Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation of DNA methylation and phenotypic traits following unilateral sexual polyploidization in <Emphasis Type="Italic">Medicago</Emphasis>

Sexual hybridization is an important generator of biodiversity and a powerful breeding tool. Hybridization can also overcome ploidy barriers when it involves 2n gametes, as in the case of unilateral sexual polyploidization (USP) that has been utilized in several crops, among which alfalfa. This research was aimed at gaining insights into the effects of USP on genome methylation and on phenotypic traits in alfalfa, an important forage species. The Methylation-Sensitive Amplified Polymorphism technique was used to estimate the cytosine methylation changes occurring in a tetraploid (2n = 4x = 32) USP progeny from crosses between a diploid Medicago sativa subsp. falcata genotype that produces 2n eggs and a cultivated tetraploid Medicago sativa subsp. sativa variety. De novo methylation or demethylation in the USP progeny were observed for 13% of the detected genomic sites, indicating that methylation changes can be relevant. USP plants showed larger surface area of the leaf epidermis cells than both parents, but this did not result in larger leaf size or higher plant biomass. They displayed significant higher ovule sterility than the tetraploid parent, but normal fertility was observed in crosses with unrelated male testers. We conclude that hybridization and sexual polyploidization resulted in novel variation in terms of remodeling of the methylation landscape as well as changes in phenotypic traits in alfalfa.     

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

Bilateral sexual polyploidization in potatoes

Summary Bilateral sexual tetraploidization was achieved by intercrossing diploid Phureja × haploid Tuberosum hybrids. Both parents contribute gametes with the somatic number of chromosomes (diplogynoids and diplandroids). More than 100 tetraploid progeny appeared in nine 2x–2x families. The tetraploids are generally more vigorous and significantly outyield their diploid full-sibs. The superiority of the tetraploids is interpreted on the basis of the mode of diplandroid (2n pollen) formation by first meiotic division restitution (FDR). Heterozygosis, epistasis and genetic diversity, if present in the parental diploid genotype, are largely maintained in the 2n gametes formed by FDR. and syngamy brings about more opportunities for heterotic responses in conjunction with polyploidization. Differences in tuber yield among tetraploid clones within families were found to be statistically significant (P <0.05). Thus, unlike somatic chromosome doubling, which can merely increase chromosome numbers, or somatic hybridization, which could in addition bring about heterosis, sexual polyploidization is also associated with genetic variability. The degree of inbreeding and the genotypic structure in the population of newly arisen tetraploids is discussed. The relationship among asexual reproduction, preservation of favorable nonadditive genetic effects by FDR. restoration of sexuality, and polyploidization is explored. The possible evolutionary significance of these factors is emphasized.     

Progenies of allotriploids of Oriental × Asiatic lilies (Lilium) examined by GISH analysis

With the aim of utilizing allotriploid (2n = 3x = 36) lily hybrids (Lilium) in introgression breeding, different types of crosses were made. First, using diploid Asiatic lilies (2n = 2x = 24), reciprocal crosses (3x − 2x and 2x − 3x) were made with allotriploid hybrids (AOA) obtained by backcrosses of F₁ Oriental × Asiatic hybrids (OA) to Asiatic cultivars (A). Secondly, the AOA allotriploids were crossed with allotetraploid (OAOA, 2n = 4x = 48), in 3x − 4x combination. Finally, the AOA allotriploids where crossed to 2n gamete producer F₁ OA hybrids (3x − 2x (2n)). Two types of triploids were used as parents in the different types of crosses, derived from: (a) mitotic polyploidization and (b) sexual polyploidization. Ploidy level of the progeny was determined by estimating the DNA values through flowcytometry as well as chromosome counting. The aneuploid progeny plants from 3x − 2x and reciprocal crosses had approximate diploid levels and in 3x − 4x crosses and 3x − 2x (2n) the progeny had approximate tetraploid levels. Balanced euploid gametes (x, 2x and 3x) were formed in the AOA genotypes. Recombinant chromosomes were found in the progenies of all crosses, except in the case of 2x − 3x crosses through genomic in situ hybridization (GISH) analyses. Recombinant chromosomes occurred in the F₁ OA hybrid when the triploid AOA hybrid was derived through sexual polyploidization, but not through mitotic polyploidization with two exceptions. Those recombinant chromosomes were transmitted to the progenies in variable frequencies.     

