2n Eggs Research Articles

Germplasm transfer from the wild tetraploid speciesSolanum acaule bitt. to the cultivated potato,S. tuberosum L. using 2N eggs

Solanum acaule Bitt. (acl1, 2n=4x=48 and 2n=6x=72) is a wild potato species in which resistance to adverse biotic and abiotic agents has been found. The tetraploid subspeciesacaule andaemulans cannot be easily crossed with the common potato,S. tuberosum L. Grouptuberosum (tbr, 2n=4x=48) because the development of the endosperm in the hybrid seed is abnormal. Since 4xacl behaves as a diploid in intra and interploidy crosses, it was postulated that the hybridization barrier withtbr could be overcome if the wild species produced 2n gametes. One hundred and ninety-seven plants of 4xacl (22 introductions) were screened for pollen of heterogeneous size; only two plants produced this type of pollen, with 3% and 5% of pollen in the large class, respectively. On the other hand, 427 plants (47 introductions) were screened for 2n eggs through controlled crosses withtbr. Forty-one plants (13 introductions) produced 219 berries of which 46 contained at least one plump seed. Seed from berries with low seed set gave rise to hexaploid hybrids possibly through the functioning of 2n eggs from 4xacl. A sample of these hybrids was successfully backcrossed as female parents totbr, giving rise to 5x BC1 progenies. These BC1 progenies could be easily back-crossed totbr, yielding tetraploid or near tetraploid BC2 plants.

Inheritance and Two Medchanisms of 2n Egg Fromation in 2x Potatoes

Inheritance and Two Medchanisms of 2n Egg Fromation in 2x Potatoes

Unreduced pollen frequencies versus hybrid production in diploid-tetraploid Vaccinium crosses

Reciprocal crosses were made between highbush Vaccinium corymbosum L. cultivars (4X) and high, medium and low frequency diplandroid pollen producing diploid clones of V. elliotti (Chapm.) and diploid pubescent forms of V. corymbosum. Pollination of 3,342 flowers produced 15 hybrids-11 tetraploids and 4 triploids. All V. elliottii hybrids were tetraploid while each successful diploid-tetraploid V. corymbosum cross produced at least one triploid. The ratio of tetraploid hybrids to 100 pollinations (H: 100P) was considered to be the best criterion for assessing crossability. Vaccinium elliottii produced a total of 8 4X hybrids while 2X V. corymbosum only produced 3. Vaccinium elliottii was more successful as a female parent and diploid V. corymbosum only produced hybrids as male parents. High frequency diplandroid clones showed a lower mean crossability as pollen parents (H: 100P=0.2) than medium frequency diplandroid clones (H: 100P=0.8). As predicted, 0% diplandroid clones wer unsuccessful as pollen parents. Unreduced pollen production was essential for tetraploid hybrid production in 4X×2X crosses. There appeared to be no consistent, inherent pattern between 2n pollen and 2n egg production in a clone nor was a tetraploid genotype effect on crossability consistently observed.

Application of interspecific sesquiploidy to introgression of PLRV resistance from non-tuber-bearing Solanum etuberosum to cultivated potato germplasm

Hybridization of synthetic allotetraploids of S. pinnatisectum with S. etuberosum (4x-EP) with S. acaule (2n = 4x = 48) resulted in two individuals that were highly fertile, in contrast to all other progenies. The unique individuals are hexaploids, 2n = 72, while the other progenies are tetraploids, 2n = 48. They are thought to be the products of a union between 2n eggs of S. acaule and normal 1n microspores of 4x-EP. The fertile hexaploids (designated 6x-AEP) produced abundant selfed seed and viable hybrids with cultivated diploid potato, S. phureja, when developing embryos were rescued from berries and cultured before transplanting to pot culture. The extreme variability in chromosome constitution of the hybrids with S. phureja and selfed progenies indicates that addition and substitution lines of etb chromosomes bearing genes of interest to breeders could easily be produced from this material. The production of sesquiploids, as the 6x-AEP hybrids are called, is discussed as a useful bridging step in the introduction of alien genes from genomes that share little homology with the cultivated genome.

Performance of tetraploid progeny derived from 2x–4x intersubspecific crosses in Dactylis glomerata L.

