2n Eggs Research Articles

Characterization of Meiotic Mutants Producing 4N Pollen and 2N Eggs in the Medicago Sativa Complex

Characterization of Meiotic Mutants Producing 4N Pollen and 2N Eggs in the Medicago Sativa Complex

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Polymerase chain reaction (PCR), with single ten-base-long nucleotide primers, was used to amplify random regions of genomic DNA from eleven diploid meiotic mutants (2n egg, 2n pollen and jumbo pollen producers) of the Medicago sativa-coerulea-falcata complex. Electrophoretic patterns of the resulting random amplified polymorphic DNA (RAPD) fragments were evaluated to develop a graphical representation of amplification products from which conserved and individual-specific markers could be readily identified. Oligonucleotide primers differed significantly in their capacity of detecting polymorphism. Jumbo pollen (jp) mutants showed higher levels of similarity, as proportion of shared amplification products, than 2n pollen mutants. A diploid plant of alfalfa, tested for the occurrence of two mechanisms of unreduced egg formation (second division restitution and apomeiosis) was analyzed to obtain genome specific fingerprints. This PCR-based characterization could be used to determine whether the apomeiotic 2n eggs develop through parthenogenesis. The application of RAPD markers for germplasm evaluation and alfalfa improvement programs is also discussed.

Abnormal macrosporogenesis in five alfalfa (Medicago sativa) mutants producing 4n pollen

An analysis of micro- and macrosporogenesis in five diploid alfalfa mutants was carried out using a stain-clearing technique. All plants produced tetranucleated microspores and jumbo pollen due to the complete failure of the postmeiotic cytokinesis as well as bi- and trinucleated macrospores. The latter was due to the absence of cytokinesis after the first and second meiotic division of macrosporogenesis. Only one out of the five clones analyzed formed tetranucleated macrospores as a consequence of the total lack of cytokinesis after both meiotic divisions. The fusion of nuclei within binucleated macrospores resulted in 2n macrospores of the SDR type, recognizable on the basis of nucleolus dimension, confirming the ability of jumbo pollen (jp) mutants to produce 2n eggs at a high frequency. Nuclear fusion was also observed within tri- and tetranucleated macrospores. Although having the same genetic background, the five clones showed significant variability in the expression of abnormal cytokinesis during macrosporogenesis.

Use of half-tetrad analysis to discriminate between two types of 2n egg formation in a potato haploid

The ratio of the first division restitution (FDR) to second division restitution (SDR) 2n eggs was estimated in 4182t, a haploid (2n = 2x = 24) of Solanum tuberosum L. that produces 2n eggs by the two modes. The segregation of three genes previously mapped relative to their centromeres, Pgm-2 (2.0 cM), Mdh-1 (33.5 cM), and 6-Pgdh-3 (30.1 cM) was analyzed in the tetraploid offspring of a 2x × 4x cross. Based on the segregation of the Pgm-2 locus, 39.7% of the progeny originated from FDR 2n eggs and 60.3% from SDR. Segregation patterns of the two distal loci within the FDR-derived 4x subpopulation indicated that the gene–centromere recombination rate during megasporogenesis was significantly reduced for Mdh-1 when compared with a previous estimate during microsporogenesis. In the SDR-derived 4x subpopulation, the gene–centromere recombination rates for Mdh-1 and 6-Pgdh-3 were not significantly different from previous estimates. Tetraploid progeny generated from one 2x × 4x cross where the 2x parent produces 2n gametes by two modes can be used to make an unbiased comparison of the potential breeding value of FDR and SDR gametes.Key words: potato, megasporogenesis, first division restitution, second division restitution, isozyme.

Production of 2n Gametes in Diploid Subspecies of Dactylis glomerata L. 2. Occurrence and Frequency of 2n Eggs

Whereas polyploidy has been found in more than half the Gramineae, the occurrence of 2n gametes, a potentially major mechanism of polyploid production, has been rarely studied. In the present work, the frequency of 2n egg production in nine diploid (n = 2x = 14) subspecies of the polyploid complex Dactylis glomerata has been estimated by determining the ploidy level of the progeny in 98 2X-4X crosses. The 2n egg origin of the 4X offspring was verified using enzyme markers. The frequency of 2n egg production in each subspecies was estimated from the number of 4X offspring, the number of florets in the seed plant and the fertility of the subspecies. The ploidy level of the progeny was determined by chromosome counts or nuclear DNA quantification using flow cytometry. Plants producing 2n eggs were detected in 47% of the fertile crosses, involving eight subspecies. Variability was related to individuals and not to subspecies. Average 2n egg frequency was 0·49% for all crosses, and 1·53% for crosses where 2n egg production was detected. Individual frequencies did not exceed 3·5%, with the exception of 26% in one plant. Triploid block was higher and fertility, frequency of high 2n gamete producers and average 2n gamete frequency were lower in the 2X-4X crosses than in the opposite 4X-2X crosses involving the same plants, previously studied. Diplogynous and diplandrous gamete production were not correlated with each other. However, the widespread occurrence of plants producing both kinds of 2n gametes in almost all the studied subspecies, as well as the presence of several individuals able to produce high rates of 2n eggs or 2n pollen, support the hypothesis that tetraploids can be formed anywhere and anytime by sexual polyploidization in diploid populations of Dactylis glomerata L.

