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Floral Transcriptome Analyses of Four Paphiopedilum Orchids with Distinct Flowering Behaviors and Development of Simple Sequence Repeat Markers

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Abstract

Paphiopedilum orchids are evergreen perennials with distinct flowering behaviors; however, little genetic information hindered the understanding of these interesting flowering patterns. In this study, the floral transcriptomes of two Paphiopedilum species, Paphiopedilum concolor and Paphiopedilum hirsutissimum, and two popular cultivars were characterized. A total of 42.4–68.5 million clean reads were generated from these four Paphiopedilum orchid floral buds. De novo assembly yielded 73,201–79,086 unigenes with average lengths of 765–848 bp for these four floral transcriptomes, of which 42.5–43.1 % were annotated in public protein databases. With 8,019 unique protein-coding genes shared, similar function classifications were found among the four orchid floral transcriptomes, and 113 Paphiopedilum floral unigenes were found to be homologous to flowering genes in Arabidopsis. Of these flowering genes, many regulatory genes, namely, MIKC, NAC, MYB, WRKY, bZIP, bHLH transcription factors, and protein kinases and plant hormone signaling-related genes were identified. Notably, one gene, PIF4 may play vital roles for regulation flowering in warm seasons. A phylogeny clustering based on MIKC MADS-box gene family among Paphiopedilum, rice, and Arabidopsis classified the 61 Paphiopedilum members into 11 subfamilies, which provided some clues on floral organ specification and development in Paphiopedilum. Differentiation expression analysis among the four floral transcriptomes demonstrated that complicated molecular functions or biological processes, such as transmembrane transport, transcription regulation, signal transduction, defense response, etc., might be responsible for the differences among these orchid floral development processes, and partial of these expression genes were validated by qRT-PCR. Finally, 20,969 EST-SSRs were identified as potential molecular markers, and 15,137 SSRs were successful for primer designing. Of 62 candidate SSR primer pairs, 51 were successfully amplified with expected sizes and exhibited polymorphisms among 14 genotypes of Paphiopedilum orchids. Our data should not only provide valuable sequence resources for novel gene discovery and marker-assisted studies in Paphiopedilum but also give us some new insights into understanding of the molecular mechanisms of flowering and floral development in Paphiopedilum.

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Abbreviations

BLAST:

Basic Local Alignment Search Tool

bp:

Base pair

cDNA:

Complementary DNA

EST:

Expressed sequence tag

FLM:

FLOWERING LOCUS M

MADS:

MCM1-AGAMOUS-DEFICIENS-SRF

MSI1:

MULTICOPY SUPPRESSOR OF IRA1

NAC:

NAM, ATAF1/2, CUC2

PCR:

polymerase chain reaction

PIF4:

PHYTOCHROME INTERACTING FACTOR4

qRT-PCR:

Real-time quantitative PCR

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Acknowledgments

This work was financially supported by the Guangzhou Municipal Science and Technology Project (No. 12C14071654) and the Guangdong Academy of Agricultural Sciences Fund (No. 201019). The authors would like to acknowledge the Shanghai Biotechnology Corporation (Shanghai) for assistance with raw data processing and the related bioinformatics analysis.

Competing Interests

The authors declare that they have no competing interests.

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    Authors and Affiliations

    1. Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China

      Dong-Mei Li, Di Zhang, Xiao-Rong Liu, Xiao-Fei Liu & Yi-Jian Lin

    2. South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou, 510650, China

      Wei Wu

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    1. Dong-Mei Li
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    2. Wei Wu
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    Correspondence to Dong-Mei Li.

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    Dong-Mei Li and Wei Wu contributed equally to this work.

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    Fig. S1

    Somatic chromosomes of four Paphiopedilum orchids. a Paphiopedilum concolor, 2n = 2x = 26. b Paphiopedilum hirsutissimum, 2n = 2x = 26. c Paphiopedilum hybrid Maudiae Red, 2n = 2x = 34. d Paphiopedilum hybrid Maudiae Green, 2n = 2x = 32 (DOC 437 kb)

    Fig. S2

    Overview of the sequencing and assembly of the four Paphiopedilum orchid floral transcriptomes sequences. a Overview of contig assembly. b Overview of scaffold assembly. c Overview of unigene assembly (DOC 432 kb)

    Fig. S3

    GO functional classification for the four Paphiopedilum orchid floral unigenes by BLASTx with E value cutoff 1.0E − 5 against the rice protein database. a Biological process. b Molecular function. c Cellular component (DOC 676 kb)

    Fig. S4

    PCR products amplified using primer pairs among 14 Paphiopedilum orchids and separated by electrophoresis on 6 % nondenaturing polyacrylamide gels. M indicates 100 bp DNA marker. Lanes 1–14 represent DNA samples from Paphiopedilum orchids P. dianthum, P. micranthum, P. hirsutissimum, Paphiopedilum hybrid Maudiae Red, Paphiopedilum hybrid Maudiae Green, P. concolor, P. wenshanense, P. bellatulum, P. areeanum, P. henryanum, P. hangianum, P. villosum, P. emersonii, and P. malipoense, respectively. P EST-SSR primer pairs (DOC 4164 kb)

    Table S1

    Primer sequences for real-time PCR (DOC 39 kb)

    Table S2

    KEGG pathway annotation of floral unigenes from the four Paphiopedilum orchids (DOC 1482 kb)

    Table S3

    Common protein-coding genes expressed in the four Paphiopedilum orchid floral buds (XLSX 589 kb)

    Table S4

    Putative flowering time-related genes in the four Paphiopedilum orchid floral transcriptomes (XLSX 81 kb)

    Table S5

    Putative transcription factor genes in the four Paphiopedilum orchid floral transcriptomes (XLSX 260 kb)

    Table S6

    MIKCc MADS-box proteins from Arabidopsis, rice, and four Paphiopedilum orchids (DOC 73 kb)

    Table S7

    List of differentially expressed genes between two among the four floral transcriptomes (XLSX 563 kb)

    Table S8

    Primer pairs designed from the EST-derived SSRs (XLSX 3678 kb)

    Table S9

    The 62 primer pairs for validation EST-SSR markers (XLSX 17 kb)

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    Li, DM., Wu, W., Zhang, D. et al. Floral Transcriptome Analyses of Four Paphiopedilum Orchids with Distinct Flowering Behaviors and Development of Simple Sequence Repeat Markers. Plant Mol Biol Rep 33, 1928–1952 (2015). https://doi.org/10.1007/s11105-015-0886-6

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