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  1. Fulltext Author Correction: Comparison of eight complete chloroplast genomes of the endangered Aquilaria tree species (Thymelaeaceae) and their phylogenetic relationships
    Hishamuddin MS, Lee SY, Ng WL, Ramlee SI, Lamasudin DU, Mohamed R
    Sci Rep, 2021 Apr 22;11(1):9160.
    PMID: 33888845 DOI: 10.1038/s41598-021-88638-1
  2. Fulltext Genetic Variability, Heritability, and Clustering Pattern Exploration of Bambara Groundnut (Vigna subterranea L. Verdc) Accessions for the Perfection of Yield and Yield-Related Traits
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Mamun A
    Biomed Res Int, 2020;2020:2195797.
    PMID: 33415143 DOI: 10.1155/2020/2195797
    Bambara groundnut (Vigna subterranea L. Verdc.) is considered an emerging crop for the future and known as a crop for the new millennium. The core intention of this research work was to estimate the variation of landraces of Bambara groundnut considering their 14 qualitative and 27 numerical traits, to discover the best genotype fitted in Malaysia. The findings of the ANOVA observed a highly significant variation (p ≤ 0.01) for all the traits evaluated. There was a substantial variation (7.27 to 41.21%) coefficient value, and 14 out of the 27 numerical traits noted coefficient of variation (CV) ≥ 20%. Yield (kg/ha) disclosed positively strong to perfect high significant correlation (r = 0.75 to 1.00; p ≤ 0.001) with traits like fresh pod weight, dry pod weight, and dry seed weight. The topmost PCV and GCV values were estimated for biomass dry (41.09%) and fresh (40.53%) weight with high heritability (Hb) and genetic advance (GA) Hb = 95.19%, GA = 80.57% and Hb = 98.52%, GA = 82.86%, respectively. The topmost heritability was recorded for fresh pod weight (99.89%) followed by yield (99.75%) with genetic advance 67.95% and 62.03%, respectively. The traits with Hb ≥ 60% and GA ≥ 20% suggested the least influenced by the environment as well as governed by the additive genes and direct selection for improvement of such traits can be beneficial. To estimate the genetic variability among accessions, the valuation of variance components, coefficients of variation, heritability, and genetic advance were calculated. To authenticate the genetic inequality, an unweighted pair group produced with arithmetic mean (UPGMA) and principal component analysis was executed based on their measurable traits that could be a steadfast method for judging the degree of diversity. Based on the UPGMA cluster analysis, constructed five distinct clusters and 44 accessions from clusters II and IV consider an elite type of genotypes that produce more than one ton yield per hectare land with desirable traits. This study exposed an extensive disparity among the landraces and the evidence on genetic relatives will be imperative in using the existing germplasm for Bambara groundnut varietal improvement. Moreover, this finding will be beneficial for breeders to choose the desirable numerical traits of V. subterranea in their future breeding program.
  3. Fulltext Author Correction: DNA fingerprinting, fixation-index (Fst), and admixture mapping of selected Bambara groundnut (Vigna subterranea [L.] Verdc.) accessions using ISSR markers system
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M, Halidu J
    Sci Rep, 2021 Aug 26;11(1):17484.
    PMID: 34446833 DOI: 10.1038/s41598-021-96946-9
  4. Fulltext Hereditary analysis and genotype × environment interaction effects on growth and yield components of Bambara groundnut (Vigna subterranea (L.) Verdc.) over multi-environments
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M
    Sci Rep, 2022 09 19;12(1):15658.
    PMID: 36123374 DOI: 10.1038/s41598-022-19003-z
    This investigation was carried out to explore G × E interaction for yield and its associated attributes in 30 Bambara groundnut genotypes across four environments in tropical Malaysia. Such evaluations are essential when the breeding program's objective is to choose genotypes with broad adaption and yield potential. Studies of trait relationships, variance components, mean performance, and genetic linkage are needed by breeders when designing, evaluating, and developing selection criteria for improving desired characteristics in breeding programs. The evaluation of breeding lines of Bambara groundnut for high yield across a wide range of environments is important for long-term production and food security. Each site's experiment employed a randomized complete block design with three replicates. Data on vegetative and yield component attributes were recorded. The analysis of variance revealed that there were highly significant (p ≤ 0.01) differences among the 30 genotypes for all variables evaluated. A highly significant and positive correlation was identified between yield per hectare and dry seed weight (0.940), hundred seed weight (0.844), fresh pod weight (0.832), and total pod weight (0.750); the estimated correlation between dry weight of pods and seed yield was 1.0. The environment was more important than genotype and G × E in determining yield and yield components.A total of 49% variation is covered by PC1 (33.9%) and PC2 (15.1%) and the genotypes formed five distinct clusters based on Ward hierarchical clustering (WHC) method. The genotypes S5G1, S5G3, S5G5, S5G6, S5G8, S5G7, S5G2, S5G4, S5G10, S5G13, S5G11, and S5G14 of clusters I, II, and III were closest to the ideal genotype with superior yield across the environments. The PCA variable loadings revealed that an index based on dry pod weight, hundred seed weight, number of total pods and fresh pod weight could be used as a selection criteria to improve seed yield of Bambara groundnut.