Phenotypic recurrent selection for 2n pollen and 2n egg production in diploid alfalfa

Phenotypic recurrent selection for 2n pollen and 2n egg production in diploid alfalfa

Gene-centromere mapping in potato by half-tetrad analysis: map distances of H1, Rx, and Ry and their possible use for ascertaining the mode of 2n-pollen formation

Diploids from the tetraploid potato varieties 'Alcmaria' and 'Pansta' and from the tetraploid CPRO genotypes Y66-13-610 and Y66-13-636 were used in half-tetrad analyses to estimate the gene-centromere map distances of the genes Rx, Ry, and H1. Employing tetraploid progeny from 2x (second division restitution) – 4x testcrosses the gene-centromere map distance of H1, conferring resistance to pathotype Ro1 of Globodera rostochiensis, was estimated to be 16.3 centimorgans (cM). For Rχ, conferring extreme resistance to potato virus X (PVX), a map distance of 33.9 cM was estimated. The gene Ry, conferring extreme resistance to potato virus Y (PVY), was estimated to be located 14.2 cM from the centromere. Using the estimated map distance for Rx, it was attempted to determine the mode of 2n-pollen formation in four diploid interspecific hybrids, including the species Solanum tuberosum, Solanum chacoense, Solanum yungasense, and Solanum phureja, by half-tetrad analysis in tetraploid progeny from 4x-2x testcrosses. The mean frequency of 8.7% nulliplex plants for Rx was outside the range of the 95% confidence intervals, for both first division restitution and second division restitution 2n pollen.Key words: nematode resistance, potato virus X resistance, potato virus Y resistance, 2n eggs, gene-centromere mapping, Solanum.

Potato germplasm enhancement with disomic tetraploid Solanum acaule. I. Efficiency of introgression

The wild potato species Solanum acaule (acl) was used as a model of a disomic tetraploid Solanum species to develop systematic methods of germplasm enhancement for disomic tetraploid species. The objective was to develop a genetically efficient method to overcome the inherent technical problems encountered in the utilization of disomic tetraploid wild species. Accessions of acl were selected from CIP's wild germplasm collection and from the collection of University of Birmingham, with emphasis on genetic attributes such as PLRV resistance and (or) PSTV resistance. Four methods were tested: (i) triploids from crosses between 4x acl × 2x potato were selected for 2n gametes production and were crossed to tetraploids or to diploids with 2n egg production; (ii) axillary buds of triploid hybrids were treated with colchicine to double chromosome numbers to generate hexaploids; (iii) in vitro chromosome doubling to obtain hexaploids from triploid hybrids; and furthermore (iv) the selected acl clones were directly crossed to tetraploid potatoes followed by a combination of second compatible pollinations with IvP 35 and subsequent embryo rescue. The combination of second compatible pollination and embryo rescue was found to be the most genetically efficient method for the utilization of the valuable genetic attributes of acl.Key words: inter-EBN crosses, ploidy manipulation, polyploid, potato breeding, wild species

Cytological Analysis and Fertility of Solanum commersonii Dun.*Solanum gourlayi Haw. Triploid Hybrids.

Meiotic analysis, pairing affinity and fertility studies were carried out in Solanum commersonii Dun. (cmm, 2n=2x=24) by Solanum gourlayi Haw. (grl, 2n=2x=24) triploid hybrids, obtained through the functioning of 2n eggs of cmm. Mean arm pairing frequency (“C”) and relative affinity (“X”) were calculated according to Alonso and Kimber (1981). The “C” value was high but the “X” value was intermediate. Pollen stainability for three triploid hybrids was 31.7, 29.5 and 2.5 respectively. Crosses between the cmm×grl triploids, used as females, and various species of Solanum with different ploidy were performed. Hybrids with 24 to 31 chromosomes were obtained from these crosses. The results of this study give and indication that the cmm and grl genomes are highly homologous. Therefore a high degree of gene exchange between the genomes of these species is expected to occurr in meiosis. The feasibility of utilizing grl as a “bridge” species, for transfering important genes of cmm to the cultivated potato, Solanum tuberosum L. are discussed.