The Dactylis glomerata L. species complex is a genetically diverse group of naturally occurring diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), and hexaploid (2n = 6x = 42) subspecies. The diploid subspecies tend to be geographically isolated and genetically distinct. Each subspecies probably possesses alleles for adaptation to specific environmental regimes. The objective of this research was to evaluate tetraploid progeny from 2x–4x intersubspecific crosses for forage yield and quality relative to their tetraploid parent. Progeny were developed by two stages of hybridization; intra- and inter-subspecific hybrids were generated from 2x–2x crosses, after which selected diploid progeny were mated, as females, to adapted tetraploid genotypes. From 14 2x–4x crosses, utilizing diploids known to produce 2n eggs, 59 tetraploid progeny were identified. These tetraploid progeny and their parents were vegetatively cloned and established in pots in two greenhouses. Forage was harvested from two replicates of each plant in each greenhouse for a total of six cuttings. Vigor, in vitro dry matter digestibility (IVDMD), and neutral detergent fiber (NDF) were determined. Parent–progeny regressions indicated evidence of heritable variation for vigor and IVDMD, but not for NDF. Favorable heterosis (relative to the tetraploid parent) on a family mean basis was significant only for IVDMD in one family. When selection among progeny within families was possible (in six families), favorable transgressive segregants were observed for all traits in at least one family and in a total of five families. The favorable performance of some tetraploid progeny, combined with the diverse nature of the species, indicate that improvements to cultivated orchard grass are possible. Selection at both the diploid and tetraploid levels will be necessary to achieve these goals.Key words: Dactylis glomerata L., gametic nonreduction, heterosis, heterozygosity, orchard grass, polysomic polyploids.

Genetic strategies to determine the mode of 2n egg formation in diploid potatoes

In this study, nineteen diploid potato clones (Solanum spp. 2n=2x=24) were identified as 2n egg producers on the basis of fruit set in 2x–4x crosses. The segregation of three genes mapped close to the centromere, Got-1 (1.1 cM), Pgm-2 (2.0 cM), and Sdh-1 (8.3 cM), were analyzed in the tetraploid offspring in these 2x–4x crosses to discriminate between First Division Restitution (FDR) and Second Division Restitution (SDR) modes of 2n egg formation. The co-dominant nature of these markers lead to more precise estimates of the recombinational frequencies as a result of completely classifying the segregating progenies. 2x–4x data revealed a predominance of SDR mechanisms occurring in 20 of the 21 families analyzed. With a SDR mode established, half-tetrad analysis (HTA) of four distal loci, 6-Pgdh-3, Mdh-1, Pgi-1, and Aps-1, revealed two SDR segregation patterns in some of the families. One pattern fit the expectations for the distal arm position. The gene-centromere map distances based upon SDR modes in the families following this pattern, were generally close to 4x−2x (FDR) estimates suggesting similar recombination rates between micro- and mega-sporogenesis. Heterozygosity transmission, on average, was 39.1%. In the other segregation pattern, in which the diploid parents were derived from S. chacoense PI 230580, higher than expected homozygosity levels were found in the female 2n gametophyte populations. A post-meiotic doubling of the reduced megaspore, which generates homozygous 2n eggs, is suggested to operate in three families. The common genetic background of the diploid clones suggested a heritable nature of this mechanism. Pooled data from these three deviant families calculated that 1.8% of the heterozygosity was transmitted to the tetraploid progeny.

The transfer of the synaptic gene (sy-2) from 1EBN Solanum commersonii Dun. to 2EBN germplasm

Crosses of a synaptic mutant (sy-2) of 2x(1EBN) S. commersonii Dun. with 2x(2EBN) S. chacoense Bitt. resulted in the production of 3x(2EBN) hybrids. Low levels of 2n egg production in these hybrids allowed subsequent crosses to be made with 4x(4EBN) cultivars, resulting in 5x(4EBN) S. commersonii-S. chacoense-Group Tuberosum hybrids. Seed set in these crosses averaged 0.22 seeds per fruit. Fifty-four unspotted seeds were produced in 1225 crosses of the 5x(4EBN) hybrids with 2x(2EBN) Group Phureja haploid extraction clones carrying dominant seed spot markers. These seeds yielded a total of 15 confirmed haploids having a diploid (2n=2x=24) or near diploid chromosome number. One haploid clone recovered displayed a synaptically abnormal phenotype similar to the original S. commersonii synaptic mutant. Preliminary test crosses of these haploids indicated that all haploids produced were 2EBN. This method for the transfer of alleles across EBN levels holds promise for the recovery of both qualitative and quantitative traits from 1EBN species. In these recovered, cross-compatible forms, clones with desired characteristics may be selected and easily incorporated into breeding programs.