  5. Fulltext Path-coefficient and correlation analysis in Bambara groundnut (Vigna subterranea [L.] Verdc.) accessions over environments
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M
    Sci Rep, 2022 01 07;12(1):245.
    PMID: 34996953 DOI: 10.1038/s41598-021-03692-z
    In a breeding program, studies of genotypic and phenotypic relationships among agricultural crop traits are useful to design, evaluate, and develop selection criteria for desirable traits. Using path coefficient analysis, the present study was executed to estimate the phenotypic, genotypic, and environmental correlation coefficients between yield and yield-related traits and to determine the direct and indirect effects of yield-related traits on yield per plant. A total of 30 genotypes of Vigna subterranea were studied under tropical conditions at two sites over two planting seasons (considered as four environments). The experiment at each site used a randomized complete block design with three replicates. Data were collected on vegetative and yield component attributes. Based on analysis of variance, pooled results showed that there were positive and highly significant differences (p ≤ 0.01) among the 30 genotypes for all attributes studied. Highly significant and positive strong correlation at phenotypic level was observed for dry seed weight (0.856), hundred seed weight (0.754), fresh pod weight (0.789), and total pod weight (0.626) with yield in kg per hectare, while moderate positive correlations were observed for harvest cut (0.360) and days to maturity (0.356). However, a perfect positive correlation was observed for the dry weight of pods with seed yield. In contrast, days to 50% flowering (- 0.350) showed a negative significant relationship with yield per hectare. The dried pod weight attribute (1.00) had a high positive direct effect on yield. Fresh pod weight had the greatest indirect effect on yield per hectare, followed by the number of total pods by dry pod weight. As a result, dry pod weight, hundred seed weight, number of total pods, and fresh pod weight could be used as selection criteria to improve the seed yield of Bambara groundnut (Vigna subterranea).
  6. Fulltext AMMI and GGE biplot analysis for yield performance and stability assessment of selected Bambara groundnut (Vigna subterranea L. Verdc.) genotypes under the multi-environmental trails (METs)
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M
    Sci Rep, 2021 Nov 23;11(1):22791.
    PMID: 34815427 DOI: 10.1038/s41598-021-01411-2
    The stability and high yielding of Vigna subterranea L. Verdc. genotype is an important factor for long-term development and food security. The effects of G × E interaction on yield stability in 30 Bambara groundnut genotypes in four different Malaysian environments were investigated in this research. The experiment used a randomized complete block design with three replications in each environment. Over multiple harvests, yield component traits such as the total number of pods per plant, fresh pods weight (g), hundred seeds weight (g), and yield per hectare were evaluated in the main and off-season in 2020 and 2021. Stability tests for multivariate stability parameters were performed based on analyses of variance. For all the traits, the pooled analysis of variance revealed highly significant (p 
  7. Molecular insight into genetic differentiation, population structure and banding pattern analysis of Bambara groundnut (Vigna subterranea [L.] Verdc.) linked with inter simple sequence repeats (ISSR)
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M, Kundu BC
    Mol Biol Rep, 2023 Sep;50(9):7619-7637.
    PMID: 37531035 DOI: 10.1007/s11033-023-08693-x
    BACKGROUND: A set of 44 selected Bambara groundnut (Vigna subterranea [L.] Verdc.) accessions was sampled from 11 distinct populations of four geographical zones to assess the genetic drift, population structure, phylogenetic relationship, and genetic differentiation linked with ISSR primers.

