Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

 The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross. Received: 22 May 1998 / Accepted: 4 June 1998     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Seawater Stress Differentially Affects Germination, Growth, Photosynthesis, and Ion Concentration in Genotypes of Jerusalem Artichoke (Helianthus tuberosus L.)

Two Jerusalem artichoke (Helianthus tuberosus L.) genotypes, NY-1 and NY-7, were subjected to different seawater concentrations (0, 10, 20, 30, 40, and 50%) for various periods of time to determine the effects on seedling growth, ion content, and photosynthetic productivity in a greenhouse. Under different seawater concentrations, sprouting rates varied greatly among the genotypes. The differences in relative growth rate (RGR), leaf chlorophyll content, total leaf area (TLA), plant dry weight (PDW), photosynthetic rate (A), stomatal conductance (g _s), and efficiency of the light harvesting of photosystem II (F _v^′/F _m^′) were significant between NY-1 and NY-7 after 12 days of stress at 40 and 50% seawater. Seawater treatments resulted in the reduction of almost all the growth parameters and coincident increases of Na⁺ and Ca²⁺ concentrations in plant tissues. Our results indicate that there is great variability for seawater tolerance among H. tuberosus varieties, and that greater photosynthesis capacity, higher RGR, and relatively higher tissue Na⁺ accumulation at high seawater concentrations appears to be associated with seawater tolerance in H. tuberosus varieties.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

The karyotype and meiosis of the 12-ploid plants—one of the offspring of the natural F₁ hybrid (Aster ageratoides subsp.ovatus (2n=36) ×Kalimeris incisa (2n=72), 2n=72)—were examined. The 2n=108 chromosomes of the 12-ploids were found to consist of 18 large chromosomes and 90 small chromosomes. In meiosis of the PMCs of the 12-ploid, chromosome configurations of 3_III+46_II+7_I, 2_III+48_II+6_I and 3_III+47_II+5_I were observed. All the univalents and trivalents were small, and among the 46–48 bivalents nine were large and the remaining 37–39 were comparatively small. The large bivalents were found to represent autosyndetic pairings, and the small bivalents and trivalents were probably formed by autosyndetic pairings. The large chromosomes of the 12-ploids were found to coincide with the large chromosomes ofovatus, and the 90 small chromosomes to correspond to small chromosomes ofovatus andK. incisa. The 12-ploids were concluded to have been produced by a fusion of an unreduced gamete of the F₁ plant and a reduced gamete ofK. incisa which was growing in proximity to the F₁s. Thus the 12-ploids were regarded to be an amphidiploid having 36 chromosomes ofovatus and 72 chromosomes ofK. incisa.     

Tissue culture and plant regeneration from sunflower (Helianthus annuus) and interspecific hybrids (H. tuberosus x H. annuus)

A method is described for the culture and regeneration of plants from callus of sunflower (Helianthus annuus) andH. annuus x H. tuberosus hybrids. Immature embryos proved to be the only explant which consistently gave regenerable cultures in all genotypes. The most responsive embryos were approximately 12 mm² in area. Genotype had a significant effect on the capacity of cultures to regenerate. Some regeneration was also obtained from cultures of tuber tissue but only from one genotype,H. tuberosus x H. annuus cross 200. None of theH. annuus accessions gave regenerable callus from root tissue. Difficulties included the premature initiation of flowering of regenerating shoots and the frequent occurence of "vitreous" plantlets which could not be transplanted successfully to soil. Some amelioration of both these problems was achieved by replacing inorganic nitrogen partially with amino acids. More effective reduction of these difficulties was accomplished by the addition of 10, 30 and 100 M phloridzin, esculin or naringin.Abbreviations BAP 6-benzylaminopurine; zeatin, trans-6-(4-hydroxy-3-methyl-but-2-enyl) aminopurine; kinetin, 6-furfurylaminopurine - IAA indole-acetic acid - NAA naphthyl acetic acid     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species

The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3–4 months and set seeds, of which 70–80% germinated. Received: 18 February 1997 / Revision received: 25 March 1997 / Accepted: 15 May 1997     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Nuclear DNA changes within Helianthus annuus L.: cytophotometric,karyological and biochemical analyses

