Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization

Abstract

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai

“…Genes involved in numerous metabolic pathways such as innate immunity, cell stress and repair system, and antioxidant defense systems have been cloned and tested to determine their mRNA expression, translational inducibility, and molecular functions. Recently, the use of a genomics platform was successfully applied in H. discus hannai , although many research groups have continuously employed ‘-omics’ platforms (e.g., genomics, proteomics, linkage map development, Bacterial Artificial Chromosome (BAC) library construction) to understand the growth, development, reproduction, molecular adaptation, or innate immune response mechanisms upon environmental stressors or pathogen challenges in the Haliotis genus [5,6,7,8,9,10,11,12,13,14]. For H. discus hannai , a genetic linkage map was constructed as a potential application of marker-assisted selection in breeding programs using amplified fragment length polymorphism (AFLP) markers [15].…”

Construction and characterization of a bacterial artificial chromosome library for Gossypium mustelinum

“…mustelinum [ 35 ], the BAC library was estimated to have a total coverage of 10.50 × genome equivalents. The probability of finding any specific locus from this BAC library is estimated to be 99.9% according to the formula N = ln(1-p)/ln(1-f/G), where P is the probability, N is the number of clones, f is the average insert size of clones, and G is the haploid genome size [ 36 ].…”

An improved suppression subtractive hybridization technique to develop species-specific repetitive sequences from Erianthus arundinaceus (Saccharum complex)

“…Repetitive sequence probes can be combined with FISH to track chromosomal inheritance and analyze karyotypes. To date, C o t-1 DNA, Bacterial Artificial Chromosome (BAC) library, Yeast Artificial Chromosome (YAC) library and chromatin immunoprecipitation (ChIP) methods have been widely applied in combination with FISH to study plant genomes [ 67 – 73 ]. These methods provide tools to obtain chromosome markers that are directly based on their genomic sequences.…”