Development and performance of balck sigatoka-resistant tetraploid hybrids of plantain (Musa spp., AAB group)

A strategy to control the black sigatoka disease (Mycosphaerella fijiensis Morelet) of plantain (Musa ssp., AAB grou) in Africa, targeting the incorporation of durable host plant resistance, was initiated at the International Institute of Tropical Agriculture (IITA). The commonly accepted intractability of plantain to genetic improvement has been challenged by the identification of 37 different, seed-fertile plantain cultivars and by the production of 250 hybrids in four years of breeding work. Twenty tetraploid hybrids have been selected for their increased black sigatoka resistance, high yields, large parthenocarpic fruits and improved ratooning. ‘Calcutta 4’ (Musa acuminata spp. burmannicoides) was the diploid male parent of 17 of the selected hybrids, which indicates that the inferior bunch characteristics of this wild banana were generally not transmitted to its tetraploid progenies. Conversely, the 4x progeny of plantain readily expressed black sigatoka resistance when crossed with ‘Calcutta 4’. Progenies of the triploid plantain cvs. ‘Obino l'Ewai’ and ‘Bobby Tannap’ differed in their black sigatoka breeding values, the former producing larger numbers of promising hybrids. Tetraploids obtained from crosses of plantain cultivars with the homozygous ‘Calcutta 4’ displayed variation in black sigatoka reaction, qualitative morphological traits and growth and yield parameters, suggesting the occurrence of segregation and recombination during the modified megasporogenesis leading to the formation of 2n eggs in the triploid female plantain parents. The variation in black sigatoka reaction among the tetraploid progenies of plantain suggests that resistance could be regulated by recessive, additive genes.

Occurrence and mechanisms of 2n egg formation in 2x potato

The occurrence of 2n eggs in 381 haploids from six tetraploid parents and in 127 plants representing five diploid wild species was detected using 2x × 4x crosses. Sixty-two percent of the haploids and 24% of the wild-species plants produced 2n eggs. Twenty-six haploids and 17 species plants that gave high seed set in 2x × 4x crosses were examined cytologically to determine the frequency and mechanisms of 2n egg formation. There was significant variation in the frequency of 2n eggs among haploids (7–57%) and among species plants (4.9–57.3%). Five mechanisms of 2n egg formation were identified: synaptic variant (genetically first division restitution); delayed meiotic division (first division restitution and second division restitution); omission of the second division (the prevalent mechanism, second division restitution); irregular anaphase II (second division restitution); and failure of second cytokinesis (second division restitution). 2n eggs can be formed by more than one mechanism within a clone. The occurrence of 2n eggs in wild species and the higher frequency of 2n eggs in haploids than in wild species indicate that sexual polyploidization has been involved in the origin and evolution of polyploid series in potato. The high frequency of 2n eggs in both haploids and diploid wild species allows generation of haploid-species hybrids that produce 2n eggs. These hybrids can then be used in the 2x × 4x and 2x × 2x breeding schemes.Key words: haploids, wild species, 2n gametes, first division restitution, second division restitution.

Potato haploid performance in 2x × 4x crosses

Nineteen haploids (2n = 2x = 24) extracted from four tetraploids ofSolanum tuberosum (2n = 4x = 48) with high 2n egg frequencies and known mode of 2n egg formation were evaluated via 4x progeny from 2x × 4x crosses (haploid × cultivar) for total tuber yield, tuber appearance and tuber set. Ninety-eight, 4x families (15-20 genotypes each) were planted in the randomized complete block design with two replications at two locations. Analyses of variance were conducted for all haploids over three common testers, and each of the four groups of haploids individually over a variable number of testers. The performance of haploids, based on general combining ability effects, was consistent for all traits, when tested with either three or more testers. Diploid clones can be evaluated in 2x × 4x crosses using three unrelated adapted 4x testers. Nonorthogonal comparisons indicated no significant differences for all traits among four groups of haploids, and between modes of 2n egg formation. Significant variation within each group of haploids indicates that selection at the haploid level should be carried out among haploids from either the same or different cultivars. Superior haploids can be utilized in 2x × 4x and 2x × 2x breeding schemes to generate 4x clones.

