Meiotic and Isozymic Analyses of Tall Fescue × Giant Fescue Hybrids and Amphiploids1

The meiotic behavior of three tall fescue (Festuca arundinacea, 2n = 6x = 42) genotypes, giant fescue (F. gigantea, 2n = 6x = 42), and their reciprocal F₁ hybrids and C₁, amphiploids was evaluated to determine the parental genomic relationships. Isozyme banding patterns were used to confirm the parental identity of the hybrids and amphiploids. At meta-phase I, the parents had predominantly bivalent pairing. The hybrids had an average of 9.51 I, 16.02 II, 0.12 III, 0.02 IV, and the amphiploids had 2.17 I, 38.82 II, 0.60 III, 0.58 IV, 0.01 V—VIII. The prevalence of bivalent pairing in both hybrids and amphiploids suggested a homoeologous relationship between the six genomes, with four of the six being more closely related. Bivalent pairing in the amphiploids indicated genetic regulation of chromosome pairing. Zymograms were obtained for acid phosphatase (ACPH), alcohol dehydrogenase (ADH), glutamate oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD) and phosphoglucoisomerase (PGI). The three tall fescue and giant fescue parents had different zymograms for ACPH, MDH, 6-PGD and PGI; thus, the tall fescue parents of the hybrids and amphiploids could be determined based on the banding patterns of these four enzymes. Phenotypes were determined for ACPH-1, PGI-2 and 6-PGD-1. ACPH-1 may be used to follow the introgression of giant fescue chromatin into a certain tall fescue genotype.     

First in vivo assessment of feeding value of Festulolium hybrids derived from Festuca arundinacea var. glaucescens and selection for palatability

This paper reports on the first results of feeding value experiments, assessed with sheep, of hybrids between tetraploid Italian ryegrass (Lolium multiflorum, 2n = 4×= 28) and a progenitor of tall fescue, Festuca arundinacea var. glaucescens (2n = 4×= 28). Three hybrid populations resulting from either high, low or no selection for palatability were compared with a variety of Italian ryegrass as control and two varieties of the cultivated hexaploid (2n = 6×= 42) tall fescue, one of which had been selected for high palatability. On average, the hybrid populations were found to be as palatable as the improved tall fescue with voluntary intake (VI), in vivo digestibility of organic matter (DOM), and net energy expressed in fodder units for milk, similar to the best palatable tall fescue and to Italian ryegrass. However, selection for improved palatability among hybrids did not result in any further improvement of other traits, except VI in the hybrid selected for high palatability which significantly exceeded VI of Italian ryegrass. In conclusion, intergeneric hybridization between L. multiflorum and F. arundinacea var. glaucescens led to an immediate increase of feeding value compared with selection for palatability in tall fescue. However, to improve feeding value of hybrids further, similar selection for high palatability appeared questionable and possibly detrimental for other agronomic traits because of complicated inheritance in tetraploid L. multiflorum×F. glaucescens hybrids.     

Cytogenetic and isozymic characterization of anther-panicle culture derived tall fescue aneuploids

Summary Chromosome associations of 44 tall fescue (Festuca arundinacea Schreb., 2n=6x=42) plants derived from anther-panicle culture of Kentucky 31 were evaluated to determine if new genetic stocks could be identified. Seventeen of the plants were euploids (21 II), two were monosomic (20II+1I), 22 were double monosomic (19II+2I) and three were triple monosomic (18II+3I). Zymograms were obtained for phosphoglucoisomerase (PGI), glutamate oxaloacetate transaminase (GOT), acid phosphatase (ACPH), malate dehydrogenase (MDH) and 6-phosphogluconate dehydrogenase (6-PGD). The zymograms were identical for GOT, ACPH, and 6-PGD but different patterns were found for MDH and PGI in some of the double monosomics and euploids from a different piece of callus than the majority of the plants.Contribution of the USDA-ARS in cooperation with the Kentucky Agricultural Experiment Station. Scientific Journal Series No. 86-3-61.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