Frequency and mechanisms of 2n egg formation in haploid tuberosum-wild species F1 hybrids

The frequency of 2n egg formation in 15 haploid Tuberosum-wild species F1 hybrids was determined by seed/fruit (S/F) following 2x × 4x crosses and cytological analyses; the mechanisms of 2n megaspore formation were detected by microscopic observations of megasporogenesis using a stainclearing technique for whole ovules. The variability among clones was from 0–102 in S/F, from 1.3 to 56.9 in percent 2n eggs, and from 2.7 to 38.3 in percent aborted ovules. There was a statistically significant correlation between S/F and cytologically determined frequency of 2n eggs. Both S/F and percent 2n eggs in particular clones varied significantly, and appeared to be affected by environment. The modifications of the meiotic process that resulted in 2n egg formation were: 1) omission of the 2nd meiotic division after a normal 1st division, 2) failure of cytokinesis at Telo II followed by nuclear restitution, and 3) synaptic abnormalities followed by nuclear restitution and a normal 2nd division. Modifications 1 and 2 are genetically equivalent to second division restitution and 3 to first division restitution. Some clones producing 2n eggs also formed a small percentage of 4n megaspores from aberrations during both meiotic divisions. The diverse mechanisms of 2n egg formation suggest the presence of independent genetic control.

The chromosomal location of a gene (msg) affecting megasporogenesis in durum wheat

An aneuploid durum wheat line (Triticum turgidum L. var. durum) having 13 chromosome pairs and 2 unpaired chromosomes at metaphase I of meiosis in pollen mother cells (i.e., monosomic for chromosomes 7A and 7D) was observed to produce some progeny plants with 2n = 40 chromosomes. These aneuploid (triploid) plants were usually weak and sterile. Triploid plants also occurred in the progeny of durum plants monosomic for chromosome 7A, or in progeny of plants that were mono-telodisomic or ditelomonotelosomic for chromosome 7Aq (13 II + 1 t II or 13 II + t II + t I) but not in the progeny of plants ditelomonotelosomic for chromosome 7Ap (13 II + t II + t 1). Therefore, there is a gene(s) on chromosome arm 7Ap that prevents the production of diploid (2n) egg cells in wheat. In the absence of 7Ap, a portion of the egg cells have 26 chromosomes, which when fertilized with a pollen nucleus with 14 chromosomes, produces progeny plants with 2n = 40 chromosomes. The data also indicated that chromosome arm 7Dp probably contains a second gene that is capable of preventing the production of triploid plants. Key words: Triticum turgidum L. var. durum, polyploidy, aneuploid, triploid, monosomic.

Evidence for the existence of endosperm balance number in the true clovers (Trifolium spp.)

The endosperm balance number (EBN) hypothesis was first advanced to explain results from interspecific crosses in Solanum and later in Impatiens. According to the EBN hypothesis, normal endosperm development following intra- or inter-specific crosses depends on having a ratio of two EBNs from the female to one EBN from the male in the endosperm tissue. EBNs may differ among related species. Successful hybrids can be obtained between species with the same EBN. The ploidy level of an individual species can be varied to modify its EBN, making it cross compatible with a species sharing its modified EBN. Interspecific crosses within Trifolium have been limited and difficult. Crosses reported in the literature, including evidence from our own study, suggest that EBN is operating in Trifolium and have been used to assign EBN numbers to some clover species. The use of 2n eggs enabled two species, differing in EBN, to be crossed. An understanding of the EBN mechanism that operates in Trifolium should make successful interspecific hybrids easier to obtain in the future.Key words: endosperm balance number, hybrids (interspecific), 2n gametes, Trifolium.