Breeding for 2n egg production in haploid × species 2x potato hybrids

The identification of diploid(2x), Tuberosum haploid × wild species hybrids (HS) which produce high frequencies of 2n eggs and have good tuber appearance is important for obtaining 4x progeny from 2x × 2x crosses in potato. More than 1700 HS clones were screened for the occurrence and frequency of 2n eggs through 2x × 4x crosses during two pollination periods. The resulting seed/fruit provides an estimate of 2n egg frequency. About 650 HS clones produced 2n eggs; 63 had 10–114 seeds/fruit; and 27 were selected for good tuber type. There was a large variation in seed set after 2x × 4x crosses, both within and between clones. Comparison of fruit and seed set between the two pollination periods using the same 595 clones indicated that environmental factor significandy affected 2n egg frequency. The mode of 2n egg formation was mainly due to omission of the second meiotic division controlled by a recessive gene (os). The gene frequency foros varied from 0.28 to 0.76 among six, 2x taxa. Both additive and dominance variance were significant for seed set after 2x × 4x crosses. Narrow sense heritability was estimated as 0.24. The average degree of dominance was equal to 1.66 which could indicate overdominance. Alternatively, this could be due to linkage disequilibrium,i.e. pseudooverdominance. However, dominance variance was greater than the additive variance indicating recurrent selection can be useful for improving the frequency of 2n egg production in the 2x population. The selected individuals, based on their phenotypic performance, should be progeny tested to identify the best parents for use in the next cycle of recombination.

Yield and tuber characteristics of 4x progeny from 2x×2x crosses

Unrelated, unselected 2x haploid-species hybrids that produce 2n eggs, by second division restitution and 2n pollen due to first division restitution were intercrossed to generate 4x progeny. The 19, 4x families were compared with seven cultivars for tuber yield, tuber appearance and tuber set. The mean yield of the 4x families was 30% higher than the mean yield of the cultivars. The best five 4x families outyielded the best five cultivars by 57% and 69% at two locations. Cultivars were superior for tuber appearance and tuber set. The high yields of the 4x families are due to genetic diversity of the 2x parents and their ability to transmit that diversity to the 4x progeny utilizing 2n gametes. The potential of the 2x×2x breeding method is dependent on developing 2x hybrid clones that form 2n eggs and possess desirable tuber types; clones producing 2n pollen and good tuber quality are available.

A new method of producing 4x hybrid true potato seed

New haploid-wild species hybrids (H-S) with desirable tuber type and 2n gamete production have been identified. They provide the opportunity for the production of inexpensive, 4x hybrid TPS from 2x×2x crosses. A proposed system to utilize plants producing either 2n eggs or 2n pollen is described. One H-S hybrid (female parent) would be selected for male fertility, self-incompatibility, high frequency of 2n eggs and no 2n pollen; an unrelated H-S (male parent) for male fertility and high 2n pollen production. In addition both parents would be selected for profuse flowering, attractiveness to bumblebees and other desirable characteristics. The male and female parents would be planted in alternate hills with bumblebees doing the pollinations. The elimination of emasculation, pollen collection, and hand pollination could reduce 4x hybrid TPS costs by more than 50 percent.

Phenotypic recurrent selection for 2n pollen and 2n egg production in diploid alfalfa

Cultivated alfalfa (Medicago sativa L.) is a polysomic polyploid with tetrasomic inheritance. In this crop the hybridization of gametes with the somatic chromosome number (2n gametes) produced by diploid hybrids is a valuable method to maximize heterozygosity. For this purpose it is necessary to obtain diploids which produce high frequencies of 2n gametes. The results of the present paper indicate that phenotypic recurrent selection was efficient in increasing the production of male and female 2n gametes in diploid alfalfa. Two distinct selections were carried out, one for 2n pollen and the other for 2n egg production and two cycles of phenotypic recurrent selection were performed. The percentage of plants producing 2n gametes and the frequency of 2n gametes per plant were improved by selection. Realized heritabilities over the two cycles of recurrent selection were 39% and 60% for 2n pollen and 2n egg production, respectively; on the whole, the responses to the selection indicate that both male and female 2n gamete production are controlled by major and minor genes.

