Green synthesis and characterization of UKMRC-8 rice husk-derived mesoporous silica nanoparticle for agricultural application.

Agriculture plays a crucial role in safeguarding food security, more so as the world population increases gradually. A productive agricultural system is supported by seed, soil, fertiliser and good management practices. Food productivity directly correlates to the generation of solid wastes and utilization of agrochemicals, both of which negatively impact the environment. The rice and paddy industry significantly adds to the growing menace of waste management. In low and middle-income countries, rice husk (RH) is an underutilized agro-waste discarded in landfills or burned in-situ. RH holds enormous potential in the development of value-added nanomaterials for agricultural applications. In this study, a simple and inexpensive sol-gel method is described to extract mesoporous silica nanoparticles (MSNs) from UKMRC8 RH using the bottom-up approach. RHs treated with hydrochloric acid were calcinated to obtain rice husk ash (RHA) with high silica purity (> 98% wt), as determined by the X-ray fluorescence analysis (XRF). Calcination at 650 °C for four hours in a box furnace yielded RHA that was devoid of metal impurities and organic matter. The X-ray diffraction pattern showed a broad peak at 2θ≈20-22 °C and was free from any other sharp peaks, indicating the amorphous property of the RHA. Scanning electron micrographs (SEM) showed clusters of spherically shaped uniform aggregates of silica nanoparticles (NPs) while transmission electron microscopy analysis indicated an average particle size of < 20 nm. Besides Energy Dispersive X-Ray which validated the chemical constituent of the silica NPs, the Fourier transform infrared (FT-IR) spectra showed peaks at 796.4 cm-1 and 1052 cm-1 corresponding to O-Si-O symmetric stretching vibration and O-Si-O asymmetric stretching, respectively. The Brunauer-Emmet-Teller (BET) analysis indicated an average pore size = 8.5 nm while the specific surface area and the pore volume were 300.2015 m2/g and 0.659078 cm3/g, respectively. In conclusion, agrowaste-derived MSN was synthesized using a simple and economical sol-gel method without the addition of surfactant reagents for controlled formation at the structural level. Owing to the MSNs' excellent physical properties, the method established herein, could be used singly (without any modifications) for the functionalization of a myriad of agrochemicals.

Introgression of the QTL qSB11-1TT conferring sheath blight resistance in rice (Oryza sativa) into an elite variety, UKMRC 2, and evaluation of its backcross-derived plants.

Introduction: Sheath blight (SB) is the most damaging fungal disease in rice caused by a soil-borne pathogenic fungus, Rhizoctonia solani Kuhn (R. solani). The disease resistance in rice is a complex quantitative trait controlled by a few major genes. UKMRC2 is a newly developed elite rice variety that possesses high yield potential but is susceptible to sheath blight disease indicating a huge risk of varietal promotion, mass cultivation, and large-scale adoption. The aim of our present study was the development of varietal resistance against R. solani in UKMRC2 to enhance its stability and durability in a wide range of environments and to validate the effects of an SB-resistance QTL on the new genetic background. Methods: In our study, we developed 290 BC1F1 backcross progenies from a cross between UKMRC2 and Tetep to introgress the QTL qSBR11-1TT into the UKMRC2 genetic background. Validation of the introgressed QTL region was performed via QTL analysis based on QTL-linked SSR marker genotyping and phenotyping against R. solani artificial field inoculation techniques. Results and Discussion: The QTL qSBR11-1TT was then authenticated with the results of LOD score (3.25) derived from composite interval mapping, percent phenotypic variance explained (14.6%), and additive effect (1.1) of the QTLs. The QTL region was accurately defined by a pair of flanking markers K39512 and RM7443 with a peak marker RM27360. We found that the presence of combination of alleles, RM224, RM27360 and K39512 demonstrate an improved resistance against the disease rather than any of the single allele. Thus, the presence of the QTL qSBR11-1TT has been validated and confirmed in the URMRC2 genetic background which reveals an opportunity to use the QTL linked with these resistance alleles opens an avenue to resume sheath blight resistance breeding in the future with marker-assisted selection program to boost up resistance in rice varieties.

Incorporating Drought and Submergence Tolerance QTL in Rice (Oryza sativa L.)-The Effects under Reproductive Stage Drought and Vegetative Stage Submergence Stresses.

