ena-STUDY-KTH-11-06-2018-16:28:04:683-509
Analysis of early bud samples in Picea abies
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. The SOC1 sub-clade of the MADS-box family of transcription factors is expanded in gymnosperms. 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 (Uddenberg et al., 2013). Here, we show through Sanger sequencing and mapping to assembled conifer genomic sequences that DAL19 produces a number of splice variants. These transcripts use alternate first and last exons, while their four central exons constitute a core region present in all splice variants. Quantitative Real-Time PCR and mRNA in situ hybridization revealed that distinct DAL19 isoforms are expressed in Picea abies meristems that will form either male or female cones, or vegetative shoots. In addition, different DAL19 transcripts also showed a cell-specific expression pattern within bud meristems. Taken together this indicates that the different DAL19 transcript isoforms perform distinct and different activities during the development of male, female, and vegetative buds in Picea abies. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read RNA sequencing. We applied this method to 42 putative MADS-box core regions in P. abies, from which we identified 1084 novel transcripts. We manually curated these transcripts to arrive at 933 potential MADS-box isoforms of 38 putative MADS-box sequences. 152 of these transcripts, 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 38 putative MADS-box sequences by mapping PacBio IsoSeq circular consensus reads derived from pooled sample sequencing to assembled transcripts.Our results indicate that the higher complexity of MADS-box transcripts identified in Picea abies results from both the expansion of the gene family in addition to the generation of numerous splice variants.
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. The SOC1 sub-clade of the MADS-box family of transcription factors is expanded in gymnosperms. 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 (Uddenberg et al., 2013). Here, we show through Sanger sequencing and mapping to assembled conifer genomic sequences that DAL19 produces a number of splice variants. These transcripts use alternate first and last exons, while their four central exons constitute a core region present in all splice variants. Quantitative Real-Time PCR and mRNA in situ hybridization revealed that distinct DAL19 isoforms are expressed in Picea abies meristems that will form either male or female cones, or vegetative shoots. In addition, different DAL19 transcripts also showed a cell-specific expression pattern within bud meristems. Taken together this indicates that the different DAL19 transcript isoforms perform distinct and different activities during the development of male, female, and vegetative buds in Picea abies. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read RNA sequencing. We applied this method to 42 putative MADS-box core regions in P. abies, from which we identified 1084 novel transcripts. We manually curated these transcripts to arrive at 933 potential MADS-box isoforms of 38 putative MADS-box sequences. 152 of these transcripts, 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 38 putative MADS-box sequences by mapping PacBio IsoSeq circular consensus reads derived from pooled sample sequencing to assembled transcripts.Our results indicate that the higher complexity of MADS-box transcripts identified in Picea abies results from both the expansion of the gene family in addition to the generation of numerous splice variants.