Analysis of meiotic behavior in selecting potential genitors among diploid and artificially induced tetraploid accessions of Brachiaria ruziziensis (Poaceae)

Sexuality is correlated with diploidy and apomixis with polyploidy in the Brachiaria genus. Brachiaria ruziziensis is a key species in Brachiaria breeding due to its obligate sexuality and intrinsic agronomic qualities. Interspecific crosses in the genus became feasible only when a few diploid accessions of B. ruziziensis were artificially tetraploidized and remained sexual. Hybridization has been done since, using natural tetraploid apomictic accessions of B. brizantha or B. decumbens as pollen donors. Twenty two accessions of B. ruziziensis from the Embrapa Beef Cattle germplasm collection (Campo Grande, MS, Brazil) were cytologically analyzed: 16 are natural diploids (2n = 2x = 18) and six are artificially induced tetraploids (2n = 4x = 36). The meiotic behavior in the 16 diploid accessions varied. The mean of meiotic abnormalities per accession ranged from zero to 24.46%. Meiotic behavior in the induced tetraploid accessions also varied with the mean of meiotic abnormalities ranging from 5.20% to 54.71%. The most common abnormalities observed in both the diploid and the tetraploid accessions, were those related to irregular chromosome segregation. In one tetraploid accession, with a high frequency of those, other irregularities involving chromosome orientation at metaphase plate and chromosome convergence to the poles, a meiotic mutation known as divergent spindle, were recorded. Meiotic behavior should be considered in selecting potential genitors for breeding.     

Relevance of unilateral and bilateral sexual polyploidization in relation to intergenomic recombination and introgression in <Emphasis Type="Italic">Lilium</Emphasis> species hybrids

Sexual polyploids were induced in diploid (2n = 2x = 24) interspecific F1 hybrids of Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) Lilium hybrids by backcrossing to Asiatic (AA) parents as well as by sib-mating of the F1 LA hybrids. A majority of the BC1 progenies were triploid and the progenies from sib-mating were tetraploid or near tetraploids. Genomic in situ hybridization (GISH) technique was applied to assess the intergenomic recombination in the BC1 populations of LA and OA hybrids obtained after unilateral sexual polyploidization. A total of 63 LA (LA × AA and AA × LA) and 53 OA hybrids were analysed. LA hybrids were originated through the functioning of 2n gametes either as 2n eggs or 2n pollen while those of OA hybrids originated through functional 2n pollen of diploid OA genotype. In both type of crosses, a majority of the progenies had originated through First Division Restitution (FDR) mechanism of functional 2n gamete either with or without a cross over. However, there were nine LA- and four OA-genotypes where Indeterminate Meiotic Restitution (IMR) was the mechanism of 2n gamete formation. Based on GISH, total amount of introgression of Longiflorum and Oriental genome into Asiatic genome was determined. Most of the BC progenies exhibited recombination and the amount of recombination was higher in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined cytologically in the 16 plants of sib-mated LA hybrids where both parents had contributed 2n gametes. Based on these results the nature of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation is discussed in the context of introgression and intergenomic recombination.     

The production of 2n pollen in rose

Based on the size differences found between haploid and diploid pollen produced by diploid and tetraploid rose cultivars, respectively, 2n pollen producers were identified in a population of 53 diploid hybrids from a cross between a dihaploid rose, derived from the haploidization of a tetraploid modern cultivar and the diploid species R. wichuraiana. Frequency of 2n pollen producers was estimated in 2002, 2003 and 2004. Highly variable frequencies were found i) within population; ii) during years of observation (between years and between different months in the same year). The variation of 2n pollen production could be related to environmental fluctuations. A cytological analysis of male meiosis was carried out in 10 hybrids randomly chosen. Among meiotic abnormalities leading to 2n pollen formation, triads (containing a 2n microspore at one pole and two n microspores at the other) resulting from abnormal spindle geometry were frequently observed. The mode of 2n pollen formation is genetically equivalent to a First Division Restitution (FDR) mechanism. FDR 2n pollen transmits a high percentage of the heterozygosity from the diploid parent – 2n pollen producer-to the tetraploid offspring.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Trifluralin-mediated polyploidization of <Emphasis Type="BoldItalic">Rosa chinensis minima</Emphasis> (Sims) Voss seedlings

