Use of tissue culture to bypass wheat hybrid necrosis

Summary Hybrid necrosis in wheat is a barrier to gene transfer in wheat breeding practice. It is based on two complementary genes, Ne1 and Ne2. Recovery mutants (Re1, Re2 and Re3) which can grow to maturity were recovered from immature embryo cultures of necrotic hybrids between T. aestivum and T. durum. Cytological observation demonstrated that Re1 had 34 chromosomes instead of 35. This indicated that one of the chromosomes carrying the Ne genes was lost. Genetic study suggested that for Re1, the lost chromosome was chromosome 5B of the durum parental line. Re mutants are male sterile but can be maintained through a young ear culture method. Re mutants could be successfully pollinated by either parental line and the BC1 progeny is partially fertile. Re mutants were repeatedly induced in about 1% of the regenerated plants from immature embryo culture. This technique provides a practical way to bypass hybrid necrosis.     

Comparative genetic analysis of the hexaploid wheat Triticum petropavlovskyi Udasz. et Migusch. and Triticum aestivum L

A poorly studied species of hexaploid wheat Triticum petropavlovskyi Udacz. et Migusch. was compared with common wheat Triticum aestivum L. by means of monosomic and genetic analyses of F2 hybrids. Triticum petropavlovskyi was found to carry 13 dominant genes determining its morphological and physiological characters and regular bivalent conjugation of chromosomes. These genes were allelic to the respective genes of common wheat and were located in the same chromosomes. The modes of gene interaction were also the same. There was simple dominance for most genes studied and complementary interaction for the genes of hybrid dwarfism and hybrid necrosis. Triticum petropavlovskyi had the following dominant genes: Hg (downy glume); Rg1 (red glume color); Hl (downy leaf); Hn (downy node); Pa (pubescent auricles); Q (speltlike ears); D1 (grass-clump dwarfism); Ne1 (hybrid necrosis); Ph1 and Ph2 (genes of bivalent conjugation preventing homoeologous chromosomes from pairing); and Vrn1, Vrn2, Vrn3, and Vrn4 (genes of the spring habit). The gene Vrn1, which caused an increase in ear emergence time and a pronounced response to vernalization, was poorly expressed. T. petropavlovskyi was earlier demonstrated to have a species-specific gene P or Eg (elongated glume), which was not allelic to the gene Eg of the tetraploid T. polonicum L. The data obtained indicate that T. petropavlovskyi has originated from T. aestivum via mutations.     

Differential response of antioxidant enzymes in leaves of necrotic wheat hybrids and their parents

The leaves of necrotic hybrid of wheat ( Triticum aestivum L.) exhibited high superoxide content associated with increased lipid peroxidation and membrane damage in earlier studies (Khanna-Chopra et al. 1998, Biochem Biophys Res Commun 248: 712–715; Dalal and Khanna-Chopra 1999, Biochem Biophys Res Commun 262: 109–112). In the present study, we investigated the activities of the antioxidant enzymes in the leaves of necrotic wheat hybrids, Kalyansona×C306 (K×C) and WL711×C306 (WL×C) and their parents at different developmental stages. The K×C hybrid exhibited more severe necrosis than WL×C. In K×C, superoxide dismutase (SOD) activity showed no increase over the parents, while WL×C showed an early increase, but it was possibly insufficient to scavenge increased superoxide. Activities of guaiacol peroxidase, ascorbate peroxidase and glutathione reductase were enhanced, while catalase exhibited a decrease in activity, with the appearance of visible necrosis in both the hybrids. The isozyme profile of the antioxidant enzymes was similar in the hybrids and their parents. One existing isoform of guaiacol peroxidase showed an early appearance in the hybrid and increased in intensity with the progression of necrosis. The results reveal a differential response of antioxidant enzymes in necrotic wheat hybrids as compared to their parents. The response differed in magnitude at developmental stages of the leaves, which might be related to the intensity of necrosis expressed by the hybrids.     

Hybrid bridges to gene flow: a case study in milkweeds (Asclepias)

