Post-zygotic gametic selection due to endosperm balance number explains unusual chromosome numbers of 3×× 2× progeny in Solanum

 Crosses between triploid and diploid genotypes are usually the best sources of trisomics in potato as well as in several other crop species. However, 3×× 2× crosses between triploid (2n=3×=36; 2EBN) Solanum commersonii-S. tuberosum hybrids and diploid (2n= 2×=24; 2EBN) genotypes gave progenies with a high number of extra chromosomes, 29–36, suggesting that only eggs with 17–24 chromosomes produced embryos that reached full development. Our hypothesis is that although triploids produce eggs with a range of chromosome numbers, 3×× 2× crosses involving a 2×(2EBN) parent favor eggs with a high chromosome number. These eggs have higher probabilities of possessing the same endosperm balance number (EBN) value (i.e. 1) of gametes produced by the 2EBN diploid parent to give the required 2:1 maternal to paternal EBN ratio in the hybrid endosperm. Under this model, trisomics are produced only if the diploid parent has an EBN of 1. Based on our results and those reported in the literature, it is proposed that in 3×(2EBN) × 2×(2EBN) crosses the endosperm balance number exercises negative selection for gametes with a low chromosome number, and a corresponding low EBN, and positive selection for gametes with a high chromosome number and EBN. Received: 2 April 1998 / Revision accepted: 27 October 1998     

The significance of genic balance to endosperm development in interspecific crosses

Summary The endosperm has played a significant role in the evolution of angiosperms because of its physiological and genetic relationships to the embryo. One manifestation of this evolutionary role is its abnormal development in interploidy crosses. It is now established that the endosperm develops abnormally in interploidy-intraspecific crosses when the maternal: paternal genome ratio deviates from 21 in the endosperm itself. We propose an Endosperm Balance Number (EBN) hypothesis to explain endosperm development in both interploidy-intraspecific and interspecific crosses. Each species is assigned an EBN on the basis of its crossing behavior to a standard species. It is the EBN which determines the effective ploidy in the endosperm of each species, and it is the EBNs which must be in a 21, maternal:paternal ratio. The EBN of a species may be determined by a few genes rather than the whole genome. This hypothesis brings most intraspecific-interploidy and interspecific crossing data under a single concept with respect to endosperm function. The implications of this hypothesis to isolating mechanisms, 2n gametes, the evolution of disomic polyploids, and reciprocal differences in seed development are discussed.     

A genetic model for the endosperm balance number (EBN) in the wild potato Solanum acaule Bitt. and two related diploid species

Solanum acaule Bitt. is a disomic tetraploid potato which has been assigned two endosperm balance numbers (EBN). It readily crosses with diploids but does not cross with other tetraploid species, although exceptions have been reported. The genetic basis of this behavior was studied in intra- and interspecific crosses involving plants of four introductions of this species and plants of one introduction of 2x S. commersonii Dun., one of 2x S. gourlayi Haw., and two of 4x S. gourlayi Haw., which have been assigned one EBN, two EBN, and four EBN respectively. Some of the pollinated pistils were used to analyze pollen-pistil compatibility reactions; the rest were left in the plants for seed production. At harvest, seeds were sorted according to size and plumpness, and the ploidy of the resulting plantlets determined from root tips. A model is proposed to explain the results of these crosses as well as the exceptions previously reported. It is based on the presence of two independent loci controlling the EBN, with two alleles in homozygosity: 1/2 and 0. This model, which is extended to cmm and grl, also explains the behavior of 3x (cmm x grl) hybrids in crosses with one-EBN, two-EBN, and four-EBN species reported in a previous work.     

