Genotyping-by-sequencing based cytoplasmic markers underpin population structural variation of perennial ryegrass

Chloroplast and mitochondrial genomes provide unique information in studying plant populations because cytoplasmic genes exhibit a different mode of inheritance and a different rate of gene mutation compared to nuclear genes. Despite this, cytoplasmic genomic contributions to plant population performance are largely unexplored because few methods are available to characterize and evaluate cytoplasmic genome-wide variations. Here we have developed cytoplasmic markers based on genotyping-by-sequencing (GBS), which enable us to characterize thousands of samples, to survey gene variants across cytoplasmic genomes, and to monitor within-population variations of chloroplast or mitochondrial origin. Using these cytoplasmic genome-wide markers we have found that within-population differentiations are evident in ryegrass (Lolium perenne), beyond the explanation of nuclear markers. Moreover, chloroplast and mitochondrial variations exhibit different patterns, with mitochondrial markers more readily reflecting the maternal origins. Application of GBS-based cytoplasmic markers should facilitate quantifying the contribution of cytoplasmic inheritance to plant performance through selective breeding or under natural selection pressure.

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.

Application of Cleaved Amplified Polymorphic Sequence Method for Analysis of Cytoplasmic Genome among Aurantioideae Intergeneric Somatic Hybrids

Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.