    METHODS AND RESULTS: The amplification of genomic DNA with 32 ISSR markers detected an average of 97.64% polymorphism while 35.15% and 51.08% polymorphism per population and geographical zone, respectively. Analysis of molecular variance revealed significant variation within population 75% and between population 25% whereas within region 84% and between region 16%. The Bidillali exposed greater number of locally common band i.e., NLCB (≤ 25%) = 25 and NLCB (≤ 50%) = 115 were shown by Cancaraki while the lowest was recorded as NLCB (≤ 25%) = 6 and NLCB (≤ 50%) = 72 for Roko and Maibergo, accordingly. The highest PhiPT value was noted between Roko and Katawa (0.405*) whereas Nei's genetic distance was maximum between Roko and Karu (0.124). Based on Nei's genetic distance, a radial phylogenetic tree was constructed that assembled the entire accessions into 3 major clusters for further confirmation unrooted NJ vs NNet split tree analysis based on uncorrected P distance exposed the similar result. Principal coordinate analysis showed variation as PC1 (15.04%) > PC2 (5.81%).

    CONCLUSIONS: The current study leads to prompting the genetic improvement and future breeding program by maximum utilization and better conservation of existing accessions. The accessions under Cancaraki and Jatau are population documented for future breeding program due to their higher genetic divergence and homozygosity.

  8. Highly divergent regions in the complete plastome sequences of Aquilaria are suitable for DNA barcoding applications including identifying species origin of agarwood products
    Hishamuddin MS, Lee SY, Syazwan SA, Ramlee SI, Lamasudin DU, Mohamed R
    3 Biotech, 2023 Mar;13(3):78.
    PMID: 36761338 DOI: 10.1007/s13205-023-03479-1
    Members of Aquilaria Lam. (Thymelaeaceae) are evergreen trees that are widely distributed in the Indomalesia region. Aquilaria is highly prized for its unique scented resin, agarwood, which is often the subject of unlawful trade activities. Survival of the tree is heavily threatened by destructive harvesting and agarwood poaching, leading to its protection under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Unfortunately, an efficient species identification method, which is crucial to aid in the conservation efforts of Aquilaria is lacking. Here, we described our search for a suitable specific DNA barcode for Aquilaria species using eight complete plastome sequences. We identified five highly variable regions (HVR) (matK-rps16, ndhF-rpl32, psbJ-petA, trnD, and trnT-trnL) in the plastomes. These regions were further analyzed using the neighbor-joining (NJ) method to assess their ability at discriminating the eight species. Coupled with in silico primer design, two potential barcoding regions, psbJ-petA and trnT-trnL, were identified. Their strengths in species delimitation were evaluated individually and in combination, via DNA barcoding analysis. Our findings showed that the combined dataset, psbJ-petA + trnT-trnL, effectively resolved members of the genus Aquilaria by clustering all species into their respective clades. In addition, we demonstrated that the newly proposed DNA barcode was capable at identifying the species of origin of six commercial agarwood samples that were included as unknown samples. Such achievement offers a new technical advancement, useful in the combat against illicit agarwood trades and in assisting the conservation of these valuable species in natural populations.

    SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03479-1.

  9. Fulltext Comparison of eight complete chloroplast genomes of the endangered Aquilaria tree species (Thymelaeaceae) and their phylogenetic relationships
    Hishamuddin MS, Lee SY, Ng WL, Ramlee SI, Lamasudin DU, Mohamed R
    Sci Rep, 2020 Aug 03;10(1):13034.
    PMID: 32747724 DOI: 10.1038/s41598-020-70030-0
    Aquilaria tree species are naturally distributed in the Indomalesian region and are protected against over-exploitation. They produce a fragrant non-timber product of high economic value, agarwood. Ambiguous species delimitation and limited genetic information within Aquilaria are among the impediments to conservation efforts. In this study, we conducted comparative analysis on eight Aquilaria species complete chloroplast (cp) genomes, of which seven were newly sequenced using Illumina HiSeq X Ten platform followed by de novo assembly. Aquilaria cp genomes possess a typical quadripartite structure including gene order and genomic structure. The length of each of the cp genome is about 174 kbp and encoded between 89 and 92 proteins, 38 tRNAs, and 8 rRNAs, with 27 duplicated in the IR (inverted repeat) region. Besides, 832 repeats (forward, reverse, palindrome and complement repeats) and nine highly variable regions were also identified. The phylogenetic analysis suggests that the topology structure of Aquilaria cp genomes were well presented with strong support values based on the cp genomes data set and matches their geographic distribution pattern. In summary, the complete cp genomes will facilitate development of species-specific molecular tools to discriminate Aquilaria species and resolve the evolutionary relationships of members of the Thymelaeaceae family.
  10. Fulltext DNA fingerprinting, fixation-index (Fst), and admixture mapping of selected Bambara groundnut (Vigna subterranea [L.] Verdc.) accessions using ISSR markers system
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M, Halidu J
    Sci Rep, 2021 Jul 15;11(1):14527.