The nature of the genetic control of Endosperm Balance Number based on aneuploid analysis of Datura

 The genetic control of Endosperm Balance Number (EBN), a mechanism of effective ploidy that controls seed development, was studied using aneuploidy. The Endosperm Balance Number hypothesis proposes that each species has an effective ploidy (EBN) in the endosperm and that it is the effective ploidies, rather than the numerical (actual) ploidies, that must be in a 2:1 maternal to paternal ratio for normal endosperm development. Experiments were conducted in Datura stramonium L. (2n=4x=48) to determine if more than one chromosome but less than the whole genome could change the EBN of the female. Triploids were crossed with tetraploids to produce aneuploids. Most plump seeds gave rise to 2n=4x=48 chromosome plants. Six plants had between 38 and 47 chromosomes. Karyotyping of these plants supported the conclusion that only two chromosomes (1.2 and 19.20), when extra, were necessary to change the EBN of the central cell. Received: 25 April 1998 / Revision accepted: 25 January 1999     

Unilateral and bilateral hybridization barriers in inter-series crosses of 4x 2EBN Solanum stoloniferum, S. pinnatisectum, S. cardiophyllum, and 2x 2EBN S. tuberosum haploids and haploid-species hybrids

Wild Mexican potato species are an important untapped source of useful variation for potato improvement. Introgression methods such as 2n gametes, chromosome doubling, and crossing with disomic 4x 2 endosperm balance number (EBN) bridge species have been used to overcome post-zygotic endosperm failure according to the EBN hypothesis. Stylar barriers can prevent zygote formation, bilaterally when zygote formation is blocked in both directions of the cross or unilaterally when zygote formation is blocked in self incompatible (SI) × self compatible (SC) crosses. In several Solanaceae species, the S-locus for SI has been implicated in interspecific incompatibility. The objectives of this research were to determine if: (1) disomic 4x 2EBN Solanum stoloniferum can be used as a bridge species for introgression of the Mexican 2x 1EBN species Solanum cardiophyllum and Solanum pinnatisectum, (2) pre- and/or post-zygotic barriers limit hybridization among EBN compatible Solanum inter-series crosses, and (3) reproductive barriers act unilaterally or bilaterally. Fruit formation and seed set was recorded for inter-pollinations of S. stoloniferum, 4x 2EBN chromosome doubled S. cardiophyllum and S. pinnatisectum, and 2x 2EBN S. tuberosum haploids (HAP) or haploid-species hybrids (H-S). In vivo pollen tube growth was analyzed for each cross combination with fluorescence microscopy. Attempts to create bridge hybrids between S. stoloniferum, and S. cardiophyllum or S. pinnatisectum were not successful. Pre- and post-zygotic barriers prevented seed formation in crosses involving S. cardiophyllum and S. pinnatisectum. Self compatibility in S. stoloniferum and S. pinnatisectum suggests that the S-locus does not contribute to the stylar barriers observed with these species. Alternatively, the presence of functional and nonfunctional (SC) S-alleles may explain interspecific incompatibility in intra- and inter-ploidy crosses. A non-stylar unilateral incongruity was discovered in H-S/HAP × S. stoloniferum crosses, indicating either a post-zygotic barrier, or a pre-zygotic barrier acting at or within the ovary. Furthermore, lack of S. stoloniferum pollen rejection may occur through absence of S. stoloniferum pollen-active genes needed to initiate pollen rejection, or through competitive interaction in S-locus heterozygous S. stoloniferum pollen. Introgression strategies using these species would benefit potato breeding by introducing genetic diversity for several traits simultaneously through co-current introgression.     

Effect of pollen source and pollen ploidy on endosperm formation and seed set in pseudogamous apomictic Paspalum notatum

 It is generally accepted that most angiosperms require an accurate balance between maternal and paternal genome contribution for endosperm development. The endosperm balance number (EBN) hypothesis postulates that each species has an effective number which must be in a 2:1 maternal to paternal ratio for normal endosperm development and seed formation. The aim of this work was to investigate the effect of different sources and ploidy levels of pollen donors on endosperm formation and seed production of aposporous tetraploid (2n=4×=40) Paspalum notatum. Hand-emasculated spikelets of an apomictic 4× plant were dusted with pollen of 2×, 4×, 5×, 6× and 8× races of the same species; 3× and 4× races of a phylogenetically closely related species, P. cromyorrhizon; and 2× and 4× races of P. simplex, a species of a different subgenus. Experiments including self-pollination as well as emasculation without pollination were conducted for controls. Results indicated that apomictic 4×P. notatum is a pseudogamous species with effective fertilization of the two unreduced (2n) polar nuclei by a reduced (n) sperm. Endosperm development and seed production occurred independently of the species or the ploidy level of the pollen donor. However, seed germination rates were significantly lower than in the self-pollinated control when the pollen donor was 3×P. cromyorrhizon or 2× and 4×P. simplex. Aposporous embryo sacs in Paspalum contribute to endosperm formation with two unreduced (2n) polar nuclei, while the male contribution is the same as in sexual plants (n). Since sexual Paspalum plants fit the EBN hypothesis, the EBN insensitivity observed in apomictic plants might be a prerequisite for the spread of pseudogamous apomixis. The EBN insensitivity could have arisen as an imprinting consequence of a high maternal genome contribution. Received: 20 February 1998 / Revision accepted: 21 October 1998     

