Shinichi Adaniya

Digital Transcriptome Analysis of Putative Sex-Determination Genes in Papaya (Carica papaya)

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 Yh) 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 Yh chromosome, implying a loss of many genes on the Yh chromosome. Nevertheless, candidate Yh 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.

Sex and ploidy of anther culture derived papaya (Carica papaya L.) plants

SummaryTo improve the efficiency of papaya anther culture, we investigated (1) hormonal medium conditions for inducing haploids or dihaploids; (2) identified the sex of established plantlets using a sex-specific DNA molecular marker and (3) estimated their ploidy by flow cytometry analysis of DNA content. Anthers with a mixture of uninucleate, mitotic, and binucleate microspores were collected from a male plant, and cultured on MS agar medium with different concentrations of CPPU and NAA. An embryo induction rate of 13.8% was attained on MS agar medium with 0.01 mg l−1 CPPU and 0.1 mg l−1 NAA. The induced embryos were subcultured on medium with 0.0025 mg l−1 CPPU. Rooting of the developed shoots was promoted by treating their basal parts with 1500 mg l−1 IBA in a 50% ethanol solution for about 10 seconds. All the embryo-derived plantlets (27 plants) were identified as female, implying that they were derived from microspores. In addition, 26 plants were determined to be triploids and one to be tetraploids. We also observed a wide range of morphological variation (e.g., in tree height and fruit size) among the established plants. Based on the results, we discussed a potential value of anther culture techniques for the breeding of papaya.

Trisomic analysis of new gene for late heading in rice, Oryza sativa L

A near isogenic line, T65-LH7 bred from a rice variety, Ketan Nangka by five times of successive backcrossing with Taichung 65 (T65) as recurrent parent was found to carry a recessive lateness gene tentatively designated as ef6(t). The present study was performed to investigate the allelic relationships between ef6(t) and other heading time genes, Ef1, Efx, ef2(t), ef3(t), ef4(t) and ef5 by allelism test and to locate the chromosomal location of ef6(t) by trisomic analysis. In allelism test, six testers carrying each of heading time genes, Ef1, Efx, ef2(t), ef3(t), ef4(t) and ef5 were used. Those testers were the near isogenic lines of T65. T65-LH7 was crossed with respective testers. Heading times in F2 and/or B1F1 plants were examined. All F2 and/or B1F1 populations derived from those crosses exhibited digenic segregations, respectively. These results suggested that ef6(t) was independent of Ef1, Efx, ef2(t), ef3(t), ef4(t) and ef5. Sub sequently, trisomic analysis of ef6(t) was performed using seven Triplo lines having extra chromosomes, 2, 4, 5, 7, 9, 10 and 12. These Triplo lines were the near isogenic lines of T65. They were used as maternal parent to cross with T65-LH7. Heading times in F2 plants obtained from self-pollination of F1 plants were observed. Among F2 plants examined only those derived from a cross between Triplo-7 and T65-LH7 showed a typical trisomic segregation manner, suggesting that ef6(t) was located on chromosome 7. Consequently, the nomenclature of the present gene should be designated as ef6.

A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

SummaryA new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO3 aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.

Host plant manipulation by fig gall midges (Diptera: Cecidomyiidae) which induce emergence projections on flower galls in the syconia of Ficus microcarpa (Moraceae)

We observed that unique projections developed from female flower galls induced by three unidentified cecidomyiid species in the syconia of Ficus microcarpa (Moraceae) on Okinawa and Amami Islands, Japan. The three cecidomyiids (sp. 1, 2 and 3) were tentatively distinguished by the differences in the shape of the projections. The projection of sp. 1 started to develop from the bottom of each gall before emergence, broke the skin of the syconium, and developed finally up to 5–6 mm in length within 6–8 h. During this period, the pupa oriented its head towards the bottom of the gall. After the projection fully elongated, the pupa pushed open the bottom of the projection with its head. The projection was easily removed from the gall at the base. The pupa quickly crawled half way out of the gall through the opening at the bottom of the projection and an adult then emerged. The projection did not develop when other hymenopteran gall inhabitants emerged. The projection was derived from plant tissues consisting of a mass of small square cells in the basal and distal portions and regularly arranged long cells in the middle portion. No projection was induced by the application of gibberellins paste to the bottom of syconia. The gall midge seemed to manipulate the fig plant to develop the projection before emergence, so that the pupa can easily pass through the sticky epidermis of the syconium. The emergence of sp. 2 and 3 could not be intensively observed.