Many diploid rose species and cultivars possess valuable traits that can be introgressed into modern tetraploid cultivars. Interspecific, interploidy crosses are possible, but triploid hybrids typically have limited fertility, hindering further breeding and selection. Tetraploidizing diploids before mating with tetraploids can alleviate fertility barriers. The efficiency of trifluralin was investigated for polyploidization of Rosa chinensis minima (2n = 2x = 14) seedlings. Treatments were trifluralin at 0.086% and 0.0086%, colchicine (0.5%), and distilled water and contained 2% dimethyl sulfoxide and a surfactant. Approximately 5 l of the treatment solution was applied to the apical meristem of seedlings (N = 337, 82–85 per treatment) in the process of cotyledon expansion. Guard cell length, pollen diameter, and root tip squashes of rooted cuttings were used to detect polyploidy in meristematic layer (L)I, LII, and LIII, respectively. Trifluralin (0.086%) was the most effective treatment for polyploidization (LI 20.2%, LII 12.9%, LIII 12.9%), followed by trifluralin (0.0086%) (LI 10.6%, LII 7.1%, LIII 4.7%) and colchicine (LI 2.4%, LII 0%, LIII 0%). Polyploidization consistently occurred from LI inward. Polyploids as a group had reduced pollen stainability and a lower leaflet length to width ratio than diploids. In addition, two diploid seedlings were identified which produce 2n pollen. Considerations in selecting germplasm and generating somatically-induced polyploids from seedlings versus clones for use in breeding are discussed.     

Relevance of sexual polyploidization for crop improvement – A review

Colchicine induced polyploids have not directly contributed for crop improvement in the past. On the other hand, the so-called natural polyploids, derived from the functioning of numerically unreduced(2n) gametes have been shown to be more relevant for crop improvement in many cases. Different types of cytological abnormalities during meiosis can give rise to 2n gametes and the genetic composition of these gametes is variable. Depending on the type meiotic abnormalities, various types of 2ngametes, such as first division restitution(FDR), second division restitution (SDR),indeterminate meiotic restitution (IMR) and post meiotic restitution (PMR) gametes,among others, have been described in recent years. For the improvement of autopolyploids such as potato, alfalfa,Vaccinium spp., and some of the fodder grasses, FDR gametes have been proved to be highly useful. However, the use of 2n gametes for the improvement of allopolyploid crops has received much less attention so far. Some of the investigations on allopolyploids, derived from Festuca-Lolium, Alstroemeria and Lilium species hybrids, have revealed that 2ngametes can be most useful for the introgression of alien genes and chromosomes into cultivars. An important feature of using sexual polyploidization in the case of allopolyploids is that introgression can be achieved through recombination due to genetic crossing-over between alien chromosomes as well as addition of alien chromosomes, which is extremely difficult or impossible to achieve in the case of colchicine induced allopolyploids. Because of the recent developments in the field of plant molecular biology, methods have become available for the analysis of 2ngametes and sexual polyploid progenies more accurately and to develop systematic breeding approaches. The methods include DNA in situ hybridization (GISH and FISH)and molecular mapping (AFLP, RFLP, RAPDs).In addition to providing basic information on the genetic and genome composition of the polyploid progenies, these methods can be potentially useful for a more efficient creation of desirable breeding material and cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

