Molecular marker systems for Oenothera genetics

Abstract

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.

Figure 1.—

Determination of diakinesis/metaphase I configuration in Oenothera strains or hybrids. Chromosome configuration of 7 pairs in strain johansen (^hjohansen·^hjohansen) (A–C), ⊙4, 5 pairs in hybrid ^hjohansen·^Gflavens (D–F), ⊙14 in strain suaveolens Grado (^Galbicans·^Gflavens) (G–I), and ⊙12, 1 pair in hybrid ^Stalbicans·^htuscaloosa. Determination in diakinesis via DAPI staining (A, D, G, J) and graphical interpretation (B, E, H, K). The respective chromosome configurations can be predicted by the chromosome formulas of the Renner complexes involved (C, F, I, L). Bivalents are labeled with roman numbers (I–VII), single chromosomes in rings with a combination of roman numbers and letters (Ia, Ib–VIIa, VIIb); so far, chromosome identity cannot be assigned directly in Oenothera. Identity of bivalent 9·8 in K was assigned indirectly since it was deduced from the chromosomal formulas of the Renner complexes involved and thus represents the free pair of ^Stalbicans·^htuscaloosa (L).

Figure 2.—

Maintenance of the permanent translocation heterozygote Oe. biennis strain suaveolens Grado. Free segregation of chromosomes and homologous recombination is suppressed in the complete permanent translocation heterozygote between the haploid Renner complexes ^Galbicans (yellow) and ^Gflavens (orange). Gametophytic lethal factors repress germination of ^Galbicans pollen, and sporophytic lethal factors eliminate homozygous ^Gflavens·^Gflavens offspring in F₁. Referring to the parental generation, identical offspring are produced in F₁. For a detailed explanation, see text.

Figure 3.—

Exchange of plastids and genome rearrangement between Oenothera strains. Repression of homologous recombination in a complete permanent translocation heterozygote and somatic segregation leading to sorting out of the two plastid types in F₁ (I and II) allow the exchange of plastomes as well as haploid genomes (A, B, and C) in F₂. For detailed explanation, see text.

Figure 4.—

Lutescent phenotype and somatic segregation of two plastome types in the F₁ generation of a cross between Oe. elata subsp. hookeri strain johansen (AA-I) and Oe. grandiflora strain tuscaloosa (BB-III), resulting in so-called hybrid variegation (A). Variegated tissue is generated if plastomes are inherited by both sexes and only one of the plastomes is incompatible with the nuclear genome. Separation of the two tissue types differing in their plastid genome results from the statistical process of sorting out, i.e., the random segregation of the two plastid types during cell divisions (Kirk and Tilney-Bassett 1978; Birky 2001). Incompatible and green tissue was correlated with a plastome type via a BamHI CAPS marker (Table 3) (B). Marker alleles rrn16-trnI_GAU I₃ and -II/III₈ were amplified from plastomes I^joh and III^tusca and digested with BamHI (lanes 2 and 3). Lane 4 shows a mixture of cleaved I^joh and III^tusca PCR products. Applying that marker to tissues of F₁ individuals, plastome I^joh exclusively correlates with the lutescent phenotype (lanes 5–9) and plastome III^tusca with green tissue (lanes 10–14).

Figure 5.—

Assignment of M40 alleles to the Renner complexes ^Stlaxans·^Stundans in the strain bauri Standard: In tuscaloosa, a single band of 500 bp was amplified (lane 1); in bauri Standard, two bands of 474 and 579 bp were detected (lane 2). The two bands of 500 and 579 bp detectable in the F₁ hybrid ^htuscaloosa·^Stundans assign the latter to ^Stundans (lane 3).

Figure 6.—

Crossing scheme to exchange plastome III of Oe. grandiflora strain tuscaloosa with plastome II of Oe. biennis strain suaveolens Grado. For a detailed description, see text.

Figure 7.—

Phenotypic and molecular discrimination of crossing intermediates and end products in the BB-II crossing program: ^Galbicans·^htuscaloosa with plastomes II^suavG or III^tusca (AB-II/III) (A). Native, compatible combination ^htuscaloosa·^htuscaloosa or hybrid ^Gflavens·^htuscaloosa with plastome III^tusca (BB-III) (B). Incompatible combination ^htuscaloosa·^htuscaloosa II^suavG (BB-II) (C). Molecular discrimination of twin hybrids and plastomes (D): ^Galbicans·^htuscaloosa (AB), heterozygous for M40 alleles A₁ and B₄, digested with SpeI (lane 1); ^htuscaloosa·^htuscaloosa (BB) homozygous for M40 allele B₄, digested with SpeI (lane 2); plastome II^suavG rrn16-trnI_GAU allele II/III₁, digested with BamHI (lane 3); and plastome III^tusca rrn16-trnI_GAU allele II/III₈ digested with BamHI (lane 4); the first lane (M) shows a 100-bp ladder (New England BioLabs, Ipswich, MA). Detailed explanations are given in the text. The weaker signals characteristic of the ^Galbicans complex in lane 1 are primer specific and independent of the complex with which ^Galbicans is associated.

Figure 8.—

Assembly and segregation behavior of the hybrid ^Stalbicans·^htuscaloosa. The F₂ population was used to identify chromosome 9·8 (linkage group 2), which segregates independently from the large, strongly linked coupling groups of the torso complexes ^Stalbicans or ^htuscaloosa (linkage group 1). Details are given in the text.