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  1. Fulltext Technical aspects in understanding effects of gamma irradiation on flower colour changes in Dendrobium Sonia
    Sakinah Ariffin, Azhar Mohamad, Ratnam, Wickneswari
    Jurnal Sains Nuklear Malaysia, 2012;24(1):91-101.
    MyJurnal
    Colour is one of the most important traits in orchids and has created great interest in breeding programmes. Gamma irradiation is an alternative way for generation of somaclonal variation for new flower colours. Phenotypic changes are usually observed during screening and selection of mutants. Understanding of targeted gene expression level and evaluation of the changes facilitate in the development of functional markers for selection of desired flower colour mutants. Four Dendrobium orchid sequences (NCBI accessions: AM490639, AY41319, FM209429 and DQ462460) were selected to design gene specific primers based on information for chalcone synthase (CHS) from NCBI database. Quantitative real-time PCR (qPCR) was used to understand flower colour expression quantitatively derived from the CHS gene activities in different flower tissues (petal and sepal) from control Dendrobium Sonia (red purple), mutant DS 35-1/M (purple pink) and mutant DS 35-WhiteA. It was found that expression of CHS gene was highest in sepals of white flowers and lowest in both sepals and petals of purple pink flowers. Genomic DNA was amplified and PCR products were sequenced, aligned and compared. Sequence variations of CHS partial gene in Dendrobium Sonia mutants with different flower colour showed that two protein positions have been changed as compared to the control. These non-synonymous mutations may have contributed to the colour alterations in the white and purple pink mutants. This paper describes important procedures to quantify gene expression such as RNA isolation (quantity and quality), cDNA synthesis and primer design steps for CHS genes.
  2. Genetic controls on starch amylose content in wheat and rice grains
    Fasahat P, Rahman S, Ratnam W
    J Genet, 2014 Apr;93(1):279-92.
    PMID: 24840849
    Starch accumulates in plants as granules in chloroplasts of source organs such as leaves (transitory starch) or in amyloplasts of sink organs such as seeds, tubers and roots (storage starch). Starch is composed of two types of glucose polymers: the essentially linear polymer amylose and highly branched amylopectin. The amylose content of wheat and rice seeds is an important quality trait, affecting the nutritional and sensory quality of two of the world's most important crops. In this review, we focus on the relationship between amylose biosynthesis and the structure, physical behaviour and functionality of wheat and rice grains. We briefly describe the structure and composition of starch and then in more detail describe what is known about the mechanism of amylose synthesis and how the amount of amylose in starch might be controlled. This more specifically includes analysis of GBSS alleles, the relationship between waxy allelic forms and amylose, and related quantitative trait loci. Finally, different methods for increasing or lowering amylose content are evaluated.
  3. Fulltext Genetic diversity of Elaeis oleifera (HBK) Cortes populations using cross species SSRs: implication's for germplasm utilization and conservation
    Ithnin M, Teh CK, Ratnam W
    BMC Genet, 2017 04 19;18(1):37.
    PMID: 28420332 DOI: 10.1186/s12863-017-0505-7
    BACKGROUND: The Elaeis oleifera genetic materials were assembled from its center of diversity in South and Central America. These materials are currently being preserved in Malaysia as ex situ living collections. Maintaining such collections is expensive and requires sizable land. Information on the genetic diversity of these collections can help achieve efficient conservation via maintenance of core collection. For this purpose, we have applied fourteen unlinked microsatellite markers to evaluate 532 E. oleifera palms representing 19 populations distributed across Honduras, Costa Rica, Panama and Colombia.

    RESULTS: In general, the genetic diversity decreased from Costa Rica towards the north (Honduras) and south-east (Colombia). Principle coordinate analysis (PCoA) showed a single cluster indicating low divergence among palms. The phylogenetic tree and STRUCTURE analysis revealed clusters based on country of origin, indicating considerable gene flow among populations within countries. Based on the values of the genetic diversity parameters, some genetically diverse populations could be identified. Further, a total of 34 individual palms that collectively captured maximum allelic diversity with reduced redundancy were also identified. High pairwise genetic differentiation (Fst > 0.250) among populations was evident, particularly between the Colombian populations and those from Honduras, Panama and Costa Rica. Crossing selected palms from highly differentiated populations could generate off-springs that retain more genetic diversity.

    CONCLUSION: The results attained are useful for selecting palms and populations for core collection. The selected materials can also be included into crossing scheme to generate offsprings that capture greater genetic diversity for selection gain in the future.

