM257 was associated with seed weight per plant. Among the four candidate genes, Ext1L/1R, coding for extensin protein/leucine-rich repeats protein, was associated with days to maturity and MAPK for transcriptional control of stress response was associated with number of panicles.

Pallavi Pawar, Churkunnu Krishnan Suresh, Shailaja Hittalmani, Keshava Murthy B.C., Hanamareddy Biradar (India) Response of Selected Recombinant Inbred Lines for Yield and Yield-Attributing Traits under Aerobic Conditions and Marker-Assisted Graphical Representation of Superior Rice Genotypes (pp 52-59)

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Original Research Paper: Rice is one of the most important food crops drastically affected by drought in the rice ecosystem. Among grain crops, rice is the single biggest user of water, requiring 2 to 3 times more water per unit of grain produced. Recognizing the water constraints to rice yield, an alternative method of rice production using available water is necessary. One such method is aerobic cultivation which requires about 50% of water as puddle cultivation. To generate new varieties suitable to aerobic cultivation, 41 early duration recombinant inbred lines developed using high-yielding (IR50) and deep-rooted (Moroberekan) parents, were evaluated for drought and yield traits under aerobic conditions. Among these, IM 65, IM71, IM84, IM108, IM122, IM151 and IM181 showed superior field traits. Furthermore, the genomic regions of IM65 (drought tolerant) and IM84 (drought susceptible) were graphically genotyped with the help of 45 SSR markers. Identification of DNA fragments responsible for drought tolerance may further help in developing introgression lines with an IR50 background and with Moroberekan-like drought tolerance.

Tracie M. Jenkins, Ming Li Wang, Noelle A. Barkley (USA) Microsatellite Markers in Plants and Insects Part II: Databases and in Silico Tools for Microsatellite Mining and Analyzing Population Genetic Stratification (pp 60-75)

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Invited Review: Nucleotide sequence information available in searchable sequence databases and the free in silico software with which to extract and analyze microsatellite data continues to grow at a rapid rate across eukaryote taxa. The sheer amount of information available means that a comprehensive or exhaustive review of databases and free bioinformatic tools lies beyond the purview of any journal review. The purpose of this review is therefore to provide targeted information aimed at helping the insect and plant biologist effectively utilize in silico resources to find, navigate and analyze empirically derived data from sequence databases. The objectives are threefold. First, since the basic characteristics of microsatellites make them the markers of choice for studies of genetic structure that underlie adaptation and evolution, these will be delineated. Second, because sequence databases are increasingly mined for microsatellites, the major databases are discussed, as well as, available programs for in silico mining of sequence databases to retrieve microsatellites for a species of interest. Lastly, a general review is given of population genetics software for in silico genetic analyses of microsatellite data to determine population genetic structure, phylogenetic relationships, and genetic diversity in a species of interest.

Mohamed Helmy, Masaru Tomita, Yasushi Ishihama (Japan) Peptide Identification by Searching Large-Scale Tandem Mass Spectra against Large Databases: Bioinformatics Methods in Proteogenomics (pp 76-85)

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Invited Review: Mass spectrometry-based shotgun proteomics approaches are currently considered as the technology-of-choice for large-scale proteogenomics due to high throughput, good availability and relative ease of use. Protein mixtures are firstly digested with protease, e. g. trypsin, and the resultant peptides are analyzed using liquid chromatography - tandem mass spectrometry. Proteins and peptides are identified from the resultant tandem mass spectra by de novo interpretation of the spectra or by searching databases of putative sequences. Since this data represents the expressed proteins in the sample, it can be used to infer novel proteogenomic features when mapped to the genome. However, high-throughput mass spectrometry instruments can readily generate hundreds of thousands, perhaps millions, of spectra and the size of genomic databases, such as six-frame translated genome databases, is enormous. Therefore, computational demands are very high, and there is potential inaccuracy in peptide identification due to the large search space. These issues are considered the main challenges that limit the utilization of this approach. In this review, we highlight the efforts of the proteomics and bioinformatics communities to develop methods, algorithms and software tools that facilitate peptide sequence identification from databases in large-scale proteogenomic studies.