Estimation of introgression rate of a fescue isozymic marker into tetraploid Italian ryegrass at early generations of backcross

The potential for introgression of Festuca glaucescens into tetraploid ryegrass Lolium multiflorum was assessed using a simplex homeoallele at the phosphoglucoisomerase isoenzyme locus (Pgi-2). Overall, the F. glaucescens species-specific marker was found to be transmitted into Italian ryegrass at a rate close to 0.5 suggesting a high rate of homeologous pairing in BC1 and of intergeneric recombination in BC2. Significant variations in transmission rate among progenies in advanced generations indicated that recombination could result from various chromosome rearrangements possibly involving more than one fescue chromosome with some selection against duplex-like introgressed genotypes. Hence, introgressing F. glaucescens genes into tetraploid ryegrass appeared as further procedure to exploit versatility in fescue species, mostly restricted so far to introgression from F.arundinacea or F. pratensis into diploid ryegrass. Implications in terms of breeding strategy and marker-assisted selection are briefly discussed in respect to the genetic control of traits of interest to be introgressed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Culture of embryos and shoot tips for chromosome doubling in Lolium perenne and sterile hybrids between Lolium and Festuca

An improved method is reported for polyploid induction in Lolium (ryegrass), and in sterile F₁ hybrids between Lolium and Festuca (fescue). Two factors greatly increased the survival rate of colchicine‐treated embryos of Lolium perenne (perennial ryegrass) germinated and cultured in vitro (1) a high concentration of sucrose (100 g/1) in the germination medium and (1), maintenance at a low temperature of 10°C for 2 weeks after treatment. The maximum success rate for chromosome doubling among survivors of perennial ryegrass was 79.1%, and for L. perenne×Festuca arundinacea F₁ hybrid embryos it exceeded 40%. The same doubling treatment also works with shoot tip culture in non‐ flowering genotypes obtained by anther culture of L. multiflorum×F. arundinacea hybrids.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Cytology and Fertility of Hybrids and Amphiploids between Aegilops caudata L. ×Triticum turgidum (L.) Thell

Interspecific hybrids and amphiploids between Aegilops caudata L. (2n = 2x = 14, CC) and Triticum turgidum (L.) Thell. conv. durum Desf M. K. (2n = 4x = 28, AABB) were produced. Such hybrids can be used to introduce desirable traits such as disease resistance into cultivated durum wheats. One of the durum parents was a ph I mutation of the cv. ‘Cappelli’ used for testing the possibility of direct introduction of alien variation into cultivated species. The amphiploids were obtained both through colchicine chromosome doubling and as natural non-reductional mciosis products. In both hybrids and amphiploids, meiotic pairing and fertility were studied. Hybrids showed varying degrees of pairing and, in addition to the one involving the ph 1 mutant, one high pairing hybrid was found (Ae. caudata× cv. ‘Capinera’). Cytological examination of microsporogenesis in amphiploids revealed a high frequency of bivalent formation. Fertility proved to be a very variable character since some of the amphiploids were almost completely sterile. The use of amphiploids in breeding programmes is discussed in relation to meiotic and fertility data.     

Cytological analyses of hybrids and derivatives of hybrids between durum wheat and Thinopyrum bessarabicum,using multicolour fluorescent GISH*

The wheat progenitors and other wild relatives continue to be important sources of genes for agronomically desirable traits, which can be transferred into durum wheat (Triticum turgidum; 2n = 4x = 28; AABB genomes) cultivars via hybridization. Chromosome pairing in durum × alien species hybrids provides an understanding of genomic relationships, which is useful in planning alien gene introgression strategies. Two durum cultivars, ‘Lloyd’ and ‘Langdon’, were crossed with diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14; JJ), to synthesize F₁ hybrids (2n = 3x = 21; ABJ) with Ph1. ‘Langdon’ disomic substitution 5D(5B) was used as a female parent to produce F₁ hybrids without Ph1, which resulted in elevation of pairing between durum and grass chromosomes – an important feature from the breeding standpoint. The F₁ hybrids were backcrossed to respective parental cultivars and BC₁ progenies were raised. ‘Langdon’ 5D(5B) substitution × Th. bessarabicum F₁ hybrids were crossed with normal ‘Langdon’ to obtain BC₁ progeny. Chromosome pairing relationships were studied in F₁ hybrids and BC₁ progenies using both conventional staining and fluorescent genomic in situ hybridization (fl‐GISH) techniques. Multicolour fl‐GISH was standardized for characterizing the nature and specificity of chromosome pairing: A–B, A–J and B–J pairing. The A–J and B–J pairing will facilitate gene introgression in durum wheat. Multicolour fl‐GISH will help in characterizing alien chromosome segments captured in the durum complement and in their location in the A and/or B genome, thereby accelerating chromosome engineering research.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

Meiosis and fertility in interspecific hybrids of Nicotiana tabacum L. ×N. glauca Grah. and their derivatives

Nicotiana glauca is of potential interest to breeders as it carries resistance to black root rot of tobacco. Cytological investigations of sexual interspecific hybrids of N. tabacum T′T′TT (2n = 4x = 48) cv. ‘Wiślica’ × N. glauca GG (2n = 2x = 24) were carried out. The analyses of chromosome association at diakinesis and metaphase I in the PMCs of amphihaploid F₁ T′TG (2n = 3x = 36) revealed low variable pairing with 0–5 bivalents. The sterile amphihaploids F₁ were converted into partial female fertile amphidiploids T′T′TTGG by chromosome doubling. Among 36 mature plants obtained, 15 were found to have chromosome numbers (2n = 6x = 72) and were verified as amphidiploids, 9 had (2n = 6x = 70 or 71) chromosomes while the remaining 12 were haploid. True amphidiploids, in spite of quite high chromosome pairing during meiosis, were very different in pollen fertility, ranging from 0% to 85%. Male fertility disturbances did not correlate with the degree of female fertility upon pollination with N. tabacum. Sesquidiploids T′TG (2n = 5x = 60) obtained from backcrossing the amphidiploids to parental tobacco showed more than 22 bivalents, 10–12 univalents and occasional multivalents that indicated the possibility of interchange events between N. tabacum and N. glauca genomes.     

Plant Regeneration and Chromosomal Stability in Tissue Cultures of the Hybrids of Elymus canadensis with Psathyrostachys juncea and Secale cereale

Callus was induced from segments of immature inflorescences of Elymus canadensis×Psathyrostachys juncea and Elymus canadensis×Secale cereale F₁ hybrids on MS medium supplemented with 2 mg/l of 2,4-D and plants were regenerated from the calluses on a hormone free MS medium. The plants regenerated from both the hybrids exhibited a high degree of stability in morphology, chromosome number and chromosome pairing. The reasons for this are discussed.     

Intergeneric Hybrids between Aegilops squarrosa and Secale cereale and their Meiotic Chromosome Behaviour

Three hybrid plants with different combinations of D and R genomes were produced from crosses between Aegilops squarrosa (2×: and 4×) and Secale cereale (2× and 4×) using embryo culture. Production frequencies of mature hybrids, having the genomes DR, DDR and DDRR, were 2.0%, 5.2% and 2.2 %, respectively, of florets pollinated. Amphidiploids were obtained directly from the cross between tetraploid parents. The majority of their morphological features were intermediate between those of the parents but, as with rye, the rachis was tough. Diploid and triploid hybrids were completely seed-sterile, whereas the amphidiploid had an average self-fertility of 4.5 % with a range of 0 to 45 %. The somatic chromosome numbers of diploids, triploids and amphidiploids were 2n = 14, 21 and 28, respectively. At meiotic metaphase I, the mean chromosome associations were 0.01 III + 0.26 II + 13.4 I in diploids, 6.96 II + 7.1 I in triploids, and 10.5 II + 7.9 I in amphidiploids. In diploids, pairing between D and R chromosomes was observed in the form of end-to-end types without chiasmata. Homologous pairing between D chromosomes was predominant in plants having two sets of D genomes. Amphidiploids showed a diploid-like chromosome behaviour. Homoeologous pairing was not observed in either triploids or amphidiploids.     

Anther Culture-Derived Regenerants from Hordeum vulgare × H. bulbosum Crosses

Hordeum bulbosum has several desirable attributes, including disease resistance, which would be worthwhile transferring to H. vulgare. Despite homoeologous chromosome pairing in the interspecific hybrids, there have been few reports of successful gene introgression between the two species. A possible explanation for this is that recombinant male gametes are at a competitive disadvantage with normal balanced gametes during post-pollination events. To circumvent this problem, the possibility of obtaining plants directly from immature pollen grains was investigated. Anthers from diploid, triploid and tetraploid H. vulgare × H. bulbosum hybrids were cultured on defined media. Only hybrids with dehiscent anthers in vivo responded in culture, and after transfer of calli and embryoids to regeneration medium, 36 albino and 12 green plants were obtained. Seven of the green regenerants survived, one of which contained 15 H. vulgare chromosomes (including one acrocentric chromosome) and one H. bulbosum chromosome. Another regenerant (Ac166) resembled a diploid H. vulgare × H. bulbosum hybrid but had partial anther dehiscence and a slightly modified chromosome constitution. Mostly normal H. vulgare progeny were obtained from crosses between H. vulgare cv.‘Emir’ and Ac166, but three plants involved chromosome additions and substitutions.     

Production and cyto-morphological analysis of intergeneric hybrids of Sorghum × Saccharum

Four intergeneric hybrids were produced by crossing sorghum [ Sorghum bicolor (L.) Moench, 2n =20] as a female parent with sugarcane ( Saccharum officinarum L., 2n = 112). The hybrids were morphologically similar to the sugarcane parent, but lacked vegetative vigour. Sorghum characters present in the hybrids included soft texture of the leaves, tight clasping of the leaf sheaths, presence of aerial roots and triangular ligule. All the hybrids were highly susceptible to natural incidence of mites. Somatic chromosome number of the hybrids ranged from 2n = 62–66, indicating n+n transmission. This is the first report of Sorghum × Saccharum hybrids with sorghum cytoplasm. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inflorescence production in an amphiploid between Lolium multiflorum (2n=28) and Festuca arundinacea (2n=84)

Summary The flowering characteristics of an amphiploid Lolium multiflorum (2n=28)× Festuca arundinacea (2n=84) have been compared with those of the parental species under field and glasshouse conditions. In field drills the number of inflorescences produced by an unselected amphiploid population was markedly less than that produced by S.170 tall fescue and S.22 Italian ryegrass. Experiments to determine the requirements for floral induction in the amphiploid showed that they were intermediate between those of the two parents. There was no indication that the hybrid was not becoming induced under field conditions and it is concluded that the competitive condition in field drills was preventing floral initiation and development. A comparison of seed from third and fourth generation stock suggested that additional multiplication through a number of generations, and subsequently growing the derived basic seed at high nitrogen levels would improve inflorescence production in the amphiploid.     