Drought and submergence have been the major constraint in rice production. The present study was conducted to develop high-yielding rice lines with tolerance to drought and submergence by introgressing Sub1 into a rice line with drought yield QTL (qDTY; QTL = quantitative trait loci) viz. qDTY3.1 and qDTY12.1 using marker-assisted breeding. We report here the effect of different combinations of Sub1 and qDTY on morpho-physiological, agronomical traits and yield under reproductive stage drought stress (RS) and non-stress (NS) conditions. Lines with outstanding performance in RS and NS trials were also evaluated in vegetative stage submergence stress (VS) trial to assess the tolerance level. The QTL class analysis revealed Sub1 + qDTY3.1 as the best QTL combination affecting the measured traits in RS trial followed by Sub1 + qDTY12.1. The effects of single Sub1, qDTY3.1 and qDTY12.1 were not as superior as when the QTLs are combined, suggesting the positive interaction of Sub1 and qDTY. Best performing lines selected from the RS and NS trials recorded yield advantage up to 4453.69 kg ha-1 and 6954 kg ha-1 over the parents, respectively. The lines were also found having great tolerance to submergence ranging from 80% to 100%, contributed by a lower percentage of shoot elongation and reduction of chlorophyll content after 14 days of VS. These lines could provide yield sustainability to farmers in regions impacted with drought and submergence while serving as important genetic materials for future breeding programs.

Drought Yield QTL (qDTY) with Consistent Effects on Morphological and Agronomical Traits of Two Populations of New Rice (Oryza sativa) Lines.

Drought has been a major limiting factor for rice production. Drought yield QTLs (qDTYs; QTLs = quantitative trait loci) were pyramided into MRQ74 and MR219 to produce drought tolerant lines. In this study, new drought tolerant MRQ74 and MR219 pyramided lines (PLs) were evaluated under drought stress (RS) and non-stress (NS) conditions to evaluate the effects of different qDTYs combinations on morphological and agronomical traits. MRQ74 PLs having qDTY12.1 possessed the best root length (RL) under both RS and NS but the effect was only significant for MR219 PLs under RS. Some qDTYs combinations also found to have consistent effect on the same trait of both populations. PLs with only qDTY12.1 showed the highest grain yield (GY) under RS in both populations which means qDTY12.1 controlled RL and caused higher GY under drought condition. The interaction of major-effect qDTY12.1 with qDTY2.2 also shows significant effect on leaf rolling (LR) of both PL populations. These qDTYs proved to be beneficial in improving traits related to drought tolerance. Selected PLs with qDTY12.1 combinations also found to have better RL and root weight (RW) under RS. Improvement of morphological and agronomical traits led to higher GY of PLs. Therefore, qDTY12.1 either is present singly or in combination with other qDTYs was the best qDTY due to its consistent effect on morphological and agronomical traits and GY across populations under RS and NS.

An integration of phenotypic and transcriptomic data analysis reveals yield-related hub genes in Jatropha curcas inflorescence.

Jatropha curcas is an oil-rich seed crop with huge potentials for bioenergy production. The inflorescence carries a number of processes that are likely to affect the overall yield potentials; floral development, male-to-female flower ratio, floral abscission and fruit set. In this study, a weighted gene co-expression network analysis which integrates the transcriptome, physical and simple sugar data of J. curcas inflorescence was performed and nine modules were identified by means of hierarchical clustering. Among them, four modules (green4, antiquewhite2, brown2 and lightskyblue4) showed significant correlation to yield factors at p≤0.01. The four modules are categorized into two clusters; cluster 1 of green4 and antiquewhite2 modules correspond to number of flowers/inflorescence, total seed weight/plant, number of seeds/plant, and number of fruits/plant, whereas cluster 2 of brown2 and lightskyblue4 modules correspond to glucose and fructose. Descriptive characterizations of cluster 1 show putative involvement in gibberellin signaling and responses, whereas cluster 2 may have been involved in sugar signaling, signal transductions and regulation of flowerings. Our findings present a list of hub genes for J. curcas yield improvement and reproductive biology enhancement strategies.

A gene co-expression network model identifies yield-related vicinity networks in Jatropha curcas shoot system.

