Identification of maternal haploids of Nicotiana tabacum aided by transgenic expression of green fluorescent protein: evidence for chromosome elimination in the N. tabacum × N. africana interspecific cross

Abstract

The Nicotiana tabacum × N. africana cross is a semilethal cross, where greater than 99 % of progeny die in the cotyledonary stage due to an interspecific lethality reaction. The low frequencies of surviving plants are mixtures of maternal tobacco haploids and aneuploid F1 hybrids. For effective use of surviving haploids in a breeding program, an efficient method is needed to distinguish them from aneuploid hybrids during the seedling stage. The first objective of this research was to investigate the use of N. africana engineered with a green fluorescent protein (gfp) transgene for distinguishing haploids from other surviving plants derived from this cross. Results demonstrated gfp expression to be a useful phenotypic marker for separation of aneuploid hybrids at the seedling stage. Microsatellite marker genotyping, flow cytometry determinations, and chromosome counts resulted in identification of progeny that exhibited intermediate DNA amounts and variable chromosome numbers donated by the N. africana parent. Three individuals with near-haploid DNA content were identified that carried N. africana markers and n = 24 + 2 or n = 24 + 3 chromosomes. The results present evidence of genetic instability in progeny from the N. tabacum × N. africana cross and suggest that, in contrast to previous belief, chromosome elimination may play at least a partial role in the generation of haploids from this cross. Additionally, microsatellite marker genotyping demonstrated a role for a gene or genes located near the end of N. tabacum chromosome H (linkage group 11) in the interspecific lethality reaction.