Summary Cytophotometric measurement of the root meristems of seedlings after Feulgen-staining reveals that large differences (up to 58.16%) in nuclear DNA content may occur in the thirty-one cultivated varieties or lines of Helianthus annuus tested. Significant variations (not exceeding 25%) in the amount of DNA, which does not differ between the root and the shoot meristems of a single seedling, are also found to exist within cultivars or lines; even seedlings obtained from seeds collected from different portions of single heads of plants belonging to a selfed line may vary one from the other in this respect. Variations in the number of chromosomes or alterations in the chromosome structure do not account for the differences observed in nuclear DNA content. Karyometric analyses demonstrate that the surface area of squashed interphase nuclei and metaphase chromosomes and the total length of the latter increase with the increase in Feulgen/DNA absorption. DNA thermal denaturation and reassociation kinetics indicate that a frequency variation in repeated DNA sequences goes hand in hand with changes in the size of the genome. These results, supporting the concept that a plant genome is highly flexible, are discussed in relation to other data to be found in the literature on the intraspecific variation in the nuclear DNA content and in relation to the way in which it is produced in H. annuus.     

Production of fertile somatic hybrid plants of sunflower and Helianthus giganteus L. by protoplast fusion

Summary Protoplasts of Helianthus giganteus and Helianthus annuus were fused using polyethylene glycol. Before fusion H.giganteus protoplasts were subjected to iodoacetic acid treatment to render them unable to divide. Fused protoplasts were cultured in V-KM medium containing benzylaminopurine and naphtaleneacetic acid. Hybrid calli were identified on the basis of their ability of embryogenic development contributed by the Helianthus giganteus parent. Fifty embryogenic calli were cultured on MS based medium without growth regulators to induce further development of somatic embryos. Elongated shoots were removed, rooted and transferred into growth chambers. Overall morphology of the plants was intermediate between the two parents. Their hybrid nature was confirmed by chromosome counting and by the analysis of esterase isozymes. The plants flowered within two to three months and later died. Thus the perennial nature of H.giganteus is a recessive trait in this interspecific hybrid. Seeds were obtained from two of the regenerated plants. From these seeds normal fertile F₂ plants could be grown.Abbreviations PEG polyethylene glycol - NAA naphtaleneacetic acid - BA benzyladenine - MS Murashige and Skoog medium - V-KM protoplast culture medium of Binding and Nehls     

Genome relationships between Hordeum vulgare L. and H. bulbosum L.

Interrelationships between H. vulgare (2x=14) and H. bulbosum (2x=14; 4x=28) were estimated on the basis of the karyotypes and the pairing behaviour of the chromosomes in diploid, triploid and tetraploid hybrids obtained with the aid of embryo culture. — A comparison of the karyotypes of the two species revealed similarities as well as differences. It was concluded that at least 4 or more of the chromosomes were similar in morphology and probably closely related. — Diploid and tetraploid hybrids are rarely obtained and their chromosome numbers tend to be unstable whereas triploid hybrids (1 vulgare + 2 bulbosum genomes) were stable and relatively easy to produce. In the diploid hybrid only 40% of the meiotic cells contained 14 chromosomes while the numbers ranged from 7 to 16 in other cells. All hybrids exhibited pairing between the chromosomes of the two species. Diploid hybrids had a mean of 5.0 and a maximum of 7 bivalents per cell in those cells having 14 chromosomes. Triploid hybrids from crosses between 2x H. vulgare and 4x H. bulbosum exhibited a mean of 1.5 and a maximum of 5 trivalents per cell. In a hexaploid sector found following colchicine treatment of a triploid the mean frequencies of chromosome associations per cell were: 5.5_I+8.0_II+0.7_III+3.7_IV+0.3_V+0.4_VI. One unstable 27 chromosome hybrid obtained from crosses between the autotetraploid forms had a mean of 1.1 and a maximum of 4 quadrivalents per cell. The chromosome associations observed in these hybrids are consistent and are taken as evidence of homoeologous pairing between the chromosomes of the two species. Interspecific hybridization between these two species also reveals that chromosome stable hybrids are only obtained when the genomes are present in a ratio of 1 vulgare2 bulbosum. Based upon the results obtained, the possibility of transferring genetic characters from H. bulbosum into cultivated barley is discussed.     