Cytogenetic analysis of interspecific hybrids in genus Citrullus (Cucurbitaceae)

Investigations on interspecific crossability in two species of the genus Citrullus were studied based on their morphology, chromosome pairing and pollen fertility in their hybrids resulting from 12 combinations(including reciprocals). These have been utilized for tracing the phylogenetic relationship between the taxa and factors responsible for their differentiation. A collective evaluation of data on cross compatibility,morphology, chromosome association and pollen fertility indicated a close phylogenetic relationship between the species C. colocynthis (wild2n = 22) and C. vulgaris (cultivated 2n = 22). Significant differences for crossability and pollen fertility were observed between hybrids of C. colocynthis × C. vulgaris and reciprocals. Meiotic analysis of F₁ hybrids revealed that these two species are freely crossable and provide normal chromosome association, involving an average of 9 to maximum 11 pairs of chromosomes. This indicated the possibility of high recombination and thereby genetic introgression of desirable traits. As both the species are of economic significance, breeding strategies can be developed for the genetic improvement of these species. This revised version was published online in August 2006 with corrections to the Cover Date.     

A New Intergeneric Hybrid between Triticum aestivum L. and Agropyron fragile (Roth) Candargy: Variation in A. fragile for Suppression of the Wheat Ph-Locus Activity

New intergeneric hybrids were obtained between Triticum aestivum L. cv. Tukuho’ (2n = 6x = 42, AABBDD) and Agropyron fragile (Roth) Candargy PGR 8097 (2n = 4x = 28, PPPP) at a frequency of 1.06 %, through the use of direct embryo culture and in ovulo embryo culture. Such hybrids could be used to transfer barley yellow dwarf virus (BYDV) resistance and winterhardiness into bread wheat. The somatic chromosome number in all the hybrid plants was 2n = 5x = 35, as expected. Considerable variation in chromosome pairing was observed among the different hybrid plants. Average meiotic chromosome configuration at metaphase I was 17.29 Is + 6.57 rod Us + 1.97 ring Us + 0.18 III + 0.03 IV + 0.002 VI. The high level of chromosome pairing in some F₁ hybrids was attributed to Ph-suppressor gene(s) present in A. fragile. The hybrids could not be backcrossed to wheat, but amphiploid seeds have been obtained by colchicine treatment.     

Cytogenetic Studies of Hybrids between Festuca gigantea Vill. and Lolium multiflorum Lam.

The meiotic behaviour of the hybrid between Festuca gigantea (2n – 6x = 42) and Festuca gigantea (2n = 6x = 42) indicates distinct structural differences between the two species. These differences are also apparent in the degree of chromosome pairing observed in Lolium multiflorum×F. gigantea compared with previous reports on the L. multiflorum×F. arundinacea hybrids. Although the L. multiflorum×F. gigantea 8x amphiploid com Dines the complementary characters of the two species and is agronomically interesting, there are some irregularities in meiotic behaviour that could affect the stability of the amphiploid. Seed fertility in the amphiploid is high and preliminary studies show evidence of a high degree of self-fertility. The pentaploid hybrid between autotetraploid L. multiflorum×F. gigantea is sufficiently fertile to be used as the pollen plant in crosses with diploid L. multiflorum (BC₁). Backcrossing the BC₃ hybrid to L. multiflorum results in mainly diploid progeny. The possibilities, of using this crossing scheme to introgress F. gigantea characters into L. multiflorum is discussed as an alternative approach to amphiploid breeding as a means o: combining specific complementary characters of the two species.     

Genotypic control of chromosome pairing in amphiploids involving the cultivated oat Avena sativa L.

Summary A genotype of the diploid species Avena longiglumis (Cw 57) has been shown to modify the genetic control of diploid-like chromosome pairing in the cultivated oat, A. sativa (2n=6x=42) leading to increased homoeologous chromosome pairing in 4x hybrids between the two species (Rajhathy & Thomas, 1974). The Cw 57 genotype has a similar effect in increasing homoeologous chromosome pairing in amphiploids combining diploid and hexaploid genomes including associations between alien chromosomes and their corresponding pairs in hexaploid species. The effect of the Cw 57 genotype is probably in altering the specificity of chromosome pairing in the early stages of meiosis. The use of the Cw 57 genotype to induce homoeologous chromosome pairing as a technique for the transfer of desirable alien variation into the cultivated oat is discussed.