We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights.

The apple (Malus × domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4 years) and a long reproductive period between the flower initiation and the mature fruit (14-16 months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.

Transgenic apple plants that overexpressed Arabidopsis FLOWERING LOCUS T (FT) under the control of the rolC promoter showed early flowering, while the introduction of FT driven by the 35S promoter induced flower development directly from transgenic apple callus in vitro, but vegetative growth was not maintained and the explant died. GFP-FT fusion proteins were detectable in transgenic apple tissues but never caused early flowering in the transformants. Under the control of the rolC promoter, the fused protein was localized in vascular tissues and fluorescence was detectable in companion cells in the stem and petiole. The transgenic apple lines that expressed AtFT driven by the rolC promoter showed differences in inflorescence architecture and floral organ number from those typical of nontransformed apple. Flowers of transgenic apple lines often contained more numerous petals, fewer stamens, and no pistils, and the pollen grains were incapable of germinating. The transgenic apple lines showed perpetual flowering without the requirement for low temperature and obvious photoperiodism. The expression patterns of six floral meristem identity genes and floral organ genes in flowers of transgenic apple lines were investigated. Among the floral meristem identity genes, expression of MdFT2 was suppressed and that of AFL2 was dramatically enhanced in the transformants. Of the floral organ genes, expression of MdMADS13, which functions as a class B gene, was unchanged from that of the flower of wild type one, whereas expression of MdMADS-NB, a possible class C gene, was suppressed.

Transformation experiments in apple (Malus × domestica Borkh.) were conducted to improve the Agrobacteriummediated transformation efficiency of many apple cultivars without using complicated procedures. A system of direct regeneration from leaf segments was used for these experiments. The effectiveness of meropenem hydrate (MEPM) as an antibiotic for Agrobacterium elimination was investigated for use of instead of cefotaxime sodium (CTX), which prevents shoot regeneration from apple leaf segments. Results show that 50 mg•L −1 of MEPM greatly enhanced the transformation efficiency. Experiments assessing the timing of kanamycin sulfate (KM) addition to the medium were also conducted to investigate the effective use of KM, the antibiotic used to select transformed cell. Addition of KM to the medium immediately after co-cultivation was completed is advantageous for the multiplication of transformed cells and for the induction of transformed shoots in spite of the low shoot regeneration rate. Based on knowledge of the timing of KM addition, the effects of pre-culture before Agrobacterium infection on the shoot regeneration medium were investigated. Pre-culture for 3-7 days improved the transformation efficiency. By applying both MEPM and the pre-culture, about 5% transformation efficiency in 'Greensleeves' was achieved on an apple cultivar without complicated procedures.

Fluorescent banding patterns of pear chromosomes were determined from samples taken from root tips of openpollinated seedlings of Pyrus pyrifolia (Burm. F) Nakai (Japanese pear), P. calleryana Decne. (Callery pear), P. pyrifolia × P. ussuriensis var. aromatica (a hybrid of Japanese pear and Iwateyamanashi), and P. mikawana Koidz. (Toyotomi Nashi). All accessions used in this study had 2n = 34 chromosomes. Chromomycin A 3 (CMA)-positive (+) bands were observed in telomeric positions of four chromosomes in all accessions. 4'-6-diamidino-2-phenylindole (DAPI)-negative bands (−) corresponded with CMA+ bands. Fluorescence in situ hybridization (FISH) was conducted using open-pollinated seedlings of 'Osa Gold' (Pyrus pyrifolia) and Toyotomi Nashi (P. mikawana) as materials. Four out of six 18S-5.8S-25S ribosomal RNA gene (rDNA) sites corresponded with CMA+/DAPI− bands. The 5S rDNA sites were detected in centromeric positions of two chromosomes. Two centromeric 5S rDNA and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH.