Construction and application of two bacterial artificial chromosome (BAC) libraries of Phalaenopsis equestris

Abstract

Phalaenopsis orchid is one of the most valuable ornamentals in the world and the development of new cultivars is economically vital to floricultural industries worldwide P equestris one of the native species orchid in Taiwan which has many attractive traits such as colorful long-lasting flowers and is one of the important parents commonly used in commercial breeding programs The genetics and genome researches on orchids will aid in the development of new cultivars to improve production The availability of a large-insert bacterial artificial chromosome (BAC) genomic library is crucial for physical mapping gene cloning and analysis of gene structure and function in Phalaenopsis orchids In this study two BAC libraries of the P equestris were constructed using the BamHI and HindIII restriction enzymes In total the two libraries contain 134 348 clones with an average insert size of 103 kb and represent approximately 12 x haploid genome equivalents Twelve gene-specific probes were used to screen the orchid BAC libraries Positive clones were identified for all 12 probes that are anchored to probes evenly distributed on the genomes of Phalaenopsis orchids Moreover the utility of both libraries was evaluated by screening for the presence of genes encoding ethylene receptor ERS1 and MADS-box that regulation of flowering time in orchids (PeSOC1) related to the Arabidopsis gene AtERS1and AtSOC1 respectively Subcloning and sequencing of BACs revealed that these BAC clones contain the corresponding genomic regions of PeERS1 and PeSOC1 The genomic sequence of the PeERS1 consists of 5 exons and 4 introns the PeSOC1 comprises 7 exons and 6 introns having the first intron of 18 413 bp and 37 171 bp respectively longer than those of ERS and SOC1 had ever been published Although the whole-genome sequence of the P equestris has been established due to the lack of reference of Phalaenopsis orchid sequences many scaffolds cannot be assembled There are 86 Mb of the unknown bases corresponding to 7% of the Phalaenopsis genome Five unknown fragments a total of 4 351 bp in intron1 of PeSOC1 that is present in the OrchidBase was successfully amended to 4 669 bp using PeSOC1 genomic sequences derived from BAC clones These results indicated that the two BAC libraries are useful genomic resources for gene cloning molecular marker development and contribution to complete the unknown bases from the NGS (next-generation sequencing) sequences in P equestris

Date of Award	2020
Original language	English
Supervisor	Wen-Luan Wu (Supervisor)