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F₂ population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (121 or 31), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.     

The effect of Secale cereale L. heterochromatin on wheat chromosome pairing

Metaphase-I chromosome association in PMCs of five F₁ hybrids 6x-triticale x T. turgidum (2n=5x=35 and genomes AABBR), and 13 plants from their backross or self offspring is reported. In wheat 18 chromosome arms and in rye 14 arms were recognized after C-banding and individually studied. Plants of backcross and F₂ showed variability for number and type of rye chromosomes, having in common the 28 durum wheat chromosomes (AABB). By testing meiotic association in plants with different rye chromosome constitutions, significant negative correlations were found. A clear negative effect of rye heterochromatin on pairing in wheat chromosomes is observed, the influence being more pronounced for large arms than for the short ones.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Prospects for interspecific hybridization of Lupinus atlanticus Gladst. with L. cosentinii Guss.

Summary Successful crossing is reported between L. atlanticus Gladst. (2n = 38) and L. cosentinii Guss. (2n = 32), using lines of both species selected for crossability followed by selection of relatively fertile progenies. In one cross, 82E75, from a single F₂ segregating plant, 22 F₃ seeds were obtained. Some other less crossable combinations were completely sterile in the f₁ or F₂. Backcrossing to both parent species was successful, but some crosses gave relatively more seed by using F₂ plants for backcrossing rather than F₂'s. It is concluded that potential exists for introgression of useful genes in both directions.     

Chromosomal location of a gene suppressing powdery mildew resistance genes Pm8 and Pm17 in common wheat (Triticum aestivum L. em. Thell.)

The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F₁ hybrids (2n=41) of Caribo x Disponent and Caribo x Helami-105 lacking chromosome 7D were resistant, whereas monosomic F₁ hybrids involving the other 20 chromosomes, as well as disomic F₁ hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.     

Successful crosses between Festuca arundinacea Schreb. and Dactylis glomerata L.

Summary Five F₁ plants have been obtained after extensive crossing between different ecotypes or varieties of Festuca arundinacea Schreb. and Dactylis glomerata L. The success did not appear to depend on specific treatments (spraying with -aminocaproic acid or gibberellic acid or pre-pollination with killed pollen from the seed parent), but the crossability is limited to exceptional plants.F₁ hybrids showed characteristics of both the parents. In four hybrids various developmental disturbances were observed (low viability, aneusomaty, absence of development of inflorescences). Only one hybrid consistently showed 2n=35 chromosomes, good viability and growth, however, it was sterile. After clonal propagation, attempts for polyploidization were started.     

Genome relationships between Hordeum procerum (6x) and H. lechleri (6x)

Investigations on the meiotic behaviour of chromosomes in interspecific hybrids (2n=6x=42) between Hordeum lechleri (6x) and H. procerum (6x) and in their component haploids have been utilized to assess the nature of pairing and the extent of genome homology between the two species. In the F₁ hybrids an average of 25 (60%) chromosomes associated at metaphase I, mostly as bivalents. A majority (60%) of the pollen mother cells (PMCs) in H. procerum haploids (2n=3x=21) displayed 21 univalents and even in the remainder, a maximum of two rod bivalents were formed resulting in an average of 0.52 bivalents per cell. In haploids of H. lechleri (2n=3x=21) however, 30% of chromosomes pair. The sum of the chromosomal associations in the component haploids represents only 17% of the complement, far below the observed frequency (60%) in the hybrids. Thus, the pairing displayed in hybrids between H. lechleri and H. procerum was mostly allosyndetic and suggestive of two genomes being common in these species.In haploid H. procerum 1/3 of the PMCs displayed a tripolar organisation of chromosomes leading to triad and hexad formation after divisions I and II respectively. The significance of hexad formation in the trihaploid H. procerum and a possible suppression of homoeologous pairing in H. procerum haploids are discussed.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.