  4. Fulltext Transcriptome analysis of reproductive tissue differentiation in Jatropha curcas Linn
    Govender N, Senan S, Mohamed-Hussein ZA, Ratnam W
    Genom Data, 2017 Sep;13:11-14.
    PMID: 28626637 DOI: 10.1016/j.gdata.2017.05.008
    Shoot and inflorescence are central physiological and developmental tissues of plants. Flowering is one of the most important agronomic traits for improvement of crop yield. To analyze the vegetative to reproductive tissue transition in Jatropha curcas, gene expression profiles were generated from shoot and inflorescence tissues. RNA isolated from both tissues was sequenced using the Ilumina HiSeq 2500 platform. Differential gene expression analysis identified key biological processes associated with vegetative to reproductive tissue transition. The present data for J. curcas may inform the design of breeding strategies particularly with respect to reproductive tissue transition. The raw data of this study has been deposited in the NCBI's Sequence Read Archive (SRA) database with the accession number SRP090662.
  5. Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon
    Song BK, Nadarajah K, Romanov MN, Ratnam W
    Cell Mol Biol Lett, 2005;10(3):425-37.
    PMID: 16217554
    The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.
  6. Fulltext Pyramiding of drought yield QTLs into a high quality Malaysian rice cultivar MRQ74 improves yield under reproductive stage drought
    Shamsudin NAA, Swamy BPM, Ratnam W, Sta Cruz MT, Sandhu N, Raman AK, et al.
    Rice (N Y), 2016 Dec;9(1):21.
    PMID: 27164982 DOI: 10.1186/s12284-016-0093-6
    BACKGROUND: With the objective of improving the grain yield (GY) of the Malaysian high quality rice cultivar MRQ74 under reproductive stage drought stress (RS), three drought yield QTLs, viz. qDTY 2.2, qDTY 3.1 , and qDTY 12.1 were pyramided by marker assisted breeding (MAB). Foreground selection using QTL specific markers, recombinant selection using flanking markers, and background selections were performed in every generation. BC1F3 derived pyramided lines (PLs) with different combinations of qDTY 2.2, qDTY 3.1 , and qDTY 12.1 were evaluated under both RS and non-stress (NS) during the dry season (DS) of 2013 and 2014 at IRRI.

    RESULTS: The GY reductions in RS trials compared to NS trials ranged from 79 to 99 %. Plant height (PH) was reduced and days to flowering (DTF) was delayed under RS. Eleven BC1F5 MRQ74 PLs with yield advantages of 1009 to 3473 kg ha(-1) under RS and with yields equivalent to MRQ74 under NS trials were identified as promising drought tolerance PLs. Five best PLs, IR 98010-126-708-1-4, IR 98010-126-708-1-3, IR 98010-126-708-1-5, IR 99616-44-94-1-1, and IR 99616-44-94-1-2 with a yield advantage of more than 1000 kg ha(-1) under RS and with yield potential equivalent to that of MRQ74 under NS were selected. The effect of three drought grain yield QTLs under RS in MRQ74 was validated. Under NS, PLs with two qDTY combinations (qDTY 2.2 + qDTY 12.1 ) performed better than PLs with other qDTY combinations, indicating the presence of a positive interaction between qDTY 2.2 and qDTY 12.1 in the MRQ74 background.

    CONCLUSION: Drought tolerant MRQ74 PLs with a yield advantage of more than 1000 kg ha(-1) under RS were developed. Differential yield advantages of different combinations of the qDTYs indicate a differential synergistic relationship among qDTYs.

