Molecular characterization of Oryza sativa L. x O. officinalis Wall ex Watt and O. sativa L. x O. ridleyi Hook derivatives through in situ hybridization

A series of experiments were conducted to develop in situ hybridization protocols using mitotic and meiotic chromosome for characterization of parental chromosomes in Oryza sativa x O. officinalis and O. sativa x O. ridleyi hybrids and backcross progenies and detection of introgressed alien chromosomes segments from wild species into rice. Meiotic chromosomes behavior and homoeologous pairing in these two interspecific hybrids was studied. Karyotypes of O. ridleyi (2n=48) was prepared from somatic chromosomes. Chromosomes of O. ridleyi were larger than O. sativa. The chromosome complement consisted of 16 metacentric, 7 submetacentric and 1 subtelocentric pairs. Karyotype analysis supported allotetraploid nature of O. ridleyi. Meiotic chromosome analysis showed partial homoeology (0-3 bivalents) between genomes of O. sativa and O. officinalis where as O. ridleyi genome was quite divergent (0-2 bivalents in a few cells) from the O. sativa. The chances of alien gene transfer into O. sativa are greater from O. officinalis as compared to O. ridleyi. The F1 hybrids showed irregular meiosis and had complete pollen and seed sterility. The parental genomes in F1 hybrids (O. sativa x O. officinalis and O. sativa x O. ridleyi) and backcross progenies were successfully identified using GISH [genomic in situ hybridization] and FISH [fluorescence in situ hybridization]. High stringency washing of slides eliminated the cross hybridization of O. officinalis probe with O. sativa and enhanced discrimination of chromosomes of parental genomes. The chromosomes of O. ridleyi could be discriminated through genomic in situ hybridization from O. sativa x O. ridleyi hybrid using ridleyi DNA as probe. Unlike O. officinalis, blocking DNA or high stringency washing were not necessary to characterize parental chromosomes of O. ridleyi and O. sativa. The 45S rDNA sites were observed by FISH on two chromosomes of O. officinalis and one chromosome of O. sativa in O. sativa x O. officinalis hybrid. None of the introgression lines derived from O. sativa x O. officinalis showed hybridization signal, possibly due to smaller size of the alien chromosome segments or high amount of competitor DNA from O. sativa or masking of hybridization signal due to background effect. The results showed that in situ hybridization could be effectively used in molecular characterization of wide-cross derivatives (F1, BC1) at different stages of cell division