Allelism of endosperm balance number (EBN) in Solanum acaule Bitt. and other wild potato species

The inheritance of endosperm balance number (EBN), a genetic, dose-dependent crossability system functioning in tuber-bearing Solanum (potato) species, was investigated for certain wild potato species having an EBN equal to one half of their ploidy. The EBN of Solanum acaule, a disomic 4(2EBN) South American species, was investigated by producing F₁ and F₂ hybrids with artificial 4x(2EBN) S. commersonii. This allowed assessment of recombination among the two genomes of disomic S. acaule and that of S. commersonii. When crossability of the hybrids with 1EBN, 2EBN and 4EBN standards was tested, no variation for EBN was detected. The apparent lack of recombination and segregation for EBN in these hybrids indicates that the genomes of S. acaule and S. commersonii carry EBN in a genetically-similar way. Combined with previous reports, these data indicate that the inheritance of EBN is similar in widely-separated taxa from South America and Mexico.     

Novel inter-series hybrids in Solanum, section Petota

Sexual hybrids between distantly related Solanum species can undergo endosperm failure, a post-zygotic barrier in inter-species hybridizations. This barrier is explained by the endosperm balance number (EBN) hypothesis, which states that parents must have corresponding EBNs for viable seed formation. Tests for inter-crossability were made involving the Mexican species Solanum pinnatisectum Dunal. (series Pinnatisecta, A^piA^pi, 1EBN), autotetraploids of this species, Solanum verrucosum Schlechtd. (series Tuberosa, AA, 2EBN), haploids (2x, 2EBN) of the South American S. tuberosum L. (series Tuberosa, A₁A₁A₂A₂, 4EBN), and F₂ haploid-species hybrids with South American species (AA, 2EBN) S. berthaultii Hawkes, S. sparsipilum (Bitter.) Juz. and Bukasov and S. chacoense Bitter. The development of hybrid endosperms was investigated for these combinations by confocal microscopy with regard to cell-division timing and tissue collapse. Novel sexual diploid (AA^pi) and triploid (AA^piA^pi) inter-series hybrids were generated from S. verrucosum × S. pinnatisectum crosses by using post-pollination applications of auxin. F₁ embryos were rescued in vitro. The hybrid status of recovered plants was verified by microsatellite marker analysis, and the ploidy was determined by chromosome counting. The application of phytohormones in inter-ploidy S. pinnatisectum × S. tuberosum crosses, however, did not delay endosperm collapse, and embryos were not formed. Other diploid, 1EBN species tested in remote hybridizations with Group Tuberosum were S. cardiophyllum Lindl., S. trifidum Correll, and S. tarnii Hawkes and Hjert., series Pinnatisecta, and S. bulbocastanum Dunal., series Bulbocastana. Based on the analysis of post-zygotic reproductive barriers among isolated species of section Petota, we propose strategies to overcome such incompatibilities.     

