Dioecy, the presence of male and female flowers on distinct individuals, has evolved independently in multiple plant lineages, and the genes involved in this differential development are just starting to be uncovered in a few species. Here, we used genomic approaches to investigate this pathway in kiwifruits (genus ). Genome-wide cataloging of male-specific subsequences, combined with transcriptome analysis, led to the identification of a type-C cytokinin response regulator as a potential sex determinant gene in this genus. Functional transgenic analyses in two model systems, and , indicated that this gene acts as a dominant suppressor of carpel development, prompting us to name it (). Evolutionary analyses in a panel of species revealed that is located in the Y-specific region of the genome and probably arose from a lineage-specific gene duplication. Comparisons with the duplicated autosomal counterpart, and with orthologs from other angiosperms, suggest that the -specific duplication and subsequent evolution of-elements may have played a key role in the acquisition of separate sexes in this species.
The effect of red-and blue-light-emitting diodes on shoot and root growth of Hybrid Franc grape, a rootstock cultivar, along with two other grape genotypes, Kadainou R-1 and Vitis ficifolia var. ganebu, were investigated in vitro. Plants cultured under red-light-emitting diodes produced the longest shoots with longer internodes for all genotypes. The chlorophyll content measured as SPAD value and leaf number per explant were highest on plants cultured under blue-light-emitting diodes in all the genotypes. Blue light was also responsible for a higher number of stomata in all the genotypes; however, there was no significant difference in size of stomata in all genotypes under the different light conditions tested. Different light-emitting diodes did not affect the rooting percentage of Hybrid Franc but red-lightemitting diodes gave a higher rooting percentage along with higher root numbers for the two other grape genotypes.
MethodsMethod S1: Screening of the expressed candidate sex determinants Developing anthers at stage 1-2, which correspond to the differentiation stage of male or female androecium (see Supplementary Figure S1), were sampled from F1 sibling vines derived from an interspecific cross, A. rufa sel. Fuchu × A. chinensis sel. FCM1, named KE population (15), planted on Kagawa University, Japan (N34.28, E134.13), in 10-22 April in 2016-2017. Total RNA was extracted using the Plant RNA Reagent (Invitrogen) and purified by phenol/chloroform extraction. Two micrograms of total RNA were processed in preparation for Illumina Sequencing, according to a previous report (15). The constructed libraries were sequenced on Illumina's HiSeq 4000 sequencer (50-bp single-end or 150-bp paired-end reads). All Illumina sequencing were conducted at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, and the raw sequencing reads were processed using custom Python scripts developed in the Comai laboratory and available online (http://comailab.genomecenter.ucdavis.edu/index.php/), as previously described (9). Male-specific Ychromosomal sequences in kiwifruit, defined MSY contigs, were comprehensively identified in previous study (15). The mRNA-Seq reads from each 5 male and female individuals from the KE population (Supplemental Table S11) (15) were used to identify the genes substantially expressed in developing anthers. The mRNA-Seq reads were aligned to the hypothetical 61 genes located on the 249 MSY contigs (Akagi et al. 2018), using the Burrows-Wheeler Aligner (BWA) (37) allowing up to ca 3% mismatches. The number of reads mapping to each contigs was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). For Friendly Boy (FrBy), which showed male-specific and anther-enriched expression, the expression patterns were further examined using various plant organs and developing anthers (stage 2a, 2b, 3a, and 3b, see Supplementary Figure S1). Method S2: Expression profiling in kiwifruit antherThe described mRNA-Seq reads from each 5 male and female individuals of the KE population were aligned to the whole CDS sequences sets in A. chinensis (27), using BWA with default parameters. The number of reads mapped to each reference sequences was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). The read counts per gene were generated from the aligned SAM files using a custom R script. Differential expression between male and female individuals was analysed in R (version 3.0.1) using the R package DESeq (Anders and Huber, 2010) (version 1.14; http://bioconductor.org/packages/release/bioc/html/DESeq.html). We conducted DESeq analysis using 5 biological replicates from male and female individuals, with the following parameters: method='per-condition' and sharingMode='maximum'. An FDR threshold of 0.1 was used to identify differentially expressed genes. Method S3: in situ RNA hybridizationRNA in ...
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