Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

New mitochondrial genome organization in three interspecific somatic hybrids of Medicago sativa including the parent-specific amplification of substoichiometric mitochondrial DNA units

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago. Received: 5 January 2001 / Accepted: 23 March 2001     

Mapping of quantitative trait loci influencing frost tolerance in Eucalyptus nitens

 Regions of the genome influencing frost tolerance in an outbred family of Eucalyptus nitens have been identified. Two QTLs present on the same linkage group, but located 40 cM apart, were identified using single-factor analysis of variance. The QTLs explained between 7.7 and 10.8% of the phenotypic variation for frost tolerance in this family. Analysis of marker loci linked to the QTLs showed one of them to have a simple mode of action with the effect segregating from the male parent in the family. For the other QTL multiple alleles were identified. This QTL showed segregation from the female parent which gave a positive effect on frost tolerance; however, an allele segregating from the male parent was identified which showed a negative interaction with the allele for increased frost tolerance. Received: 10 May 1997 / Accepted: 2 June 1997     

Asymmetric protoplast fusions between wild species and breeding lines of potato – effect of recipients and genome stability

 The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation. Received: 30 April 1998 / Accepted: 24 July 1998     

Organelle DNA analysis of Solanum and Brassica somatic hybrids by PCR with ’universal primers’

In order to set up a quick and easy procedure for determining the cytoplasmic composition of somatic hybrids, we tested a set of ’universal primers’ for plastidial and mitochondrial DNA on 13 genotypes belonging to the following species: Nicotiana tabacum, Solanum commersonii, Solanum tuberosum, Solanum etuberosum, Solanum phureja, Brassica oleracea, Brassica rapa, ’Anand’ CMS B. rapa, ’Chiang’ CMS B. oleracea, and ’Ogura’ CMS B. oleracea. Such primers are homologous to conserved coding sequences and amplify polymorphic intergenic or intronic regions. cpDNA polymorphism within Solanum and Brassica spp. was found with two and four primer pairs, respectively. The primers for the intergenic region between the trnF and trnV genes gave polymorphism among several tested species and were used in S. commersonii (+) S. tuberosum somatic hybrids,and B. oleracea (+) ’Anand’ CMS B. rapacybrids. Two primer pairs for mtDNA revealed polymorphism between S. commersonii and S. tuberosum, and one showed intraspecific polymorphism in S. tuberosum. The primer pair for the intergenic region between the rps14 and cob genes (pumD) showed a fragment of about 1.5 kb in S. tuberosum and S. phureja. A shorter fragment and no amplification were found in S. etuberosum and S. commersonii, respectively, suggesting frequent intrageneric rearrangements in this genome region. All Brassicaceae evidenced a fragment about 150-bp longer than in S. tuberosum. The same primers were also used with interspecific Solanum spp. somatic hybrids. Both PCR with pumD primers and hybridization with rpl5/rps14 genes indicated lack of linkage between rpl5/rps14 and cob genes in S. commersonii. Compared to direct visualization of restricted organellar DNA or Southern analysis with labelled probes, amplification of cpDNA and mtDNA with universal primers, followed by electrophoresis of either entire or restricted amplified fragments, is a simpler, more rapid and less expensive method to determine the organelle genome composition of interspecific Solanum and Brassica somatic hybrids. Received: 2 August 2000 / Accepted: 22 September 2000     

Chondriome-type characterization of potato: mt α, β, γδɛ and novel plastid-mitochondrial configurations in somatic hybrids

 One hundred and eighty dihaploid clones used for protoplast fusions, and 144 tetraploid German potato cultivars were analysed for their cytoplasms using 11 homologous mt DNA-probes, and were classified as mitochondrial (mt) types α, β, γ, δ, and ɛ according to their RFLP patterns. From the 4x cultivars, 79 had the typical mt-type β of Solanum tuberosum being different from the 46 cvs which had the mt-α type and 19 others with mt-γ. A dendrogram shows their relationships to other Solanum species. The distantly related mt-ɛ was only found in di-haploids, and particularly in clones deriving from Solanum phureja and Solanum andigena. Accessory mt types will be actualized on website (http://www.edv.agrar.tu-muenchen.de/pbpz/ mm/mt/al1.htm). In order to evaluate the genetic potential of novel plastid-mitochondrial configurations we have analyzed four representative populations, which derive from different fusion-combination classes: [α (+) β], [α (+) γ], [α (+) δ] and [α (+) ɛ]. On the mitochondrial expression level, hybrids from an [α (+) ɛ] fusion could be distinguished by in-organello translation from [α (+) β] hybrids, and other di-haploids, by an additional translation product of 15 kDa. In fusion parents with mt-α and -γ an additional atp6 reading frame is detectable in sub-stoichiometric amounts by the use of specific PCR primers. The gene differs from the original 211 bp 3′ from the stop codon. Novel RFLP-patterns in 10% of the somatic hybrids were due to a high-rate replication of this pre-existing parental genome region. A second characteristic for somatic hybrids was the partial addition of parental mt sub-genomes. The major part of them revealed a new organization in their mt genomes at the mt-type characteristic loci rpl5, rps14, cob, rps10, coxI and rpl2, which contain recombination-specific repeats homologous to Petunia spp. and Nicotiana. A schematic model for the formation of novel mitochondrial genomes in potato somatic hybrids is provided. Received: 7 November 1998 / Accepted: 30 November 1998     

Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997     

Somatic cell hybridization of ’oxy’ CMS Brassica juncea (AABB) with B. oleracea (CC) for correction of chlorosis and transfer of novel organelle combinations to allotetraploid brassicas

Alloplasmic lines of cultivated Brassica species with B. oxyrrhina cytoplasm are male-sterile and suffer from severe chlorosis. We developed male-sterile lines corrected for chlorosis by fusing protoplasts of CMS B. juncea (AABB) with ’oxy’ cytoplasm and normal B. oleracea (CC). A large number of male-sterile AABBCC somatic hybrids with desirable organelle combinations, i.e. chloroplasts of B. oleracea and mitochondria with recombinant genomes, were recovered. While no recombination was observed in the chloroplast genome, the mitochondrial genome showed extensive recombination that resulted in the appearance of totally novel banding patterns in some of the hybrids. Hybrids with a parental-type mitochondrial genome as well as recombinant patterns close to either of the parental types were also obtained. Using AABBCC somatic hybrids as bridging material, we transferred the desirable organelle combinations to B. juncea (AABB), B. napus (AACC), and B. carinata (BBCC). Many of these lines are now at advanced stages of backcrossing and show stable inheritance of the CMS character and do not suffer from chlorosis. Received: 9 August 1999 / Accepted: 14 September 1999     

Freezing tolerance and tuber production in selfed and backcross progenies derived from somatic hybrids between Solanum tuberosum L. and S. commersonii Dun.

 Selfed and backcross progenies developed from tetraploid somatic hybrids between Solanum tuberosum (tbr) and S. commersonii (cmm) were characterized for nonacclimated freezing tolerance (NA) and acclimation capacity (ACC) (two independent genetic components of freezing tolerance) under controlled environments. The segregation covered 28% and 71% of the parental range for NA and ACC, respectively, with the distribution skewed toward the tbr parent. Therefore, ACC appeared to be relatively easier to recover in the segregating generation. Some first backcross progeny had greater freezing tolerance than the cultivated parent primarily through the increase in ACC. When grown in the field, the improved freezing tolerance observed in the selfed progeny under controlled conditions was confirmed. Among NA, ACC, and freezing tolerance after acclimation (AA, which is the cumulative performance of NA and ACC), AA exhibited the highest correlation coefficient with field frost tolerance. In addition to freezing tolerance, vine maturity and tuber traits including tuber yield, tuber number per plant, mean tuber weight, and specific gravity were also segregating. No significant correlation between undesirable tuber traits and freezing tolerance was detected. Vine maturity and freezing tolerance were significantly correlated, so more careful selection for earliness was necessary in incorporating freezing tolerance. Yield comparable or superior to the backcross parent Wis AG 231 and an early Canadian cultivar, ‘Sable’, was found in many backcross progeny and some selfed progeny. The observed high yield can be attributed to the increase in mean tuber weight as well as tuber number. Moreover, a high portion of progeny had a specific gravity higher than 1.085, and some greater than 1.1. The implications derived from this study in breeding for freezing tolerance and further use of these materials are discussed. Received: 22 August 1998 / Accepted: 4 January 1999     

Molecular marker-assisted introgression of the wild Solanum commersonii genome into the cultivated S. tuberosum gene pool