The implications of 2n egg gametes in nobilization and breeding of sugarcane

Nobilization in sugarcane is viewed from a genetic angle considering the significance of 2n gametes. The inbreeding coefficients resulting from 2n gametes, for first and second nobilizations are worked out. The implication of 2n gametes to sugarcane breeding are discussed.

Bilateral sexual tetraploidization in red clover

Twenty-four diploid red clover (Trifolium pratense L.) plants, from the cultivars 'Arlington', 'Florex', and 'Redman', and C760, a Wisconsin synthetic, were tested for 2n egg production by crossing them with tetraploid plants. Four plants were identified and selected as 2n egg producers. These plants were then crossed with a known producer of 2n pollen in an attempt to obtain tetraploid plants through bilateral sexual polyploidization. Thirteen percent of the seed obtained in one of the 2x–2x crosses were tetraploid. The female plant produced an estimated 0.14 2n eggs per 1000 flowers in the cross. In a second experiment, bilateral sexual polyploidization was attempted, using 14 plants which had not been previously tested for 2n egg production. Two of these plants produced one and two tetraploids each, representing 2n egg frequencies of 3.33 and 5.00 2n eggs per 1000 flowers, respectively. It seems likely that it will be possible to use bilateral sexual polyploidization to tetraploidize red clover germplasm in the future.Key words: tetraploid red clover, Trifolium pratense. 2n gametes.

Production of 2n Pollen in Red Clover1

The success of tetraploid red clover (Trifolium pratense L.) in Europe has stimulated interest in developing tetraploid red clover adapted to the United States. The use of 2n gametes to produce tetraploid alfalfa (Medicago sativa L.) and potato (Solanum tuberosum L.) from diploid germplasm suggests that 2n gametes may be an alternative to chemical treatment. Diploid plants that produce 2n eggs are known to occur in red clover, and plants that produce 2n pollen have been reported. The objectives of this study were to search for red clover plants producing 2n pollen, and to determine the cytological origin of such pollen. Six hundred plants, belonging to six different diploid (2n = 2x = 14) cultivars, were screened for 2n pollen production by examining dry pollen samples with the aid of a microscope. Eighteen plants (3%) produced at least 1% 2n pollen and were classifted as 2n pollen producers. Individual plants ranged from 1 to 84% in frequency of 2n pollen production. Pollen mother cell analyses revealed that 2n pollen resulted from parallel or tripolar spindles during anaphase II of microsporogenesis. This mechanism is genetically equivalent to a first division restitution (FDR) mode of gamete formation. One 2n pollen producer proved to be a synaptic mutant. Normally, synaptic mutants are male sterile, but fertility is restored by parallel spindles in this plant. Because the lack of homologous pairing limits genetic recombination, this plant can produce gametes with the same genetic constitution as the sporophyte. It is apparent from the results that 2n gametes may be useful in red clover polyploidization programs.

The use of Endosperm Balance Number and 2n gametes to transfer exotic germplasm in potato

A triploid hybrid (2n=3x=36) between a colchicine-induced 4x(2EBN) Solanum brevidens (a non-tuber-bearing species) and 2x(2EBN) S. chacoense (a tuber-bearing species) was used as a vehicle for germplasm transfer to S. tuberosum Group Tuberosum. The use of 2n gametes from the triploid allowed the unique opportunity for transferring exotic germplasm from Series Etuberosa to Gp. Tuberosum material. The triploid hybrid used had a pollen stainability of less than 0.1%. Observations of microsporogenesis revealed that metaphase I pairing configurations were primarily 12 bivalents and 12 univalents with occasional trivalents. Anaphase I separations were irregular, often with lagging univalents. Meiotic observations and pollen morphology suggest that the stainable pollen produced by the hybrid was 2n=3x=36. A single pentaploid hybrid (2n=5x=60) was produced by the fertilization of a rare 2n egg from the triploid with a normal male gamete from the clone 'Wis AG 231' (2n=4x=48). Limited crosses to other 1, 2 and 4EBN species and cultivars were unsuccessful. The pentaploid hybrid had a more regular meiosis than the triploid and dramatically improved pollen stainability (37% stainable pollen). Stylar blocks prevented estimates of male fertility in crosses. Female fertility in 47 crosses with nine cultivars averaged 19 seeds per fruit. Although S. brevidens is non-tuber-bearing, and the triploid produced only stolons, the pentaploid hybrid tuberized well under field conditions, despite being very late. Results suggest that the tuberization response is a dosage and/or threshold effect. This approach to the incorporation of 1EBN germplasm indicates the utility of the EBN concept coupled with 2n gametes. Further, it demonstrates a means for the introgression of 1EBN species genes into Gp. Tuberosum material.

