The genome, transcriptome and miRNome of the Phalaenopsis yield insights into floral organ development and flowering regulation

Autor: Huang, Jian-Zhi, 黃建誌
Rok vydání: 2016
Druh dokumentu: 學位論文 ; thesis
Popis: 104
Phalaenopsis represents an economically important orchid among floricultural crops. The predicted Phalaenopsis genome size is approximately 1.34 to 8.12 gigabases (Gb), which is distributed across 19 chromosomes. In this study a high-quality draft genome sequence of winter flowering Phalaenopsis ‘KHM190’ cultivar is reported which was sequenced to 90-fold coverage using next generation sequencing technology and de novo assembly. The estimated genome size of KHM190 is about 3.1-Gb, which covers 89.9% of the genomic region and 59.74% of the genome represented as repetitive elements. The 89.5 Gb RNA-seq and 113 million sRNA-seq reads were generated to identify 41,153 protein-coding genes and 188 miRNA families. A draft genome for Phalaenopsis pulcherrima ‘B8802’, a summer flowering species, has also been generated via resequencing. Comparison of the genome data from the two Phalaenopsis accessions (KHM190 and B8802) allowed the discovery of 691,532 SNPs (single nucleotide polymorphisms). RNA sequencing analysis of Phalaenopsis flowering tissues suggests a potential connection between gibberellin pathway and flowering genes during the reproductive phase change induced by cool temperature. In addition, the key role of PhAGL6b in the regulation of labellum organ development involves alternative splicing in big lip mutant is revealed. Phalaenopsis has a zygomorphic floral structure, including three outer tepals, two lateral inner tepals and a highly modified inner median tepal called labellum or lip. The regulation of its organ development remains unclear. This study generated RNA-seq and sRNA-seq reads with the Illumina platform for floral organs of the Phalaenopsis wild-type and peloric mutant. A total of 43,552 unigenes were obtained with mean length of 1081 nt and median length of 532 nt. Pair-wise comparison of sepals, petals and labellum between peloric mutant and its wild-type revealed 1,838, 758 and 1,147 unigenes, respectively, with significant differential expression. Furthermore, 27 transcripts were selected with differential expression patterns a one-by-one comparison of each transcript performed by real-time PCR and RNA-seq. PhAGL6a (CUFF.17763), PhAGL6b (CUFF.17763.1), PhMADS1 (CUFF.36625.1), PhMADS4 (CUFF.25909) and PhMADS5 (CUFF.39479.1) were significantly upregulated in the lip-like petal of the peloric mutant. In addition, PhAGL6a, PhAGL6b and PhMADS4 were strongly expressed in the labellum and significantly upregulated in lip-like petals and lip-like sepals of peloric-mutant flowers that were validated by real-time RT-PCR. In addition, PhAGL6b and PhMADS4 were significantly downregulated in the labellum of the big lip mutant, with no change in expression of PhAGL6a. PhAGL6a PhAGL6b, and PhMADS4 might play crucial roles in the development of the labellum in Phalaenopsis. miRNAs are a type of noncoding RNA that play essential roles in regulating the molecular machinery at transcriptional and post-transcriptional levels. miRNA regulation in floral organ development has not been reported in Phalaenopsis. Using the next generation sequencing technology, six small RNA libraries were obtained, and reads between 4,800,239 to 6,997,280 were generated from floral organs of both wild-type and peloric mutant of Phalaenopsis. A total of 397 conserved miRNA families and 3,054 unique mature new candidate miRNAs were identified that belong to 463 families. Comparison of miRNA expression patterns among different floral organs revealed 297, 237 and 284 conserved miRNAs that are significantly differentially expressed in both the wild-type and peloric mutant. Furthermore, 20 miRNAs were selected with differential expression patterns for real-time PCR analysis in floral organs of both genotypes. miR393, miR390 and miR167 were upregulated in lip-like petals of peloric-mutant flowers. The predicted target genes of these miRNAs are mainly involved in auxin signaling pathway, which suggests the involvement of auxin-mediated responses to affect petal development such as peloric flowers.
Databáze: Networked Digital Library of Theses & Dissertations
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