A breeding scheme involving ploidy and EBN manipulations was set up to overcome the interspecific barriers existing between the cultivated Solanum tuberosum and the wild species S. commersonii. Three backcross generations were obtained that were analyzed by means of molecular markers in order to verify the occurrence of recombination between homeologous chromosomes, the extent of the wild genome carried in each backcross, and the efficiency of introgressing useful genes. Twenty commersonii-specific RFLPs provided evidence for recombination on 5 out of 12 chromoso-mes; for the others no more than 1 commersonii-specific marker was found. Thirty-four commersonii-specific RAPDs and 61 commersonii-specific AFLPs were used to estimate the wild genome content in the BC₁, BC₂, and BC₃ generations. The mean value of commersonii-specific markers was 93% and 91% in the BC₁, 74% and 76% in the BC₂, and 31% and 26% in the BC₃ using RAPDs or AFLPs, respectively. Efficiency of the breeding scheme was evaluated by monitoring across these progenies the introgression of S. commersonii resistance to tuber soft rot caused by Erwinia carotovora. Eleven and five resistant genotypes were found among BC₂ and BC₃ hybrids, respectively. The same progenies were also evaluated for chromosome number and tuber traits. For all analyzed traits except stolon length, all BC₂ and BC₃ hybrids resembled the tuberosum type. In order to choose the best genotypes to obtain the following backcrosses, we performed, in each generation, a negative molecular-assisted selection against the wild genome combined with selection for resistance to Erwinia spp. and other traits. Received: 3 April 2000 / Accepted: 31 August 2000     

Fertile Solanum tuberosum+S. commersonii somatic hybrids as sources of resistance to bacterial wilt caused by Ralstonia solanacearum

 The wild potato relative Solanum commersonii is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum. To overcome sexual incompatibilites due to differences in ploidy and endosperm balance numbers, somatic hybrids were made that combine the S. tuberosum and S. commersonii genomes. The resulting somatic hybrid plants are vigorous, but their disease resistance level and their fertility was unknown. We therefore tested the S. commersonii and S. tuberosum source material cv Superior, potato cv Atlantic and six somatic hybrid lines for resistance to a virulent strain of R. solanacearum (race 3, biovar 2) at 28°C. As expected, S. commersonii was significantly more wilt-resistant than the cultivated potatoes. In five of the six somatic hybrid lines, disease resistance levels were similar to that of the resistant S. commersonii parent. The resistance level of the sixth somatic hybrid was intermediate, significantly different from both S. commersonii and S. tuberosum. In controlled crosses, the somatic hybrids in this study proved both to be male- and female-fertile and were self-compatible. More importantly, the somatic hybrids can be crossed with S. tuberosum to produce viable seeds. Received: 23 June 1998 / Accepted: 13 October 1998     

Isozyme evidence of the parental origin and possible fertility of the hybrid Potamogeton×fluitans Roth

 The hybrid Potamogeton×fluitans Roth is thought to have resulted from hybridization between P. lucens L. and P. natans L. This hybrid has only been recorded at a few locations in the British Isles. At most of these sites the number of individuals found have been quite low. The one exception to this being a population in the Moors River, Dorset and South Hampshire, England, where a substantially larger population exists. Using seven different enzyme systems, this study provided support for the putative parental origin of this hybrid. In addition, the population in the Moors River was shown to contain numerous multi-locus phenotypes suggesting that these individuals may be partially fertile and may backcross to one of its parental species, or alternatively undergo selfing or crossing to produce an F₂ generation. Received December 1, 2000 Accepted June 20, 2001     

Non-random mating in classical lekking grouse species: seasonal and diurnal trends

This paper is the first to integrate both field and theoretical approaches to demonstrate that fertility benefits can be a direct benefit to females mating on the classical lek. Field data collected for male sharp-tailed grouse (Tympanuchus phasianellus), a classical lekking species, revealed potential fertility benefits for selective females. Adult males and individuals occupying centrally located territories on the lek were found to have significantly larger testes than juveniles and peripheral individuals. Further, using empirical data from previously published studies of classical lekking grouse species, time-series analysis was employed to illustrate that female mating patterns, seasonal and daily, were non-random. We are the first to show that these patterns coincide with times when male fertility is at its peak. Received: 26 February 1999 / Revised: 13 December 1999 / Accepted: 15 March 2000     

5′-Anchored simple-sequence repeat primers are useful for analysing potato somatic hybrids

Somatic hybrids between Solanum tuberosum cv 'Brodick' and S. sanctae-rosae were selected for having elevated chromosome numbers, intermediate morphology and potato cyst nematode resistance (derived from S. sanctae-rosae). DNA was extracted from this material and subjected to inter-SSR PCR directed by oligonucleotide primers comprising 5′-anchored di- and tri-nucleotide repeat motifs. PCR products were separated by polyacrylamide gel electrophoresis and visualised by silver staining. An average of 30.31 bands per primer were generated in each parent and, on average, 8.19 of these bands were polymorphic. S. sanctae-rosae-specific bands were observed in the hybrid material as was evidence of deletions, substitutions and rearrangements. The relative merits of this technique for the analysis of somatic hybrid material are discussed. Received: 23 July 1996 / Revision received: 22 October 1996 / Accepted: 22 March 1999