Abstract. Siregar UJ, Maulana MI, Suharsono UW. 2017. Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis). Biodiversitas 18: 1150Biodiversitas 18: -1158. Agarwood is one of non timber forest products (NTFP) which has high economic value. Diminishing agarwood trees in natural forest due to intensive harvesting has shifted the production paradigm to forest plantation. In order to support agarwood tree improvement, investigation on the agarwood tree genomes and genes involved in agarwood production is highly needed through genomic library construction. This research aimed at establishing cloning protocols for genomic library construction of agarwood tree (Aquilaria malaccensis) until sequencing process. Genomic DNA was isolated using DNeasy Plant Mini Kit from Qiagen, then was digested with EcoR1, and was ligated to pGem®-T Easy vector system before it was finally transformed and was cloned to E.coli strain DH5α. Confirmation of DNA inserts was done using PCR colony with universal primer of SP6 and T7, plasmid isolation and also PCR plasmid and plasmid DNA digestion used Quick Plasmid Miniprep Kit from Thermofisher. Cloned A. malaccensis DNA fragments were sequenced and BLAST at NCBI site. The cloning successfully obtained five white colonies which contain inserted A. malaccensis DNA fragments with the size of 136-225 bp. PCR colony, plasmid isolation, PCR plasmid, and plasmid DNA digestion had confirmed the existence of inserted A. malaccensis DNA genome inside the bacteria cells. Sequencing and BLAST showed that DNA inserts from colony number 6, 8, and 9 were not similar with A. malccensis sequence that was recorded in NCBI, indicating that the genomic region in this library construction is different from the genomic DNA sequences of A. malaccensis in NCBI.
Most ectotherms obey the temperature-size rule, meaning they grow larger in a colder environment. This raises the question of how the interplay between genes and temperature affect the body size of ectotherms. Despite the growing body of literature on the physiological life-history and molecular genetic mechanism underlying the temperature-size rule, the overall genetic architecture orchestrating this complex phenotype is not yet fully understood. One approach to identify genetic regulators of complex phenotypes is Quantitative Trait Locus (QTL) mapping. Here, we explore the genetic architecture of body size phenotypes, in different temperatures using Caenorhabditis elegans as a model ectotherm. We used 40 recombinant inbred lines (RILs) derived from N2 and CB4856, which were reared at four different temperatures (16°C, 20°C, 24°C, and 26°C) and measured at two developmental stages (L4 and adult). The animals were measured for body length, width at vulva, body volume, length/width ratio, and seven other body-size traits. The genetically diverse RILs varied in their body-size phenotypes with heritabilities ranging from 0.20 to 0.99. We detected 18 QTL underlying the body-size phenotypes across all treatment combinations, with the majority clustering on Chromosome X. We hypothesize that the Chromosome X QTL could result from a known pleiotropic regulator – npr-1 – known to affect the body size of C. elegans through behavioral changes. In conclusion, our findings shed more light on multiple loci affecting body size plasticity and allow for a more refined analysis of the temperature-size rule.
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