    PMID: 34267249 DOI: 10.1038/s41598-021-93867-5
    As a new crop in Malaysia, forty-four Bambara groundnut (Vigna subterranea L. verdc.) genotypes were sampled from eleven distinct populations of different origins to explore the genetic structure, genetic inconsistency, and fixation index. The Bambara groundnut, an African underutilized legume, has the capacity to boost food and nutrition security while simultaneously addressing environmental sustainability, food availability, and economic inequalities. A set of 32 ISSRs were screened out of 96 primers based on very sharp, clear, and reproducible bands which detected a total of 510 loci with an average of 97.64% polymorphism. The average calculated value of PIC = 0.243, RP = 5.30, H = 0.285, and MI = 0.675 representing the efficiency of primer set for genetic differentiation among the genotypes. The ISSR primers revealed the number of alleles (Na = 1.97), the effective number of alleles (Ne = 1.38), Nei's genetic diversity (h = 0.248), and a moderate level of gene flow (Nm = 2.26) across the genotypes studied. The estimated Shannon's information index (I = 0.395) indicates a high level of genetic variation exists among the accessions. Based on Nei's genetic dissimilarity a UPMGA phylogenetic tree was constructed and grouped the entire genotypes into 3 major clusters and 6 subclusters. PCA analysis revealed that first principal component extracted maximum variation (PC1 = 13.92%) than second principal component (PC2 = 12.59%). Bayesian model-based STRUCTURE analysis assembled the genotypes into 3 (best ΔK = 3) genetic groups. The fixation-index (Fst) analysis narrated a very great genetic diversity (Fst = 0.19 to 0.40) exists within the accessions of these 3 clusters. This investigation specifies the effectiveness of the ISSR primers system for the molecular portrayal of V. subterranea genotypes that could be used for genetic diversity valuation, detection, and tagging of potential genotypes with quick, precise, and authentic measures for this crop improvement through effective breeding schemes.
  11. Fulltext Genetic analysis and selection of Bambara groundnut (Vigna subterranea [L.] Verdc.) landraces for high yield revealed by qualitative and quantitative traits
    Khan MMH, Rafii MY, Ramlee SI, Jusoh M, Al Mamun M
    Sci Rep, 2021 Apr 07;11(1):7597.
    PMID: 33828137 DOI: 10.1038/s41598-021-87039-8
    As a crop for the new millennium Bambara groundnut (Vigna subterranea [L.] Verdc.) considered as leading legumes in the tropical regions due to its versatile advantages. The main intent of this study was to find out the high yielding potential genotypes and considering these genotypes to develop pure lines for commercial cultivation in Malaysia. Considering the 14 qualitative and 27 quantitative traits of fifteen landraces the variation and genetic parameters namely, variability, heritability, genetic advance, characters association, and cluster matrix were determined. ANOVA revealed significant variation for all the agronomic traits (except plant height). Among the accessions, highly significant differences (P ≤ 0.01) were found for almost all the traits excluding fifty percent flowering date, seed length, seed width. The 16 traits out of the 27 quantitative traits had a coefficient of variation (CV) ≥ 20%. A positive and intermediate to perfect highly significant association (r = 0.23 to 1.00; P 
  12. Fulltext Recent Strategies for Detection and Improvement of Brown Planthopper Resistance Genes in Rice: A Review
    Sani Haliru B, Rafii MY, Mazlan N, Ramlee SI, Muhammad I, Silas Akos I, et al.
    Plants (Basel), 2020 Sep 14;9(9).
    PMID: 32937908 DOI: 10.3390/plants9091202
    Brown planthopper (BPH; Nilaparvata lugens Stal) is considered the main rice insect pest in Asia. Several BPH-resistant varieties of rice have been bred previously and released for large-scale production in various rice-growing regions. However, the frequent surfacing of new BPH biotypes necessitates the evolution of new rice varieties that have a wide genetic base to overcome BPH attacks. Nowadays, with the introduction of molecular approaches in varietal development, it is possible to combine multiple genes from diverse sources into a single genetic background for durable resistance. At present, above 37 BPH-resistant genes/polygenes have been detected from wild species and indica varieties, which are situated on chromosomes 1, 3, 4, 6, 7, 8, 9, 10, 11 and 12. Five BPH gene clusters have been identified from chromosomes 3, 4, 6, and 12. In addition, eight BPH-resistant genes have been successfully cloned. It is hoped that many more resistance genes will be explored through screening of additional domesticated and undomesticated species in due course.