Gábor Gyulai, Zoltán Szabó, Barna Wichmann, András Bittsánszky (Hungary), Luther Waters Jr. (USA), Zoltán Tóth (Hungary), Fenny Dane (USA) Conservation Genetics – Heat Map Analysis of nuSSRs of aDNA of Archaeological Watermelons (Cucurbitaceae, Citrullus l. lanatus) Compared to Current Varieties (pp 86-96)

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Original Research Paper: Seed remains of watermelon (Citrullus lanatus lanatus) were excavated from two sites dating from the 13^th (Debrecen) and 15^th centuries (Budapest) Hungary. Morphological characterization, aDNA (ancient DNA) extraction, microsatellite analyses, and in silico sequence alignments were carried out. A total of 598 SSR fragments of 26 alleles at 12 microsatellite loci of DNAs were detected in the medieval and current watermelons. A heat map analysis using doubledendrograms based on microsatellite fragment patterns revealed the closest similarity to current watermelons with red flesh (13^th CENT) and yellow flesh (15^th CENT) colors. In silico studies on cpDNA and mtDNA of watermelon revealed new data on Citrullus genome constitution. The results provide new tools to reconstruct and ‘resurrect’ extinct plants from aDNA used for conservation genetics.

Othman E. Othman, Fawzia A. Zayed, Ali A. El Gawead, Medhat R.A. El-Rahman (Egypt) Genetic Polymorphism of Three Genes (PRL, K-CN and PIT-1) Associated with Milk Traits in Egyptian Buffalo (pp 97-101)

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Original Research Paper: In dairy farm animals, the principal goal of selection is the improvement of milk yield and composition. The genes encoding milk protein and hormones are excellent candidate genes for linkage analysis with quantitative trait loci (QTLs) because of their biological significance on the quantitative traits of interest. Prolactin (PRL), a polypeptide hormone with multiple functions, secreted mainly by the anterior pituitary gland, shows many biological activities and plays essential roles in reproduction, lactation and immune functions. Casein proteins and their genetic variants have been reported as important factors associated with lactation perfor­mance, milk composition and the efficiency of cheese yield. Genetic variants of the bovine kappa-casein (K-CN) gene are associated with milk protein content and have a significant influence on rennet clotting time, firmness and cheese yield of milk. The pituitary-specific transcription factor (PIT-1)gene is responsible for pituitary development and hormone-secreting gene expression in mammals. PIT-1 was studied as a candidate genetic marker for growth, carcass and also for milk yields traits. Genomic DNA extracted from 100 healthy buffaloes was amplified using primers that were designed for the cattle PRL, K-CN and PIT-1 gene sequences. The amplified fragments of PRL (294-bp), K-CN (530-bp) and PIT-1 (451-bp) were digested with RsaI, HindIII and HinfI restriction enzymes, respectively. All tested buffaloes were genotyped as GG for PRL, BB for K-CN and for PIT-1.

Muhammad Youssef (Egypt) Khirshyat 1.0: a Simple Micro-Program for Some Molecular Biology Protocols (pp 102-105)

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Research Note: Khirshyat 1.0 is a simple, dynamic and informative micro-program designed mainly to provide scientists, researchers and students in research systems and universities in developing countries, a useful tool on some molecular biology protocols. The program includes the most commonly used molecular markers (AFLP, SRAP and RAPD), PCR optimization, oligonucleotide properties, calculations of concentration and quantity using the molecular weight of most commonly used chemicals, plant DNA extraction, DNA concentration and purity, primer preparation, polyacrylamide gel electrophoresis and silver nitrate staining protocols. It accelerates the process of preparing stocks, buffers, solutions as well as provides important tips and general information with simple illustrations about the most common protocols. The program and documentation are freely available online.