SSR identification and marker development for sago palm based on NGS genome data.
ABSTRACT: Sago palm (Metroxylon sagu Rottb.) is one of the most productive carbohydrate-producing crops. Unfortunately, only limited information regarding sago palm genetics is available. This study aimed to develop simple sequence repeat (SSR) markers using sago palm NGS genomic data and use these markers to evaluate the genetic diversity of sago palm from Indonesia. De novo assembly of partial sago palm genomic data and subsequent SSR mining identified 29,953 contigs containing 31,659 perfect SSR loci and 31,578 contigs with 33,576 imperfect SSR loci. The perfect SSR loci density was 132.57/Mb, and AG, AAG and AAAT were the most frequent SSR motifs. Five hundred perfect SSR loci were randomly selected and used for designing SSR primers; 93 SSR primer pairs were identified. After synteny analysis using rice genome sequences, 20 primer pairs were validated using 11 sago palm accessions, and seven primers generated polymorphic alleles. Genetic diversity analysis of 41 sago palm accessions from across Indonesia using polymorphic SSR loci indicated the presence of three clusters. These results demonstrated the success of SSR identification and marker development for sago palm based on NGS genome data, which can be further used for assisting sago palm breeding in the future.
Project description:Sago palm, or Metroxylon sagu, is a hardy and versatile plant that is able to tolerate many stresses, biotic and abiotic, during its growth. It is one of the plants that are able to grow in waterlogged area where others could not. Apart from that sago palm is also a source of starch, contributes economically to the people and an important export for the state of Sarawak. Despite the importance of sago palm especially in the production of starch and its ability to withstand stresses, so far, not many molecular studies have been reported on sago palm. To study the characters in sago palm, transcriptome analysis was conducted where it would give a better understanding of the plant development through gene expression. Here, we report the construction of a cDNA library and preliminary expressed sequence tags analysis from the young leaves of sago palm. A total of 434 clones were sequenced with inserts ranging from 1,000 to 3,000 bps with primary and amplified titers of 8 × 105 and 1.0 × 109 pfu/ml, respectively. Clustering of these sequences resulted in a set of 372 tentative unigenes comprising 340 singletons and 32 contigs. The database was also annotated with BLAST2GO which showed that majority of the transcripts were involved in primary metabolism and stress tolerance.
Project description:<b>Background:</b> Giant gourami ( <i>Osphronemus goramy, Osphronemidae</i>), belonging to gurami sago strain, is an important economic fish species that was newly released for domestication in 2018 in Indonesia. The present study aimed to determine the growth, production and feed conversion efficiency of gurami sago strain in different aquaculture systems. <b>Methods:</b> A mean of 240 juveniles were stocked (initial weight mean, 54.53 g and length 13.88 cm) into concrete ponds, ?oating net cages and earthen freshwater ponds (12 m <sup>3</sup>) with three replicates of each. The juveniles were fed a floating commercial pellet diet containing 30% crude protein and 5% crude lipids. Feed was supplied at 3% of fish biomass per day throughout the 90 days of the experiment. The research was conducted in the area surrounding Lake Maninjau of Indonesia. <b>Results:</b> After 90 days, the mean weight of ?sh reared in concrete ponds was 166.86 g, floating net cages was 179.51 g and earthen freshwater ponds was 149.89 g. The mean final biomass was 37.64 kg for concrete ponds, 41.27 kg for floating net cages, and 33.72 kg for earthen freshwater ponds. The specific growth rates (%/day) for concrete ponds, floating net cages and earthen freshwater ponds were 0.67, 0.75 and 0.62, respectively. The feed conversion rates were 1.45 for concrete ponds, 1.30 for floating net cages and 1.87 for earthen freshwater ponds. The net yields (kg m <sup>-3</sup>) were 2.05 for concrete ponds, 2.27 for floating net cages, and 1.73 for earthen freshwater ponds. The exponents (b) of the length-weight relationship were calculated for concrete ponds (1.0146), floating net cages (1.2641), and earthen freshwater ponds (1.0056). <b>Conclusion:</b> The study showed that the growth performance, production and feed conversion efficiency of the gurami sago strain were the best found in floating net cages and considered a new aquaculture system in the future.