  13. Bacterial leaf blight resistance in rice: a review of conventional breeding to molecular approach
    Chukwu SC, Rafii MY, Ramlee SI, Ismail SI, Hasan MM, Oladosu YA, et al.
    Mol Biol Rep, 2019 Feb;46(1):1519-1532.
    PMID: 30628024 DOI: 10.1007/s11033-019-04584-2
    Breeding for disease resistant varieties remains very effective and economical in controlling the bacterial leaf blight (BLB) of rice. Breeders have played a major role in developing resistant rice varieties against the BLB infection which has been adjudged to be a major disease causing significant yield reduction in rice. It would be difficult to select rice crops with multiple genes of resistance using the conventional approach alone. This is due to masking effect of genes including epistasis. In addition, conventional breeding takes a lot of time before a gene of interest can be introgressed. Linkage drag is also a major challenge in conventional approach. Molecular breeding involving markers has facilitated the characterization and introgression of BLB disease resistance genes. Biotechnology has brought another innovation in form of genetic engineering (transgenesis) of rice. Although, molecular breeding cannot be taken as a substitute for conventional breeding, molecular approach for combating BLB disease in rice is worthwhile given the demand for increased production of rice in a fast growing population of our society. This present article highlights the recent progress from conventional to molecular approach in breeding for BLB disease resistant rice varieties.
  14. Fulltext Effects of Grafting on Morphophysiological and Yield Characteristic of Eggplant (Solanum melongena L.) Grafted onto Wild Relative Rootstocks
    Musa I, Rafii MY, Ahmad K, Ramlee SI, Md Hatta MA, Oladosu Y, et al.
    Plants (Basel), 2020 Nov 15;9(11).
    PMID: 33203189 DOI: 10.3390/plants9111583
    Grafting is regarded as an integral component of sustainable vegetable production. It is important in the management of soil-borne diseases, and reports suggest that grafting with viable rootstocks can enhance crop growth and yield. This research was conducted using splices and cleft grafting techniques to investigate graft compatibility among varieties of high yielding eggplant scion (MCV1, MCV2, CCV1, CCV2, CCV3, NCV, and TCV) grafted onto wild rootstocks (MWR, BWR, and TWR) to study their morphophysiological and yield characteristics. High yielding scions grafted onto wild relative rootstocks were compared with two controls including self-grafted and non-grafted. All the scion had a high rate of germination (≥95%) and remarkable graft success (100%) was recorded in MCV1, MCV2, and TCV using the cleft techniques. Generally, the use of rootstocks resulted in higher total and marketable fruit yield compared to the non-grafted and self-grafted scion plants, respectively. In particular, MWR and TWR rootstock conferred the highest vigour to the scion, resulting in the highest values recorded for total and marketable fruit yield, number of fruits per plant and average fruit weight. A similar result was obtained in fruit length and diameter, where long and wide fruits were observed in scions grafted onto MWR and TWR rootstocks, respectively. Grafting of high yielding eggplant scion onto resistant MWR, BWR and TWR eggplant rootstock was found to be beneficial for eggplant cultivation. The remarkable compatibility and vigour of the rootstock with scion led to the improvement in total and marketable yield of the fruits. As such, it can be concluded that the use of wild relative rootstocks of eggplant species can be a valuable method of improving eggplant production.
  15. Fulltext Genetic Diversity Assessment of MPOB-Senegal Oil Palm Germplasm Using Microsatellite Markers
    Myint KA, Yaakub Z, Rafii MY, Oladosu Y, Samad MYA, Ramlee SI, et al.
    Biomed Res Int, 2021;2021:6620645.
    PMID: 33997027 DOI: 10.1155/2021/6620645
    Molecular characterization of oil palm germplasm is crucial in utilizing and conserving germplasm with promising traits. This study was conducted to evaluate the genetic diversity structures and relationships among 26 families of MPOB-Senegal oil palm germplasm using thirty-five microsatellite markers. High level of polymorphism (P = 96.26%), number of effective allele (N e = 2.653), observed heterozygosity (H o = 0.584), expected heterozygosity (H e = 0.550), total heterozygosity (H T = 0.666), and rare alleles (54) were observed which indicates that MPOB-Senegal germplasm has a broad genetic variation. Among the SSR markers, sMo00053 and sMg00133 were the most informative markers for discrimination among the MPOB-Senegal oil palm germplasm for having the highest private alleles and the rare alleles. For selection and conservation, oil palm populations with high rare alleles and Nei's gene diversity index should be considered as these populations may possess unique genes for further exploitation.
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