Identification and frequency of tetraploid progeny from 2x–4x and 4x–2x crosses in Dactylis

The utilization of diploid germ plasm in the improvement of tetraploid orchard grass (Dactylis glomerata L. ssp. glomerata Hayek) requires some means of polyploidization. Studies in several different crop species have shown that sexual polyploidization has the advantage of transmitting a large proportion of heterozygosity from the parent to the offspring. In tetrasomic tetraploids, maximum heterozygosity is a necessary requirement for maximum heterosis. The objective of this research was to investigate the occurrence of 2n eggs and 2n pollen in several sources of wild diploid germ plasm, by quantifying the frequency of 4x genotypes among the progeny of interploidy crosses. Pollen diameter was determined in 110 diploid genotypes. These genotypes were then ranked for five descriptive statistics based on diameter (maximum, 75th percentile, median, arithmetic mean, mode). The eight top-ranking genotypes in four of the five categories were crossed to male-sterile 4x seed parents. Randomly selected diploid genotypes were also crossed as seed parents to 4x genotypes. Tetraploid progeny were recovered from both types of crosses. Of 57 2x genotypes, 15 produced 2n eggs. Of the 8 large pollen diameter genotypes, 6 produced 4x progeny in relative frequencies of 3–100%. The success of recovery was dependent on the genotype of the pollen parent as well as the seed parent. Of the 6 genotypes producing 2n pollen, 2 also produced 2n eggs, resulting in the recovery of tetraploids from selfed progeny.Key words: germ-plasm utilization, interploidy crosses, forage grasses, plant introductions.

Use of Ipomoea trifida (HBK.) G. Don germ plasm for sweet-potato improvement. 2. Fertility of synthetic hexaploids and triploids with 2n gametes of I. trifida, and their interspecific crossability with sweet potato

Twenty-two synthetic hexaploids and 33 triploids with 2n pollen production of Ipomoea trifida were used in crosses to estimate their male and female fertility and their crossability with sweet potato, Ipomoea batatas (L.) Lam. Several important conclusions were drawn. (i) The fertility of pollen of the triploid plants was confirmed, which suggested they are truly functional 2n pollen. (ii) The average male fertility of hexaploid plants was higher than that of triploid with 2n pollen production plants. (iii) 2n egg production was observed in several triploid genotypes, (iv) From a polycross, 3079 seeds with normal shapes were produced. These represent a population of hexaploid I. trifida with a wide genetic base, and they may be important material for sweet-potato improvement, (v) A total of 3275 seeds were obtained from crosses with six sweet-potato female parents, of which only 60 germinated, indicating the existence of an interspecific barrier. The implications of 2n gametes for breeding and evolution of sweet potato are also discussed.Key words: Ipomoea batatas, Ipomoea trifida, 2n gametes, sexual polyploidization, triploid.

Cytological evidences of SDR-FDR mixture in the formation of 2n eggs in a potato diploid clone

Macrosporogenesis and microsporogenesis were investigated in a diploid S. tuberosum x S. chacoense potato hybrid, characterized by more than 50% 2n egg formation. Fifty-five percent of dyad formation of 2n macrospores is ascribed to two meiotic abnormalities: omission of the second meiotic division, occurring at a frequency of 38%, and irregular spindle axis orientation at metaphase I at a frequency of 16%: These abnormalities give origin to a mixture of 2n eggs, composed of mostly second division restitution (SDR) and a small portion of first division restitution (FDR). Microsporogenesis showed rare dyads of 2n microspores depending on parallel spindles observed in anaphase II.

Backcrossing Tetraploidy into Diploid Medicago Falcata L. Using 2n Eggs

Chromosome manipulation may be useful in germplasm enhancement where wild and cultivated forms are at different ploidy levels. In this study, tetraploidy was backcrossed into a diploid Medicago falcata L. synthetic (2x WISFAL‐1) in a series of five 2x‐4x backcrosses using 2x WISFAL‐1 as the recurrent parent and the 4x M. sativa L. as the nonrecurrent parent. Since nearly all 2x M. falcata plants produced a low background frequency of 2n eggs, it was necessary to pollinate only =500 2x flowers with 4x pollen to obtain one to five tetraploid hybrids per 2x plant. The new tetraploid form of M. falcata, designated 4x WISFAL‐1, contains 98% M. falcata germplasm and 2% M. sativa. The broad‐based synthetic 4x WISFAL‐1 has all the characteristics of 2x WISFAL‐1 except that it has larger vegetative and reproductive organs, and crosses directly with cultivated alfalfa. The 4x WISFAL‐1 can be used for gene transfer from M. falcata to cultivated alfalfa. The yellow flower color of 4x WISFAL‐1 also can be used as a codominant flower‐color marker at the tetraploid level. Two narrow‐based tetraploid populations were developed by backcrossing to PI 231731 and PI 258754, respectively, using 4x WISFAL‐1 as the nonrecurrent parent. Thus, 4x WISFAL‐1 is also useful in germplasm enhancement of individual diploid Pis by elevating them to the tetraploid level for direct crossing with cultivated M. sativa.