Project description:Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated from Geobacillus sp. strain ARM, was overexpressed in Escherichia coli system and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60-80 °C, with a half-life of 4 h and in a pH range 6-9. The immobilized enzyme could be stored at 10 °C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing.
Project description:Physalis peruviana, commonly known as Cape gooseberry, is an Andean Solanaceae fruit with high nutritional value and interesting medicinal properties. In the present study we report the development and characterization of microsatellite loci from a P. peruviana commercial Colombian genotype. We identified 932 imperfect and 201 perfect Simple Sequence Repeats (SSR) loci in untranslated regions (UTRs) and 304 imperfect and 83 perfect SSR loci in coding regions from the assembled Physalis peruviana leaf transcriptome. The UTR SSR loci were used for the development of 162 primers for amplification. The efficiency of these primers was tested via PCR in a panel of seven P. peruviana accessions including Colombia, Kenya and Ecuador ecotypes and one closely related species Physalis floridana. We obtained an amplification rate of 83% and a polymorphic rate of 22%. Here we report the first P. peruviana specific microsatellite set, a valuable tool for a wide variety of applications, including functional diversity, conservation and improvement of the species.
Project description:The red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) is one of the most dangerous pests of major cultivated palms including coconut, oil palm, and sago. The larval stage of the weevil causes the most destruction of the palms as it completely destroys the palm cabbage. In this study, the larvae were given three different diets-coconut cabbage, oil palm cabbage, and sago stem, under laboratory conditions for food consumption and developmental time experiment. The protein profiles of the digestive systems of the larvae fed on these three diets were also determined. Although the coconut diet was the most consumed by RPW larvae compared to oil palm and sago diets, the growth rate of RPW larvae on oil palm diet was however significantly shorter than those on the coconut and sago diets: the RPW only need 1 mo and 9 d to complete the larval duration. Proteins profiling of eight 2-DE gel protein spots that range 50-20 kDa were identified by mass spectrometry sequence analysis. Based on the Matrix Science Software, the most dominant protein was cationic trypsin. However, based on the NCBI BLAST tool, aminopeptidase N was the most dominant enzyme. This finding can lead to the development of pest control strategies based on the antinutritional protease inhibitors as potential biocontrol agents. Urgent action to find effective control methods should be taken seriously as this weevil is presumed to be one of the serious pests of oil palm industry in Malaysia.
Project description:Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (?5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both "Zheda 23" and "Zheda 83". Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.
Project description:BACKGROUND: Brachiaria ruziziensis is one of the most important forage species planted in the tropics. The application of genomic tools to aid the selection of superior genotypes can provide support to B. ruziziensis breeding programs. However, there is a complete lack of information about the B. ruziziensis genome. Also, the availability of genomic tools, such as molecular markers, to support B. ruziziensis breeding programs is rather limited. Recently, next-generation sequencing technologies have been applied to generate sequence data for the identification of microsatellite regions and primer design. In this study, we present a first validated set of SSR markers for Brachiaria ruziziensis, selected from a de novo partial genome assembly of single-end Illumina reads. RESULTS: A total of 85,567 perfect microsatellite loci were detected in contigs with a minimum 10X coverage. We selected a set of 500 microsatellite loci identified in contigs with minimum 100X coverage for primer design and synthesis, and tested a subset of 269 primer pairs, 198 of which were polymorphic on 11 representative B. ruziziensis accessions. Descriptive statistics for these primer pairs are presented, as well as estimates of marker transferability to other relevant brachiaria species. Finally, a set of 11 multiplex panels containing the 30 most informative markers was validated and proposed for B. ruziziensis genetic analysis. CONCLUSIONS: We show that the detection and development of microsatellite markers from genome assembled Illumina single-end DNA sequences is highly efficient. The developed markers are readily suitable for genetic analysis and marker assisted selection of Brachiaria ruziziensis. The use of this approach for microsatellite marker development is promising for species with limited genomic information, whose breeding programs would benefit from the use of genomic tools. To our knowledge, this is the first set of microsatellite markers developed for this important species.