Abnormal meiosis in alfalfa, Medicago sativa: cytology of 2N egg and 4N pollen formation

The cytological mechanism of 2n egg formation was studied in several diploid (2n = 2x = 16) and tetraploid (2n = 4x = 32) clones of cultivated alfalfa (Medicago sativa L.). The comparison of normal megasporogenesis with megasporogenesis that produced 2n eggs was made using an ovule clearing technique with methyl salicylate. Developmental sequences in the formation of n and 2n eggs were the same through anaphase II. Following anaphase II in 2n egg formation cytokinesis occurred only in the micropylar diad, not in the chalazal diad. Micropylar megaspores disintegrated leaving a functional unreduced megaspore of the second division restitution (SDR) type at the chalazal end. The two nuclei in the megaspore can fuse prior to the mitotic divisions or during the first two mitotic divisions. The SDR mechanism of 2n egg formation was confirmed in selected diploid clones by comparing half-tetrad analysis of 2n eggs with half-tetrad analysis of a known first division restitution (FDR) 2n pollen producer. During the study of 2n egg formation in tetraploid alfalfa, several diploid clones were found which produced octoploid (2n = 8x = 64) progeny from 4x-2x crosses. Pollen development in the diploids was normal through telophase II. Cytokinesis was absent, however, and the four telophase nuclei fused to produce a 4n pollen grain. The fusion of a 2n (2n = 4x = 32) egg and a 4n (4n = 4x = 32) pollen grain produced the octoploid progeny.

Breeding for apomixis in potato: Pursuing a utopian scheme

Apomictic propagation in potato holds a great promise for the new technology of growing potatoes from botanical seeds in stead of tubers. It is explained that apomixis results from the combination of abnormal meiosis, abnormal fertilization and parthenogenesis. An ideal scheme is given for growing potatoes from true seeds. This scheme remains utopian when autonomous apomixis is not available. Autonomous apomixis occurs when the reduction division is lacking (no gene recombination, no reduction of chromosome number) and the resulting 2n egg cell develops parthenogenetically without fertilization of the central nucleus. The first best approach is pseudogamous apomixis, which differs from autonomous apomixis in the necessity of fertilization of the central nucleus. Second best is the parthenogenetic development of 2n eggs, since these have originated through an abnormal type of reduction division. This type is characterized by the lack of formation of a reduction cell wall and fusion of the two spindles at metaphase II of meiosis (first division restitution). Apomixis in the narrow sense, either pseudogamous or autonomous, is defined. It has not been found in potato species or species hybrids. On the basis of our knowledge about characteristic features of existing apomictic species, about genetics and evolution of apomixis and about the occurrence of particular elements of apomixis in potato the possibility of introducing apomixis into potato from existing elements is discussed along with methods to induce missing elements. Some tentative approaches are suggested which in the long run may lead to the detection of potato genotypes with apomictic propagation.