The plant shoot system consists of reproductive organs such as inflorescences, buds and fruits, and the vegetative leaves and stems. In this study, the reproductive part of the Jatropha curcas shoot system, which includes the aerial shoots, shoots bearing the inflorescence and inflorescence were investigated in regard to gene-to-gene interactions underpinning yield-related biological processes. An RNA-seq based sequencing of shoot tissues performed on an Illumina HiSeq. 2500 platform generated 18 transcriptomes. Using the reference genome-based mapping approach, a total of 64 361 genes was identified in all samples and the data was annotated against the non-redundant database by the BLAST2GO Pro. Suite. After removing the outlier genes and samples, a total of 12 734 genes across 17 samples were subjected to gene co-expression network construction using petal, an R library. A gene co-expression network model built with scale-free and small-world properties extracted four vicinity networks (VNs) with putative involvement in yield-related biological processes as follow; heat stress tolerance, floral and shoot meristem differentiation, biosynthesis of chlorophyll molecules and laticifers, cell wall metabolism and epigenetic regulations. Our VNs revealed putative key players that could be adapted in breeding strategies for J. curcas shoot system improvements.

Identification of functionally important microRNAs from rice inflorescence at heading stage of a qDTY4.1-QTL bearing Near Isogenic Line under drought conditions.

A cross between IR64 (high-yielding but drought-susceptible) and Aday Sel (drought-tolerant) rice cultivars yielded a stable line with enhanced grain yield under drought screening field trials at International Rice Research Institute. The major effect qDTY4.1 drought tolerance and yield QTL was detected in the IR77298-14-1-2-10 Backcrossed Inbred Line (BIL) and its IR87705-7-15-B Near Isogenic Line (NIL) with 93.9% genetic similarity to IR64. Although rice yield is extremely susceptible to water stress at reproductive stage, currently, there is only one report on the detection of drought-responsive microRNAs in inflorescence tissue of a Japonica rice line. In this study, more drought-responsive microRNAs were identified in the inflorescence tissues of IR64, IR77298-14-1-2-10 and IR87705-7-15-B via next-generation sequencing. Among the 32 families of inflorescence-specific non-conserved microRNAs that were identified, 22 families were up-regulated in IR87705-7-15-B. Overall 9 conserved and 34 non-conserved microRNA families were found as drought-responsive in rice inflorescence with 5 conserved and 30 non-conserved families induced in the IR87705-7-15-B. The observation of more drought-responsive non-conserved microRNAs may imply their prominence over conserved microRNAs in drought response mechanisms of rice inflorescence. Gene Ontology annotation analysis on the target genes of drought-responsive microRNAs identified in IR87705-7-15-B revealed over-representation of biological processes including development, signalling and response to stimulus. Particularly, four inflorescence-specific microRNAs viz. osa-miR5485, osa-miR5487, osa-miR5492 and osa-miR5517, and two non-inflorescence specific microRNAs viz. osa-miR169d and osa-miR169f.2 target genes that are involved in flower or embryonic development. Among them, osa-miR169d, osa-miR5492 and osa-miR5517 are related to flowering time control. It is also worth mentioning that osa-miR2118 and osa-miR2275, which are implicated in the biosynthesis of rice inflorescence-specific small interfering RNAs, were induced in IR87705-7-15-B but repressed in IR77298-14-1-2-10. Further, gene search within qDTY4.1 QTL region had identified multiple copies of NBS-LRR resistance genes (potential target of osa-miR2118), subtilisins and genes implicated in stomatal movement, ABA metabolism and cuticular wax biosynthesis.

Association between QTLs and morphological traits toward sheath blight resistance in rice (Oryza sativa L.).

Sheath blight is considered the most significant disease of rice and causes enormous yield losses over the world. Breeding for resistant varieties is the only viable option to combat the disease efficiently. Seventeen diverged rice genotypes along with 17 QTL-linked SSR markers were evaluated under greenhouse conditions. Pearson's correlation showed only the flag leaf angle had a significant correlation with sheath blight resistance under greenhouse screening. Multivariate analysis based on UPGMA clustering and principal component analysis (PCA) indicated that the flag leaf angle, flag leaf length, and plant compactness were significantly associated with the following SSR marker alleles: RM209 (116,130), RM202 (176), RM224 (126), RM257 (156), RM426 (175), and RM6971 (196), which are linked to the SB QTLs: QRlh11, qSBR11-3, qSBR11-1, qSBR9-1, qShB3-2, and qSB-9. A Mantel test suggested a weak relationship between the observed phenotypes and allelic variation patterns, implying the independent nature of morphological and molecular variations. Teqing and Tetep were found to be the most resistant cultivars. IR65482-4-136-2-2, MR219-4, and MR264 showed improved resistance potentials. These results suggest that the morphological traits and QTLs which have been found to associate with sheath blight resistance are a good choice to enhance resistance through pyramiding either 2 QTLs or QTLs and traits in susceptible rice cultivars.