Project description:Sago palm is an important agricultural starch producing plant which contributes to the economics of Malaysia where the trunk is the main plant part which the starch is stored. However, there are sago palms that planted on peat soil failed to develop its trunk even after 17 years been planted. This phenomenon is known as 'non-trunking', which eliminates the economic value of the plants. Numeral researches have been done to address the phenomenon but the molecular mechanisms of sago palm respond toward the responsible stresses are still lacking. Therefore, in this study we collected samples from Trunking (Normal) and Non-Trunking sago palm leaf samples which planted on peat soil for total RNA extraction followed by next generation sequencing using BGISEQ-500 platform. The raw reads were cleaned and de novo assembled using TRINITY software package. Total of 40.11 Gb bases were sequenced from the sago palm leaf samples and after assembled producing 102,447 unigenes, with N50 score 1,809 bp and GC ratio of 44.34%. Unigenes by aligning with 7 (NR, NT, GO, KOG, KEGG, SwissProt & InterPro) functional databases and 65,523 (63.96%) unigenes were annotated. Functional annotation results in the detection of 46,335 Coding DNA sequences by Transdecoder. 30,039 Simple-sequence repeats distributed on 21,676 Unigenes were detected using Primer3 software, and 2,355 Transcription Factor coding Unigenes were predicted using getorf and hmmseach software. A total of 492 up-regulated and 444 down-regulated at least two-fold differential expressed gene were detected using DESeq2 algorithm with 95% confidence. Overall design: Comparison between trunking and non-trunking sago palm leaf tissue grant ID: RDCRG/CAT/2019/23 grant title: Sarawak Research Development Council Grant name of the funding source: Sarawak Research Development Council (SRDC) Grant information: grant ID: F07/TOC/1743/2018 grant title: Tun Openg Sago Chair Grant UNIMAS name of the funding source: Sarawak Government
Project description:We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of 'N67-10' and 'Hawaiian Smooth Cayenne'. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders' rights.
Project description:Role of gut microbiome in obesity and type 2 diabetes (T2D) became apparent from several independent studies indicating that gut microbiome modulators like prebiotics may improve microbiome perturbations (dysbiosis) to ameliorate metabolic derangements. We herein isolate water soluble, nondigestible polysaccharides from five plant-based foods (acorn, quinoa, sunflower, pumpkin seeds and sago) and assess their impact on human fecal microbiome and amelioration of high-fat-diet (HFD)-induced obesity/T2D in mice. During polysaccharide isolation, purification, biochemical and digestion resistance characterization, and fermentation pattern by human fecal microbiome, we select acorn- and sago-derived prebiotics (on the basis of relatively higher purity and yield and lower protein contamination) and examine their effects in comparison to inulin. Prebiotics treatments in human fecal microbiome culture system not only preserve microbial diversity but also appear to foster beneficial bacteria and short-chain fatty acids (SCFAs). Feeding of acorn- and sago-derived prebiotics ameliorates HFD-induced glucose intolerance and insulin resistance in mice, with effects comparatively superior to those seen in inulin-fed mice. Feeding of both of novel prebiotics as well as inulin increases SCFAs levels in the mouse gut. Interestingly, gut hyperpermeability and mucosal inflammatory markers were significantly reduced upon prebiotics feeding in HFD-fed mice. Hypothalamic energy signaling in terms of increased expression of pro-opiomelanocortin was also modulated by prebiotics administration. Results demonstrate that these (and/or such) novel prebiotics can ameliorate HFD-induced defects in glucose metabolism via positive modulation of gut-microbiome-brain axis and hence could be useful in preventing/treating diet-induced obesity/T2D.