Spontaneous interchanges in females of Drosophila melanogaster

Herein is described an attempts to establish chromosome pairing-interchange relationships in Drosophila melanogaster female. For this purpose, the formation of half-translocations was studied in XXY and XX females bearing compounds of the second pair of autosomes. With respect to XXY females, it was expected that the free Y chromosome would pair with these compounds and that half-translocations involving 2L would arise. In as much as compound chromosomes in XX females had no partner for pairing, the formation of half-translocations involving 2L was not expected.Half-translocations were registered in the F1 from crosses of XX and XXY females to b j pr cn/T(Y;2)C males. The cross was designed to permit the detection of very rarely occurring non-homologue interchanges.Offspring number was 335 in XX females and 550 in XXY females. The majority of offspring consisted of individuals arisen from the spontaneous restitution of compounds and the formation of 2n egg cells. Based on phenotype, the offspring of XX females contained 4 individuals with half-translocations involving 2L; there were 48 such flies among the offspring of XXY females. As confirmed by progeny analysis, 38 half-translocations occurred in XXY females and none in XX females. Of the 31 spontaneous interchanges in XXY females 28 were recorded between the Y and the left compound, one between the Y and the right compound, and one between the X and the left compound. Non-homologue interchanges were of oogonial origin judging by the fact that individuals with half-translocations arose in clusters. Unlike Y - left compound interchanges, the interchanges between autosomal compounds seem to be of meiotic origin.

Selection for 2n gametes and tuberization inSolanum chacoense

A breeding scheme is outlined which broadens the genetic base of the potato by incorporation of germplasm fromS. chacoense (Argentina) with Group Phureja and haploids of Group Tuberosum. Selection for adaptation to cultivation and production of 2n gametes is carried out at the diploid level, with the benefits of disomic ratios.S. chacoense provides increased genetic diversity, vegetative vigor and profuse flowering. About one-fifth of the 654 plants examined produced 2n eggs; clones with high 2n egg production may be obtained either by screening within particular introductions or by intercrossing known 2n egg producers. Only 16 of 1,212 plants screened produced 2n pollen —one by first division restitution. There is great variation among introductions and individuals for tuberization in the greenhouse. Selection for early tuberization in the field may be difficult due to extremely long stolons, but it would be facilitated by incorporation of the short stolon characteristic from the topiary mutant typeS. infundibuliforme. Male sterile Tuberosum haploids hybridize withS. chacoense to give male fertile interspecific F1 progeny, some of which produce 2n eggs or 2n pollen.

Polyploid evolution via 2n gametes

Polyploid evolution is evident in several important taxonomic series of the tuber-bearing Solanums. Polyploids can result from the functioning of 2n gametes following both intra and inter-ploidy matings. The bilateral and unilateral sexual polyploidizations (BSP and USP, respectively) are greatly facilitated by the existence of genetically determined 2n gametes. Many species, in series containing polyploids, have individuals with 2n gametes. The genetic basis of 2n pollen formation has been established in Phurejahaploid Tuberosum hybrids, and the genetic basis of 2n egg formation is being investigated in these hybrids andS. chacoense. Preliminary results indicate that relatively few genes may be involved. FDR and to a lesser extent SDR 2n gametes avoid the inbreeding in the polyploid that is maximal with somatic doubling. The vigor, fertility and competitive ability of a founder polyploid are dependent on minimizing inbreeding depression. Even more important, polyploids, particularly from FDR 2n gametes, inherit the beneficial epistatic interactions of the parent(s). Genetically determined 2n gametes make systematic sexual polyploidization a recurring phenomenon in two ways. For example, initial BSP of two diploids gives rise to a tetraploid, and thereafter USP ensures continuing contact between ploidy levels resulting in unidirectional introgression. Triploids can also be involved in polyploid evolution if they produce 2n gametes by FDR. Artificial hybridizations indicate that 2n gametes, by overcoming crossing barriers between various ploidy levels, provide the opportunity for continuous gene flow.

Genetic evidence for mode of 2n pollen formation andS-locus mapping in potatoes

Two diploid clones heterozygous for the geney (yellow tuber flesh), and producing diplandroids (2n pollen) by parallel spindles (ps) and premature cytokinesis 1 (pc1), respectively, were crossed to eight white-fleshed cultivers. Frequencies of nulliplex progeny obtained from these two sets of independent crosses indicate that there are two distinct genetic modes of 2n pollen formation. Diplandroids formed by ps are genetically equivalent to first division restitution (FDR) gametes and those formed by pcl are equivalent to second division restitution (SDR) gametes. Selfed seeds were obtained from 12 diploid clones which form 2n pollen by FDR and producing 2n eggs (diplogynoids) as well. Selfing four SDR diploids did not yield any seed although three of the four clones produced a fair number of 2n eggs. These results suggested that theS-locus is very close to the centromere.