Abstract
Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.
Highlights
In plants, members of the MADS-box gene family play important roles during diverse aspects of plant development and have been implicated in regulating e.g., floral transition and floral meristem and organ identity
To assess if other novel MADS-box transcripts exist that are similar to the transcripts we discovered in DEFICIENS AGAMOUS LIKE 19 (DAL19), we applied our assembly method to reads from total mRNA sequencing (RNA-seq) data from nine meristematic bud samples to reconstruct likely transcript isoforms
Our analysis shows that all forms of alternative splicing occur in the MADSbox gene family in P. abies
Summary
Members of the MADS-box gene family play important roles during diverse aspects of plant development and have been implicated in regulating e.g., floral transition and floral meristem and organ identity (see e.g., O’Maoileidigh et al, 2014 and references within). MADS is an acronym for the four founding members of this gene family: MCM1 from Saccharomyces cerevisiae, AGAMOUS from Arabidopsis thaliana, DEFICIENS from Antirrhinum majus, and SRF from Homo sapiens (Schwarz-Sommer et al, 1990). Evolutionary studies in seed plants have demonstrated that angiosperms and gymnosperms share orthologous MADS-box genes, and that these orthologs in many cases are involved in similar biological processes. MADS-box genes regulating carpel development in angiosperms have orthologous genes in gymnosperms involved in female cone development (Tandre et al, 1995; Winter et al, 1999)
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