  7. A transgressive brown rice mediates favourable glycaemic and insulin responses
    Karupaiah T, Aik CK, Heen TC, Subramaniam S, Bhuiyan AR, Fasahat P, et al.
    J Sci Food Agric, 2011 Aug 30;91(11):1951-6.
    PMID: 21480266 DOI: 10.1002/jsfa.4395
    BACKGROUND: We evaluated glycaemic response of a brown rice variant (BR) developed by cross-breeding. Subjects (n = 9) consumed 50 g carbohydrate equivalents of BR, white rice (WR) and the polished brown rice (PR) in comparison to 50 g glucose reference (GLU) in a cross-over design. Plasma glucose and insulin at 0, 15, 45, 60, 90, 120 and 180 min were measured and incremental area under the curve (IAUC) and indices for glucose (GI) and insulin (II) calculated.
    RESULTS: BR compared to PR or WR produced the lowest postprandial glycaemia (GI: 51 vs 79 vs 86) and insulinaemia (II: 39 vs 63 vs 68) irrespective of amylose content (19 vs 23 vs 26.5%). Only BR was significantly different from GLU for both plasma glucose (P = 0.012) and insulin (P = 0.013) as well as IAUC(glu) (P = 0.045) and IAUC(ins) (P = 0.031). Glycaemic and insulinaemic responses correlated positively (r = 0.550, P < 0.001). Linear trends for IAUC(glu) and IAUC(ins) indicated a greater secretion of insulin tied in with a greater glycaemic response for WR (r(2) = 0.848), moderate for PR (r(2) = 0.302) and weakest for BR (r(2) = 0.122).
    CONCLUSION: The brown rice variant had the lowest GI and II values but these advantages were lost with polishing.
  8. Fulltext Marker assisted pyramiding of drought yield QTLs into a popular Malaysian rice cultivar, MR219
    Shamsudin NA, Swamy BP, Ratnam W, Sta Cruz MT, Raman A, Kumar A
    BMC Genet, 2016;17:30.
    PMID: 26818269 DOI: 10.1186/s12863-016-0334-0
    Three drought yield QTLs, qDTY 2.2, qDTY 3.1, and qDTY 12.1 with consistent effect on grain yield under reproductive stage drought stress were pyramided through marker assisted breeding with the objective of improving the grain yield of the elite Malaysian rice cultivar MR219 under reproductive stage drought stress. Foreground selection using QTL specific markers, recombinant selection using flanking markers, and background selection were performed. BC1F3-derived lines with different combinations of qDTY 2.2 , qDTY 3.1, and qDTY 12.1 were evaluated under both reproductive stage drought stress and non-stress during the dry seasons of 2013 and 2014 at IRRI.
  9. Mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis)
    Ng CH, Lee SL, Ng KK, Muhammad N, Ratnam W
    J Genet, 2009 Apr;88(1):25-31.
    PMID: 19417541
    The mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean+/- s.e. t(m) = 0.86+/-0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%-70%), followed by among pods within inflorescence (29%-37%), and the least variation among inflorescences within tree (1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.
  10. Fulltext RNA-seq data of the Jatropha curcas L. shoot system
    Govender N, Senan S, Mohamed-Hussein ZA, Isa MNM, Yaakob Z, Ratnam W
    Data Brief, 2018 Dec;21:71-74.
    PMID: 30338276 DOI: 10.1016/j.dib.2018.09.081
    Jatropha curcas L. or the physic nut is a monoecious shrub belonging to the Euphorbiaceae family. The plant is an ideal feedstock for biodiesel production; oil-rich seed (37-42%), has a broad range of growth habitat such as arid, semi-arid and tropical and a relatively feasible process for conversion of crude oil into biodiesel. The major constraint affecting the success of large-scale J. curcas plantation is seed yield inconsistency. Numerous research projects conducted on J. curcas with integrated genetic, genomic and transcriptomic approaches have been applied on the leaf, apical meristem, flower, root and fruit tissues. However, to date, no genomics data of J. curcas shoot system are publicly available, despite its importance in understanding flowering, fruiting and seed set qualities targeted for yield improvement. Here, we present eighteen sets of shoot and inflorescence transcriptomes generated from J. curcas plants with contrasting yields. Raw reads of the RNA-seq data are found in NCBI׳s Sequence Read Archive (SRA) database with the accession number SRP090662 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP090662). This transcriptomic data could be integrated with the present genomic resources for in depth understanding of J. curcas reproductive system.
  11. The 172-kb genomic DNA region of the O. rufipogon yld1.1 locus: comparative sequence analysis with O. sativa ssp. japonica and O. sativa ssp. indica
    Song BK, Hein I, Druka A, Waugh R, Marshall D, Nadarajah K, et al.
    Funct Integr Genomics, 2009 Feb;9(1):97-108.
    PMID: 18633654 DOI: 10.1007/s10142-008-0091-x
    Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1.
  12. Fulltext Molecular characterization and comparative sequence analysis of defense-related gene, Oryza rufipogon receptor-like protein kinase 1
    Law YS, Gudimella R, Song BK, Ratnam W, Harikrishna JA
    Int J Mol Sci, 2012;13(7):9343-9362.
    PMID: 22942769 DOI: 10.3390/ijms13079343
    Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression.
  13. Fulltext Association Mapping of Yield and Yield-related Traits Under Reproductive Stage Drought Stress in Rice (Oryza sativa L.)
    Swamy BPM, Shamsudin NAA, Rahman SNA, Mauleon R, Ratnam W, Sta Cruz MT, et al.
    Rice (N Y), 2017 Dec;10(1):21.
    PMID: 28523639 DOI: 10.1186/s12284-017-0161-6
    BACKGROUND: The identification and introgression of major-effect QTLs for grain yield under drought are some of the best and well-proven approaches for improving the drought tolerance of rice varieties. In the present study, we characterized Malaysian rice germplasm for yield and yield-related traits and identified significant trait marker associations by structured association mapping.