2n gametes in potato species and their function in sexual polyploidization

Summary The extent and pattern of polyploidy in the tuber-bearing Solanums varies among the many taxanomic series that have been identified in this subsection of Solanum. While several series appear to be entirely diploid, others exhibit a range of ploidy levels from 2x to 6x, and some contain only polyploid species.In many diploid, triploid and tetraploid species 2n gametes (gametes or gametophytes with the sporophytic chromosome number) have been detected. Both 2n eggs and 2n pollen occur. 2n gametes provide the opportunity for unilateral and bilateral sexual polyploidization. The genetic determination and consequences of sexual polyploidization strongly suggest that 2n gametes have been the major instrument in the polyploid evolution of the tuber-bearing Solanums. Somatic doubling of species and interspecific hybrids appears to be of very limited importance.New evidence for the occurrence of 2n eggs and 2n pollen in many species is reported, and data from the literature are added to illustrate the widespread distribution of 2n gametes throughout the subsection. A very high correlation is found between polyploidy and 2n gametes, and its significance is discussed. Proof is presented for the occurrence of alleles governing 2n pollen production in the cultivated tetraploids, providing additional evidence for the hypothesis that 2n gametes have been involved in their origin.Multiple unilateral and bilateral sexual polyploidizations are proposed for the origin of the cultivated tetraploids: this accounts for the large variability encountered in this group, which closely resembles that of the related diploids. Similar evolutionary pathways are hypothesized for the other polyploid complexes in the subsection.A scheme is proposed in which participation of both 2n and n gametes link together all ploidy levels in the tuber-bearing Solanums, thus overcoming the ploidy barriers and providing for gene flow throughout the sympatric species of the subsection.Paper No. 2093 from the Laboratory of Genetics. Research supported by the College of Agricultural and Life Sciences, a gift from Frito-Lay, Inc., and grants from NIH (GM 15422) and The International Potato Center. Plant materials, unpublished chromosome counts and technical help of IR-1 are gratefully acknowledged.     

The inheritance of a basal branching type in guar

Summary Eight different genotypes of the F₁ hybrids between Oriental × Asiatic lily (Lilium) hybrids (2n = 2x = 24) were treated with nitrous oxide (N₂O) gas under pressure for 24 and 48 hours. At the time of treatment, all plants possessed early meiotic stages in the anthers of the oldest flower buds. The mature flowers from treated plants were monitored for fertility through pollen germination in vitro as well as by using them in crosses with diploid Asiatic hybrids (2n = 2x = 24) both as male and female parents. In five out of the eight genotypes of OA hybrids there was evidence for the production of 2n pollen which germinated in vitro from either one or both treatments. The 2n pollen from three genotypes was successfully used in crosses. In two cases, the treated plants were successfully used as female parents which indicated the formation of 2n (or 2x) egg cells. From an analysis of 41 sexual polyploid progenies obtained from N₂O treated plants it was shown that they were all euploids consisting of 34 triploids (2n = 3x = 36) and seven tetraploids (2n = 4x = 48). A detailed cytological analysis of 12 progeny plants through genomic in situ hybridization (GISH) proved that N₂O had induced first division restitution gametes in most cases and in two cases they produced gametes through indeterminate meiotic restitution. There was evidence for intergenomic recombination in three cases.     

Unreduced 3x gamete formation of allotriploid hybrid derived from the cross of Primula denticulata (4x)?×?P. rosea (2x) as a causal factor for producing pentaploid hybrids in the backcross with pollen of tetraploid P. denticulata

A triploid hybrid, which was obtained from interspecific crosses between tetraploid Primula denticulata (2n = 4x = 44) and P. rosea (2n = 2x = 22), successfully produced 11 plants by backcrossing with pollen of tetraploid P. denticulata. Analysis of ploidy level using flow cytometry and chromosome counting in the 11 BC₁ plants revealed that all progeny had much larger DNA contents and chromosome number than both parents. In this triploid-tetraploid (3x–4x) crossing, progeny was predominantly true or near pentaploid presumably produced by the fertilization between true or near triploid female gamete produced from triploid hybrid and diploid pollen of tetraploid P. denticulata. These results suggest that unreduced (3x) or near triploid female gametes were partially produced by single step meiosis, either first-division restitution or second-division restitution process. The zygotes formed by the fertilization between true or near triploid egg produced by single step meiosis in triploid hybrid and diploid pollen produced by normal meiosis of tetraploid P. denticulata might be the only survivors in embryogenesis.     

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

To integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.     

Screening <Emphasis Type="Italic">A. ventricosa</Emphasis> populations for 2n gametes

Sexual polyploidization via the formation of 2n gametes has been acknowledged as the most significant evolutionary mode of polyploidization among plant species. The present study was conducted in order to determine whether 2n gametes are present in the C-genome diploid Avena ventricosa Bal. ex Coss., a species that contributed to the evolution of the cultivated hexaploid species (Avena sativa L). Individual plants belonging to four different Cypriot populations, were screened for pollen grain size variation with the aim to distinguish 2n gametes. Avena ventricosa ARI00-845 was identified to produce large pollen grains at a low percentage (1.21%). Subsequent analysis using flow cytometry confirmed the presence of 2n gametes in the pollen. Cytogenetic analyses of pollen mother cells revealed cells with twice the typical chromosome number at metaphase I (i.e., 28 chromosomes). We postulate that irregularities in cell wall formation preceding meiosis could have contributed to the mode of chromosome doubling.     