Dosage effects in the endosperm of diplosporous apomictic Tripsacum (Poaceae)

 Imprinting in the endosperm of angiosperms, a phenomena by which expression of alleles differs depending on whether they originate from the male or female parent, has been shown to explain most failure of interploidy or interspecific crosses in plants. Because of imprinting, seeds develop normally only if a specific dosage is represented in the endosperm, with the relative contributions of genomes in the ratio of two maternal doses to one paternal dose (2m:1p). In Tripsacum, a wild relative of maize, all polyploids reproduce through the diplosporous type of apomixis. Diplospory results from meiotic failure in megasporocytes that develop into eight-nucleate unreduced female gametophytes. The male gametophytes remain unaffected. Flow cytometry was used to determine ploidy levels in the endosperm of both apomictic and sexual Tripsacum accessions. In both cases, fertilization appeared to involve only one sperm nucleus. Therefore, endosperm of apomictic Tripsacum develops normally even though the ratio of genomic contributions deviates from the normal 2m:1p ratio. Ratios of 2:1, 4:1, 4:2, 8:1 and 8:2 were observed, depending on both the ploidy level of the parents and the mode of reproduction. Thus, specific dosage effects are seemingly not required for endosperm development in Tripsacum. These findings suggest that evolution of diplosporous apomixis might have been restricted to species with few or no imprinting requirements, and the findings have strong implications regarding the transfer of apomixis to sexually reproducing crops. Received: 17 February 1997 / Revision accepted: 7 July 1997     

Introgression of resistance to root-knot nematodes from wild Central American Solanum species into S. tuberosum ssp. tuberosum

 Crossing experiments were conducted to introduce resistance to the root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from various polyploid Central American Solanum spp. into the cultivated potato, S. tuberosum ssp. tuberosum. The most effort was put into producing tetraploid hybrids through inter-EBN (Endosperm Balance Number) crosses. From the crosses of tetraploid S. tuberosum (4 EBN) with tetraploid S. stoloniferum and S. fendleri (both 2 EBN), few seeds were derived that led to viable plants. In vitro culture of immature seeds also yielded several hybrid plants. From crosses of diploid S. tuberosum (2 EBN) with hexaploid S. hougasii (4 EBN) four hybrids were obtained through in vitro culture. Backcrosses were made with selected hybrids and a variable number of seeds was produced depending on the hybrid genotype. The successful introgression of resistance into backcross populations is shown. A scheme is presented for the introgression of traits at a tetraploid level from allotetraploid Solanum species into autotetraploid S. tuberosum through sexual crosses. The relevance of EBN for potato breeding is discussed. Received: 25 November 1996 / Accepted: 14 February 1997     

Ploidy level manipulations in potato through sexual hybridisation

There is no better use of sexual reproduction in regard to breeding and genetic research than the ploidy level manipulations possible in the potato and its relatives. Unique reproductive characteristics of tuber‐bearing Solanum species make possible: the production of gametes with unreduced chromosome number; the presence of an endosperm dosage system that regulates success of interploidy/interspecific crosses; the possibility to easily extract maternal haploids following crosses with S. phureja. This paper reviews results obtained in scaling genomic multiples up and down in potato, and relates these manipulations to breeding strategies for the genetic improvement of the cultivated potato. Several ploidy series have been developed, ranging from the monoploid to the hexaploid level. Cultivated tetraploids were scaled down to the diploid and monoploid level by haploidy. Scaling upward was achieved by sexual polyploidisation via 2n gametes that resulted in triploid, tetraploid, pentaploid, and hexaploid genotypes with a broad genetic base. Altogether, the success of ploidy level manipulations constitutes further proof that sexual polyploidisation played an important role in the polyploid evolution of Solanum species, and supports the idea that gene flow can be relatively easily accomplished through interploid and bridge crosses.     

Genetic control of Endosperm Balance Number (EBN): three additive loci in a threshold-like system