    RESULTS: The drought screening was successful in screening germplasm with a yield reduction of up to 60% and heritability for grain yield under drought was up to 78%. There was a wider phenotypic and molecular diversity within the panel, indicating the suitability of the population for quantitative trait loci (QTL) mapping. Structure analyses clearly grouped the accessions into three subgroups with admixtures. Linkage disequilibrium (LD) analysis revealed that LD decreased with an increase in distance between marker pairs and the LD decay varied from 5-20 cM. The Mixed Linear model-based structured association mapping identified 80 marker trait associations (MTA) for grain yield (GY), plant height (PH) and days to flowering (DTF). Seven MTA were identified for GY under drought stress, four of these MTA were consistently identified in at least two of the three analyses. Most of these MTA identified were on chromosomes 2, 5, 10, 11 and 12, and their phenotypic variance (PV) varied from 5% to 19%. The in silico analysis of drought QTL regions revealed the association of several drought-responsive genes conferring drought tolerance. The major-effect QTLs are useful in marker-assisted QTL pyramiding to improve drought tolerance.

    CONCLUSION: The results have clearly shown that structured association mapping is one of the feasible options to identify major-effect QTLs for drought tolerance-related traits in rice.

  14. Fulltext Changes in Mitochondrial Epigenome in Type 2 Diabetes Mellitus
    Low HC, Chilian WM, Ratnam W, Karupaiah T, Md Noh MF, Mansor F, et al.
    Br J Biomed Sci, 2023;80:10884.
    PMID: 36866104 DOI: 10.3389/bjbs.2023.10884
    Type 2 Diabetes Mellitus is a major chronic metabolic disorder in public health. Due to mitochondria's indispensable role in the body, its dysfunction has been implicated in the development and progression of multiple diseases, including Type 2 Diabetes mellitus. Thus, factors that can regulate mitochondrial function, like mtDNA methylation, are of significant interest in managing T2DM. In this paper, the overview of epigenetics and the mechanism of nuclear and mitochondrial DNA methylation were briefly discussed, followed by other mitochondrial epigenetics. Subsequently, the association between mtDNA methylation with T2DM and the challenges of mtDNA methylation studies were also reviewed. This review will aid in understanding the impact of mtDNA methylation on T2DM and future advancements in T2DM treatment.
  15. Tropical and subtropical Asia's valued tree species under threat
    Gaisberger H, Fremout T, Kettle CJ, Vinceti B, Kemalasari D, Kanchanarak T, et al.
    Conserv Biol, 2021 Dec 05.
    PMID: 34865262 DOI: 10.1111/cobi.13873
    Tree diversity in Asia's tropical and subtropical forests is central to nature-based solutions. Species vulnerability to multiple threats, which affect provision of ecosystem services, is poorly understood. We conducted a region-wide, spatially explicit assessment of the vulnerability of 63 socioeconomically important tree species to overexploitation, fire, overgrazing, habitat conversion, and climate change. Trees were selected for assessment from national priority lists, and selections were validated by an expert network representing 20 countries. We used Maxent suitability modeling to predict species distribution ranges, freely accessible spatial data sets to map threat exposures, and functional traits to estimate threat sensitivities. Species-specific vulnerability maps were created as the product of exposure maps and sensitivity estimates. Based on vulnerability to current threats and climate change, we identified priority areas for conservation and restoration. Overall, 74% of the most important areas for conservation of these trees fell outside protected areas, and all species were severely threatened across an average of 47% of their native ranges. The most imminent threats were overexploitation and habitat conversion; populations were severely threatened by these factors in an average of 24% and 16% of their ranges, respectively. Our model predicted limited overall climate change impacts, although some study species were likely to lose over 15% of their habitat by 2050 due to climate change. We pinpointed specific natural areas in Borneo rain forests as hotspots for in situ conservation of forest genetic resources, more than 82% of which fell outside designated protected areas. We also identified degraded areas in Western Ghats, Indochina dry forests, and Sumatran rain forests as hotspots for restoration, where planting or assisted natural regeneration will help conserve these species, and croplands in southern India and Thailand as potentially important agroforestry options. Our results highlight the need for regionally coordinated action for effective conservation and restoration.
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