Digital transcriptome analysis of putative sex-determination genes in papaya (Carica papaya)

Abstract

Papaya (Carica papaya) is a trioecious plant species that has male, female and hermaphrodite flowers on different plants. The primitive sex chromosomes genetically determine the sex of the papaya. Although draft sequences of the papaya genome are already available, the genes for sex determination have not been identified, likely due to the complicated structure of its sex-chromosome sequences. To identify the candidate genes for sex determination, we conducted a transcriptome analysis of flower samples from male, female and hermaphrodite plants using high-throughput SuperSAGE for digital gene expression analysis. Among the short sequence tags obtained from the transcripts, 312 unique tags were specifically mapped to the primitive sex chromosome (X or Y(h)) sequences. An annotation analysis revealed that retroelements are the most abundant sequences observed in the genes corresponding to these tags. The majority of tags on the sex chromosomes were located on the X chromosome, and only 30 tags were commonly mapped to both the X and Y(h) chromosome, implying a loss of many genes on the Y(h) chromosome. Nevertheless, candidate Y(h) chromosome-specific female determination genes, including a MADS-box gene, were identified. Information on these sex chromosome-specific expressed genes will help elucidating sex determination in the papaya.

Figure 1. Flower samples for the Ht-SuperSAGE analysis.

The papaya flowers used for the RNA extraction and Ht-SuperSAGE analysis (P1: male 7-mm flower; P2: male 20-mm flower; P3: female 7-mm flower; P4: female 20-mm flower; P5: hermaphrodite 7-mm flower; and P6: 20-mm hermaphrodite flower).

Figure 2. Functional categories of the genes annotated from the SC-tags.

Genes corresponding to 456 SC-tags were categorized according to their function, based on their annotation as described in the Methods section. “No match” indicates the tags that had no significant similarities to any non-redundant protein sequences in the databases. “Unknown function” indicates those genes for which the functions of their encoded proteins could not be determined. The SC-tags with different annotations were grouped into the corresponding categories.

Figure 3. Confirmation of sex-dependent gene expression for Cp2671, which encodes a putative MADS-box protein.

Figure 4. Structure and expression analysis of the genes corresponding to the Cp3177 tag, which encodes a putative monodehydroascorbate reductase (MDAR).

A) RT-PCR analysis of the MDAR gene corresponding to the Cp3177 tag. P1 to P6 correspond to the flower samples indicated in Figure 1. An actin gene was used as a constitutively expressed control gene . B) A list of the polymorphic tags and their counts for the MDAR genes in the Ht-SuperSAGE data. The polymorphic sequences among the tags are underlined. C) The regions flanking the Cp3177 tag on the BAC clones 46O19 (X chromosome) and 90D06 (Y^h chromosome). The arrows indicate the locations of the PCR primers used for amplification. The black regions represent the predicted exons. An insertion of the retroelement sequence was observed in the 90D06 sequence (Y^h chromosome). D) Genomic PCR amplification of the MDAR gene in each sex type. The smaller band was equally amplified in all of the sex types. The larger bands, indicating the insertion of retroelements, were only observed in males and hermaphrodites.