Summary The genetic control of Endosperm Balance Number (EBN) was investigated by a complete diallel of four exceptional diploid Solanum commersonii-S. chacoense hybrids (1 1/2 EBN) and backcrosses to their species parents, S. commersonii (1 EBN) and S. chacoense (2 EBN). Crosses in which the female parent had a higher EBN value than the male, S. chacoense (2 EBN)XF₁ (11/2 EBN) and F₁ (11/2 EBN)X S. commersonii (1 EBN), produced viable seed to aborted seed ratios of 11.1 and 11.3, respectively, and had average to small sized viable seed. Crosses in which the female parent had a lower EBN value than the male, S. commersonii (1 EBN)XF₁ (11/2 EBN) and F₁ (11/2 EBN)XS. chacoense (2 EBN), produced viable seed to aborted seed ratios of 1 7.9 and 1 6.7, respectively, and had average to large sized viable seeds. The results of these crosses appear to be consistent with the relative EBN values of the male and female parent. A model is proposed for the system regulating endosperm development. The assumptions of this model are: (1) three unlinked loci control the system; (2) the loci are homozygous within a species; (3) the genes have additive effects and are of equal strength within a species; (4) the genes within S. chacoense have twice the effect with respect to endosperm regulation as those within S. commersonii; and (5) a slight excess maternal dosage will produce the qualitative effect of small but viable seed. This model, in which quantitative genes operate in a dosage dependent system bears many similarities to classical, threshold-type genetic models.     

Allelism of Endosperm Balance Number (EBN) in Mexican tuber-bearing Solanum species

Summary Endosperm Balance Number (EBN) is a genetic, dose-dependent crossability system functioning in tuber-bearing Solanum species. Each species has been assigned 1EBN, 2EBN, or 4EBN. Species thus designated cross only within their EBN group. Doubling of chromosome number also doubles the EBN. The ploidy: EBN ratio is not consistent among Solanum species. Some diploids are 2EBN while others are 1EBN. Some tetraploids are 4EBN while others are 2EBN. Species from Mexico typically have EBNs that are one-half of their ploidy [e.g. 2x(1EBN), 4x(2EBN)]. Hybrids of Mexican species and a South American species, 2x(1EBN) S. Commersonii, and its 4x(2EBN) colchicine derivative were made and crossed to 1, 2, and 4EBN standard testers to determine the relationship of the genetic organization of EBN among and within these species. Diploid hybrids crossed only to 1EBN standard testers. Hybrids of 4x(2EBN) S. commersonii and 4x(2EBN) Mexican species crossed almost exclusively to 2EBN standard testers. Complex tetraploid hybrids involving S. commersonii, S. stenophyllidium (a Mexican diploid), and Mexican tetraploids of series Longipedicellata also crossed only to 2EBN testers. The apparent lack of recombination and segregation for EBN in these hybrids indicates that the genomes of the Mexican diploid and tetraploid species carry EBN in a way genetically similar to that of the South American species S. Commersonii.Cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service and the Wisconsin Experiment Station. Supported in part by the USDA/Cooperative States Research Service Competitive Grant No. 83-CRCR-1-1253     

The role of 2n gametes and endosperm balance number in the origin and evolution of polyploids in the tuber-bearing Solanums

Polyploidization has played a major role in the origin and evolution of polyploid species. In this article we outline the unique characteristics of 2n gametes and implications of their participation in the evolution of polyploid Solanum species. The genetic consequences of 2n gametes indicate that sexual polyploidization results in greater variability, fitness, and heterozygosity than does somatic doubling. Further, the mechanisms of 2n gamete formation and the frequency of 2n gamete-forming genes in present polyploids and their ancestral species provide additional evidence of their involvement. Equally important is the endosperm, via the endosperm balance number (EBN) incompatibility system, in complementing the role of 2n gametes. In fact, the EBN system acts as a screen for either 1n or 2n gametes, depending on the EBN and chromosome numbers of parental species. EBN in combination with 2n gametes maintains the ploidy integrity of diploid ancestral species, while providing the flexibility for either unilateral or bilateral sexual polyploidization.     

Genetic control of Endosperm Balance Number (EBN) in the Solanaceae based on trisomic and mutation analysis.

The genetic control of Endosperm Balance Number (EBN) was studied using trisomics and induced mutation. In order to induce and detect a change in a factor determining the EBN of the male, pollen from tetraploid Datura was irradiated and used to pollinate diploids. No triploids were produced in over 70 000 fertilizations. At least 70 would have been expected if the deletion of the function of a single gene could change the EBN. An attempt was made to find a particular chromosome that could alter the EBN of the female when it was present as the extra chromosome in 2x + 1 x 4x crosses. In tests of trisomics in both Datura and Solanum (potato) no chromosome could be found that changed the EBN. Therefore, it is concluded that more than one gene and more than one chromosome is involved in determining EBN. Key words : crossing barriers, endosperm, Datura, potato, speciation.     