Evaluating Crossbred Red Rice Variants for Postprandial Glucometabolic Responses: A Comparison with Commercial Varieties.

Consumption of white rice predisposes some Asian populations to increased risk of type 2 diabetes. We compared the postprandial glucometabolic responses to three newly-developed crossbred red rice variants (UKMRC9, UKMRC10, UKMRC11) against three selected commercial rice types (Thai red, Basmati white, Jasmine white) using 50-g carbohydrate equivalents provided to 12 normoglycaemic adults in a crossover design. Venous blood was drawn fasted and postprandially for three hours. Glycaemic (GI) and insulin (II) indices, incremental areas-under-the-curves for glucose and insulin (IAUCins), indices of insulin sensitivity and secretion, lactate and peptide hormones (motilin, neuropeptide-Y, orexin-A) were analyzed. The lowest to highest trends for GI and II were similar i.e., UKMRC9 < Basmati < Thai red < UKMRC10 < UKMRC11 < Jasmine. Postprandial insulinaemia and IAUCins of only UKMRC9 were significantly the lowest compared to Jasmine. Crude protein and fiber content correlated negatively with the GI values of the test rice. Although peptide hormones were not associated with GI and II characteristics of test rice, early and late phases of prandial neuropeptide-Y changes were negatively correlated with postprandial insulinaemia. This study indicated that only UKMRC9 among the new rice crossbreeds could serve as an alternative cereal option to improve diet quality of Asians with its lowest glycaemic and insulinaemic burden.

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage.

BACKGROUND: Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing. RESULTS: Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3-5.7 %), cell transport (13.2-18.4 %), response to stress (10.5-11.3 %), lignin catabolic process (3.8-5.3 %), metabolic processes (32.1-39.5 %), oxidation-reduction process (9.4-13.2 %) and DNA replication (5.7-7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes. CONCLUSIONS: The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Molecular characterization of a cellulose synthase gene (AaxmCesA1) isolated from an Acacia auriculiformis x Acacia mangium hybrid.

Cellulose is the major component of plant cell walls, providing mechanical strength to the structural framework of plants. In association with lignin, hemicellulose, protein and pectin, cellulose forms the strong yet flexible bio-composite tissue of wood. Wood formation is an essential biological process and is of significant importance to the cellulosic private sector industry. Cellulose synthase genes encode the catalytic subunits of a large protein complex responsible for the biogenesis of cellulose in higher plants. The hybrid Acacia auriculiformis x Acacia mangium represents an important source of tree cellulose for forest-based product manufacturing, with enormous economic potential. In this work, we isolate the first cellulose synthase gene, designated AaxmCesA1, from this species. The isolated full-length AaxmCesA1 cDNA encodes a polypeptide of 1,064 amino acids. Sequence analyses revealed that AaxmCesA1 cDNA possesses the key motif characteristics of a CesA protein. AaxmCesA1 shares more than 75 % amino acid sequence identity with CesA proteins from other plant species. Subsequently, the full-length AaxmCesA1 gene of 7,389 bp with partial regulatory and 13 intron regions was also isolated. Relative gene expression analysis by quantitative PCR in different tissues of the Acacia hybrid, suggests the involvement of the AaxmCesA1 gene in primary cell wall synthesis of rapidly dividing young root cells. Similarity analyses using Blast algorithms also suggests a role in primary cell wall deposition in the Acacia hybrid. Southern analysis predicts that AaxmCesA1 is a member of a multigene family with at least two isoforms in the genome of the Acacia hybrid.

Development of high-throughput SNP-based genotyping in Acacia auriculiformis x A. mangium hybrids using short-read transcriptome data.

