Evaluation of Crossability between Nicotiana benthamiana and Nicotiana excelsior

Wild tobacco species in the Nicotiana section Suaveolentes are promising genetic resources to introduce their disease resistance to cultivated tobacco, Nicotiana tabacum. However, hybrid lethality is observed in hybrid seedlings from crosses between most Suaveolentes species and N. tabacum. In particular, N. benthamiana belonging to the section Suaveolentes produces only viable hybrids after crossing with N. tabacum. In the present study, crossability between N. benthamiana and N. excelsior (section Suaveolentes) was investigated to test the possible usefulness of N. benthamiana as the bridge parent to transfer desirable genes of N. excelsior to N. tabacum via bridge crossing. After reciprocal crosses using three accessions of N. benthamiana and N. excelsior each, several crossing barriers such as cross-incompatibility, seed abortion, and male and female hybrid sterility were observed. Although reciprocal hybrids between N. benthamiana and N. excelsior showed a high degree of chromosome pairing in meiosis, univalents and multivalents, as well as chromosome bridges and lagging chromosomes, were observed. These meiotic abnormalities were thought to cause hybrid sterility. The possible usefulness of reciprocal hybrids between N. benthamiana and N. excelsior is discussed.

Genetic Mapping of the HLA1 Locus Causing Hybrid Lethality in Nicotiana Interspecific Hybrids

Hybrid lethality, a postzygotic mechanism of reproductive isolation, is a phenomenon that causes the death of F1 hybrid seedlings. Hybrid lethality is generally caused by the epistatic interaction of two or more loci. In the genus Nicotiana, N. debneyi has the dominant allele Hla1-1 at the HLA1 locus that causes hybrid lethality in F1 hybrid seedlings by interaction with N. tabacum allele(s). Here, we mapped the HLA1 locus using the F2 population segregating for the Hla1-1 allele derived from the interspecific cross between N. debneyi and N. fragrans. To map HLA1, several DNA markers including random amplified polymorphic DNA, amplified fragment length polymorphism, and simple sequence repeat markers, were used. Additionally, DNA markers were developed based on disease resistance gene homologs identified from the genome sequence of N. benthamiana. Linkage analysis revealed that HLA1 was located between two cleaved amplified polymorphic sequence markers Nb14-CAPS and NbRGH1-CAPS at a distance of 10.8 and 10.9 cM, respectively. The distance between these markers was equivalent to a 682 kb interval in the genome sequence of N. benthamiana.

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here, we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (Gossypium davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.

Two Nicotiana occidentalis accessions enable gene identification for Type II hybrid lethality by the cross to N. sylvestris

Hybrid lethality, meaning the death of F1 hybrid seedlings, has been observed in many plant species, including Nicotiana. Previously, we have revealed that hybrids of the selected Nicotiana occidentalis accession and N. tabacum, an allotetraploid with S and T genomes, exhibited lethality characterized by the fading of shoot color. The lethality was suggested to be controlled by alleles of loci on the S and T genomes derived from N. sylvestris and N. tomentosiformis, respectively. Here, we extended the analysis of hybrid lethality using other two accessions of N. occidentalis identified from the five tested accessions. The two accessions were crossed with N. tabacum and its two progenitors, N. sylvestris and N. tomentosiformis. After crosses with N. tabacum, the two N. occidentalis accessions yielded inviable hybrid seedlings whose lethality was characterized by the fading of shoot color, but only the T genome of N. tabacum was responsible for hybrid lethality. Genetic analysis indicated that first-mentioned N. occidentalis accession carries a single gene causing hybrid lethality by allelic interaction with the S genome.

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (G. davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.

Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

Analysis of the possible cytogenetic mechanism for overcoming hybrid lethality in an interspecific cross between Nicotiana suaveolens and Nicotiana tabacum

Hybrid lethality is a type of reproductive isolation in which hybrids die before maturation, due to the interaction between the two causative genes derived from each of the hybrid parents. The interspecific hybrid of Nicotiana suaveolens × Nicotiana tabacum is a model plant used in studies on hybrid lethality. While most of the progeny produced from such a cross die, some individuals grow normally and mature. Separately, a technique for producing mature hybrids by artificial culture has been developed. However, the mechanism by which hybrids overcome lethality, either spontaneously or by artificial culture, remains unclear. In the present study, we found that some hybrids that overcome lethality, either spontaneously or by artificial culture, lack the distal part of the Q chromosome, a region that includes the gene responsible for lethality. Quantitative polymerase chain reaction results suggested that the distal deletion of the Q chromosome, detected in some hybrid seedlings that overcome lethality, is caused by reciprocal translocations between homoeologous chromosomes. The results showed that chromosomal instability during meiosis in amphidiploid N. tabacum as well as during artificial culturing of hybrid seedlings is involved in overcoming hybrid lethality in interspecific crosses of the genus Nicotiana.

Pre- and Postzygotic Barriers Associated with Intergeneric Hybridization between Aronia melanocarpa (Michx.) Elliott x Pyrus communis L. and ×Sorbaronia dippelii (Zabel) CK Schneid. x Pyrus communis

Intergeneric hybridization between Aronia and Pyrus may provide a pathway for developing novel fruit types with larger, sweeter fruits, while maintaining the high levels of biologically health-promoting compounds present in Aronia fruits. Here we describe a deleterious genetic incompatibility, known as hybrid necrosis or hybrid lethality, that occurs in intergeneric F1 hybrids of Aronia melanocarpa x Pyrus communis and ×Sorbaronia dippelii x Pyrus communis. Pollination experiments revealed that maternal A. melanocarpa and ×S. dippelii pistils are compatible with pollen from P. communis. Controlled pollinations using different mating combinations resulted in varying levels of fruit and seed set. Because every combination produced at least some viable seeds, prezygotic incompatibility does not appear to be present. We attempted to recover putative intergeneric progeny via either in vitro germination or in vitro shoot organogenesis from cotyledons. Progeny of putative hybrids from A. melanocarpa x P. communis only survived for a maximum of 14 days before succumbing to hybrid lethality. Regeneration of ×S. dippelii x P. communis was successful for two seedlings that have been maintained for an extended time in tissue culture. These two seedlings have leaf morphologies intermediate between the two parental genotypes. We also confirmed their hybrid status by using AFLPs and flow cytometry. Putative intergeneric hybrids were grown out ex vitro before showing symptoms of hybrid necrosis and dying after 3 months. Eventually micrografts failed, ultimately showing the same symptoms of hybrid necrosis. These results show that intergeneric hybridization is possible between Aronia and related genera in the Rosaceae, but there are postzygotic barriers to hybridity that can prevent the normal growth and development of the progeny.