An interpretation of genomic balance in seed formation in interspecific hybrids of Solanum

Summary To investigate the mechanisms of seed failure in intraspecific and interspecific crosses of Solanum two diploid, S. commersonii and Group Phureja, and one tetraploid species, S. acaule, species were crossed and the seeds were analyzed for embryo and endosperm development. Many seeds of certain crosses observed seven days after pollinations were found to contain abnormal embryos and degenerating endosperms. In some cases seeds contained an embryo with no endosperm, or an endosperm with no embryo. Other interspecific crosses which were predicted to fail actually produced seeds with normally developed embryos and endosperms. To further characterize the intra- and interspecific embryos and endosperms the nuclear DNA was measured. There are several ways to explain the ploidy levels of embryos and endosperms among the crosses, the occurrence of unreduced gametes in some cases and genomic instability in other cases. The latter resulted in chromosome loss at meiotic and mitotic divisions. Genomic balance in interspecific seeds is critical to both embryo and endosperm development.Scientific Journal Series Article No. 14636 of the Minnesota Experiment Station     

Mitochondrial DNA variation in cultivated and wild potato species (Solanum spp.).

The European cultivated potato, Solanum tuberosum subsp. tuberosum, has 6 related cultivated species and more than 200 wild relatives. In Solanum spp., studies of cytoplasmic organelles have been mainly confined to the plastid DNA composition of cultivated and wild species. In this study, 53 genotypes of 30 potato species belonging to the subsections Estolonifera and Potatoe, 2 tomato species, and a black nightshade genotype were examined using PCR markers to evaluate mitochondrial DNA diversity and assess whether mtDNA variability was correlated with series classification, geographical origin, ploidy, and endosperm balance number (EBN). The markers used revealed interspecific mtDNA variability in Solanum spp. and identified 13 different haplotypes. Intraspecific variability was also observed in a few species and genomic regions. Cluster analysis allowed arrangement of the 13 haplotypes into 7 subgroups, and statistical association tests showed significant relationships between mitochondrial patterns detected by molecular analysis and ploidy, EBN, and geographical origin. On the whole, the evolutionary patterns for the genomic regions analyzed reflected the species relationships established on the basis of morphological and molecular (nuclear and plastidial DNA) data. The mtDNA variability shown is also important for better characterization of genetic resources for potato breeding.     

Interploid St. Augustinegrass [<Emphasis Type="Italic">Stenotaphrum secundatum</Emphasis> (Walt.) Kuntze] hybrids recovered by embryo rescue

St. Augustinegrass is one of the most important warm season turfgrasses in the southern United States because of its shade tolerance. Most cultivars are diploids (2n = 2x = 18) and are susceptible to various diseases and insects. Polyploid cultivars in the species have some resistance to pests, but most lack cold tolerance. In this study, eight polyploid genotypes were crossed with six diploid cultivars to transfer pest resistance to the diploids. Because interploid crosses often result in aborted seed, it was necessary to use in vitro techniques. Using embryo rescue, 268 plants were recovered from 2,463 emasculated and pollinated florets (10.88% crossability). Because of the heterogeneous nature of the species, these purported hybrids could not be verified by phenotype. DNA markers were used for hybrid identification. A subset of 25 plants from crosses between the aneuploid cultivar Floratam (2n = 4x = 32) and five diploid cultivars were analyzed using 144 expressed sequence tags–simple sequence repeats (EST-SSRs) developed from buffelgrass cDNA sequence data. Chi-square tests for paternal-specific markers revealed that all analyzed progeny were true F₁ hybrids and none originated from self-fertilization or unintended outcrossing. In addition to identifying DNA polymorphism, the EST-SSRs revealed that genetic variation exists among all analyzed cultivars and is not partitioned between ploidy levels. The findings demonstrate that these embryo rescue techniques will enable the entire spectrum of St. Augustinegrass genetic variation to be better used through the recovery of interploid hybrids.