Bilateral polyploidization inDactylis glomerata L. subsp.lusitanica: occurrence,morphological and genetic characteristics of first polyploids

Summary InDactylis glomerata L. subsp.lusitanica, triploid and tetraploid plants were obtained by bilateral sexual polyploidization in crosses between diploid parents known to produce 2n gametes. The polyploid and diploid progeny were compared for allozyme diversity (allele number and heterozygosity), phenological (pollen fertility, inflorescence emergence date), cellular (stomatic cell size) and morphological characters (vegetative biomass, seed weight, total seed number per plant, panicle number and seed number per panicle length) in four contrasting growing conditions. Stomatal cell size and the total number of alleles were significantly lower in diploids than in polyploids which sometimes recovered more than two distinct alleles at a locus via 2n gametes possessing heterozygous loci. Pollen fertility was similar in diploids and tetraploids, but significantly lower in the triploids. Seed weight was significantly higher in polyploids, although the average number of seeds per panicle length and the total number of seeds was higher in the diploid sibs. No phenological differences occurred among ploidy levels whereas such differences were observed inDactylis in all the natural areas of sympatry between diploids and their related polyploids. Our results provide evidence for an instantaneous effect of polyploidization on certain traits and suggest that natural selection would act subsequently on several other traits to allow the establishment of new polyploids.     

AFLP fingerprinting in Medicago spp.: Its development and application in linkage mapping

Cultivated alfalfa (Medicago sativa L., 2n= 4x= 32) is one of the most important forage crops in temperate climates. The genus Medicago includes diploid species that are a valuable source of wild germplasm for studying the reproductive system of alfalfa and its abnormalities. A linkage map of an apomeiotic mutant of Medicago falcata (L.) Arcang. (2n= 2x= 16) that spanned 368.6 cM and included 29 amplified fragment length polymorphism (AFLP), 35 random amplified polymorphic DNA (RAPD) and three restriction fragment length polymorphism (RFLP) loci was constructed using a one-way pseudo-testcross mapping strategy. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. In general: (1) highly informative and reproducible RAPD and AFLP fingerprints were generated and several genome-specific primers selected; (2) of 67 marker loci mapped, 51 were arranged in 11 main linkage groups and eight additional couples of linked marker loci were detected; (3) mapping of an F₁ population theoretically allowed a better estimation of linkage distances since it avoided segregation distortion (x² analyses revealed segregation distortion in only 5.2% of marker loci); (4) the high frequency of unlinked marker loci obtained suggests that, in this alfalfa genotype, DNA markers are distributed throughout the genome. This type of genetic map should find application and prove useful in marker-assisted selection and map-based breeding programmes in meiotic mutants of alfalfa for which there is a lack of suitable genetic markers.     

Effect of selection and temperature stress on the production of 2n gametes in Lotus tenuis

Five Lotus tenuis genotypes showing 2n pollen production greater than 5% and producing 4x progenies in 4x(male sterile Lotus corniculatus) × 2x crosses were found in different natural populations. Meiotic analyses of these plants showed some second metaphases with disoriented spindles, i.e. parallel and tripolar spindles which resulted in diads and triads, respectively. Because useful genes for winter growth and resistance to powdery mildew from L. tenuis can be introgressed into L. corniculatus via 2n gametes, a programme of selection was started to increase the occurrence of 2n gamete production in L. tenuis. Controlled crosses were performed between genotypes producing 2n gametes and the progenies were checked for 2n pollen production and meiotic aberration, following the same methodologies as used for the parental plants. Although in some progeny plants the frequency of 2n gamete production increased remarkably, the overall progeny level of 2n gamete production remained unchanged. This suggested that different genes for meiotic aberration exist in natural populations and/or differential seed-setting ability exists between genotypes. Diallel crosses between selected genotypes confinned this hypothesis. Evaluation of 2n pollen production of clones grown under different temperature regimes (warm vs. temperate) showed that, in L. tenuis, the effects of genotype, temperature and genotype × temperature interaction are significant. In particular, high temperature increases the level of 2n gamete production in L. tenuis.