BACKGROUND: Next Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in Acacia auriculiformis and Acacia mangium. Like other non-model species, SNP detection and genotyping in Acacia are challenging due to lack of genome sequences. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of A. auriculiformis x A. mangium hybrids. RESULTS: We identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two Acacia hybrid mapping populations using Bowtie against 7,839 de novo transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our in silico SNP detection approach is highly accurate (100%) compared to the traditional in vitro approach (44%). Further validation of 96 SNPs using Illumina GoldenGate Assay gave an overall assay success rate of 89.6% and conversion rate of 37.5%. We explored possible factors lowering assay success rate by predicting exon-intron boundaries and paralogous genes of Acacia contigs using Medicago truncatula genome as reference. This assessment revealed that presence of exon-intron boundary is the main cause (50%) of assay failure. Subsequent SNPs filtering and improved assay design resulted in assay success and conversion rate of 92.4% and 57.4%, respectively based on 768 SNPs genotyping. Analysis of clustering patterns revealed that 27.6% of the assays were not reproducible and flanking sequence might play a role in determining cluster compression. In addition, we identified a total of 258 and 319 polymorphic SNPs in A. auriculiformis and A. mangium natural germplasms, respectively. CONCLUSION: We have successfully discovered a large number of SNP markers in A. auriculiformis x A. mangium hybrids using next generation transcriptome sequencing. By using a reference genome from the most closely related species, we converted most SNPs to successful assays. We also demonstrated that Illumina GoldenGate genotyping together with manual clustering can provide high quality genotypes for a non-model species like Acacia. These SNPs markers are not only important for linkage map construction, but will be very useful for hybrid discrimination and genetic diversity assessment of natural germplasms in the future.

Characterization of microRNAs expressed during secondary wall biosynthesis in Acacia mangium.

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants.

Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing.

BACKGROUND: Acacia auriculiformis × Acacia mangium hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in Acacia hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants. RESULTS: We sequenced transcriptomes of A. auriculiformis and A. mangium from non-normalized cDNA libraries synthesized from pooled young stem and inner bark tissues using paired-end libraries and a single lane of an Illumina GAII machine. De novo assembly produced a total of 42,217 and 35,759 contigs with an average length of 496 bp and 498 bp for A. auriculiformis and A. mangium respectively. The assemblies of A. auriculiformis and A. mangium had a total length of 21,022,649 bp and 17,838,260 bp, respectively, with the largest contig 15,262 bp long. We detected all ten monolignol biosynthetic genes using Blastx and further analysis revealed 18 lignin isoforms for each species. We also identified five contigs homologous to R2R3-MYB proteins in other plant species that are involved in transcriptional regulation of secondary cell wall formation and lignin deposition. We searched the contigs against public microRNA database and predicted the stem-loop structures of six highly conserved microRNA families (miR319, miR396, miR160, miR172, miR162 and miR168) and one legume-specific family (miR2086). Three microRNA target genes were predicted to be involved in wood formation and flavonoid biosynthesis. By using the assemblies as a reference, we discovered 16,648 and 9,335 high quality putative Single Nucleotide Polymorphisms (SNPs) in the transcriptomes of A. auriculiformis and A. mangium, respectively, thus yielding useful markers for population genetics studies and marker-assisted selection. CONCLUSION: We have produced the first comprehensive transcriptome-wide analysis in A. auriculiformis and A. mangium using de novo assembly techniques. Our high quality and comprehensive assemblies allowed the identification of many genes in the lignin biosynthesis and secondary cell wall formation in Acacia hybrids. Our results demonstrated that Next Generation Sequencing is a cost-effective method for gene discovery, identification of regulatory sequences, and informative markers in a non-model plant.

Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

BACKGROUND: Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. RESULTS: In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing. Differential expression of the small RNAs between secondary xylem tissues with contrasting lignin content suggests that a cascade of miRNAs play an interconnected role in regulating the lignin biosynthetic pathway in Acacia species. CONCLUSIONS: Our study critically demonstrated the roles of small RNAs during secondary wall formation. Comparison of the expression pattern of small RNAs between secondary xylem tissues with contrasting lignin content strongly indicated that small RNAs play a key regulatory role during lignin biosynthesis. Our analyses suggest an evolutionary mechanism for miRNA targets on the basis of the length of their 5' and 3' UTRs and their cellular roles. The results obtained can be used to better understand the roles of small RNAs during lignin biosynthesis and for the development of gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability. For the first time, small RNAs were proven to play an important regulatory role during lignin biosynthesis in A. mangium.