Natural hybridization occurs throughout areas of sympatry for the North American milkweeds Asclepias exaltata and A. syriaca (Asclepiadaceae), even though the formation of F1 hybrid seed is a rare event. For introgressive hybridization to proceed, F1 and advanced hybrids must be released from reproductive barriers and successfully mate with one or both parental species. I investigated the mating system of natural hybrids between A. exaltata and A. syriaca in three populations in Shenandoah National Park, Virginia. Allozyme data and a maximum-likelihood procedure were used to estimate the frequency of six genotypic classes (parentals, F1, F2, and backcrosses) of the hybridizing populations, the pollinia received by hybrid plants, and the paternal parents of seeds produced by hybrids. F1 hybrids, backcross A. syriaca, and parental A. syriaca individuals were common in three hybrid populations. Even though self-pollinations and interhybrid pollinations were common, F2 seed production and the occurrence of F2 individuals were rare in hybrid populations. Hybrid plants received more pollen from A. syriaca than A. exaltata, which resulted in the production of more backcross-A. syriaca seed than backcross-A. exaltata seed. Asclepias exaltata was rare in the hybrid populations, but A. exaltata pollinia were received by hybrids and this species sired between 15% and 36% of the seeds produced on hybrids. The potential for introgression with A. exaltata populations is lower because this species is unsuccessful as the maternal parent in interspecific and backcross hand-pollinations. The asymetry of hybridization with A. syriaca as the maternal parent is further supported by the incorporation of maternally inherited chloroplast DNA markers in hybrids. Hybrid milkweeds frequently backcross with both parental species and may be released from the reproductive barriers that limit the formation of F1 hybrids in natural populations. The direction of interspecific gene flow and introgression in milkweeds is influenced by the reproductive biology of hybrids, the constituency of the surrounding population, and failure of some crosses to produce seeds. Finally, introgressive hybridization remains an important evolutionary force even when the initial formation of F1 hybrids in natural populations is rare.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Molecular mapping of hybrid necrosis genes Ne1 and Ne2 in hexaploid wheat using microsatellite markers

Hybrid necrosis is the gradual premature death of leaves or plants in certain F₁ hybrids of wheat (Triticum aestivum L.), and it is caused by the interaction of two dominant complementary genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively. To date, molecular markers linked to these genes have not been identified and linkage relationships of the two genes with other important genes in wheat have not been established. We observed that the F₁ hybrids from the crosses between the bread wheat variety ‘Alsen’ and four synthetic hexaploid wheat (SHW) lines (TA4152-19, TA4152-37, TA4152-44, and TA4152-60) developed at the International Maize and Wheat Improvement Center (CIMMYT) exhibited hybrid necrosis. This study was conducted to determine the genotypes of TA4152-60 and Alsen at the Ne1 and Ne2 loci, and to map the genes using microsatellite markers in backcross populations. Genetic analysis indicated that Alsen has the genotype ne1ne1Ne2Ne2 whereas the SHW lines have Ne1Ne1ne2ne2. The microsatellite marker Xbarc74 was linked to Ne1 at a genetic distance of 2.0 cM on chromosome arm 5BL, and Xbarc55 was 3.2 cM from Ne2 on 2BS. Comparison of the genetic maps with the chromosome deletion-based physical maps indicated that Ne1 lies in the proximal half of 5BL, whereas Ne2 is in the distal half of 2BS. Genetic linkage analysis showed that Ne1 was about 35 cM proximal to Tsn1, a locus conferring sensitivity to the host selective toxin Ptr ToxA produced by the tan spot fungus. The closely linked microsatellite markers identified in this study can be used to genotype parental lines for Ne1 and Ne2 or to eliminate the two hybrid necrosis genes using marker-assisted selection. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Hybrid necrosis in triticale and the expression of necrosis genes in allopolyploids

Summary The occurrence in triticale of four different genes causing hybrid necrosis is described: Ne1 and Ne2 in the B genome of wheat and Ner1 and Ner2 in the rye genome. Hybrid necrosis develops due to dominant complementary interaction of two genes. This interaction in triticale, however, may take place not only between genes belonging to the same genome but also between genes of different genomes. In triticale, these genes can cause hybrid necrosis in four different combinations. The inheritance of the phenomenon in triticale is, therefore, more complicated than it is in wheat or rye. To avoid hybrid necrosis in triticale, attention should be paid that no necrosis genes are introduced into the primary triticale stocks from the wheat and rye parents. The expression of necrosis genes is influenced by the level of ploidy. Any additional genome — A, B, D, or R — may exert a suppressing effect on the expression of necrosis genes. Therefore, when identifying genotypes of triticale with regard to their necrosis genes, the level of ploidy has to be accounted for. Moreover, the present results illustrate that gene expression in polyploids is not only determined by interactions with other single genes but that it may also be modified by the total genotype of the respective individual.     

Sexual reproduction of cordgrass hybrids (Spartina foliosa x alterniflora) invading tidal marshes in San Francisco Bay

We genetically analysed cordgrass plants and seedlings throughout the San Francisco, California, USA, estuary and found that hybrids between exotic Spartina alterniflora and native Spartina foliosa are the principal cordgrass invaders and colonizers. We hypothesized that this was due to higher seed set and siring ability by hybrids relative to the native species; too few alien parents remained in San Francisco Bay for our comparative studies. Hybrid seed comprised 91% to 98% of that set in the marsh study plants over the 2 years of the study. Total viable pollen production by hybrid plants was 400 times that of the native plants. Seed and pollen production were highly skewed towards a few hybrid genotypes. In addition to seed produced by hybrid plants, hybrid seed was produced by S. foliosa due to hybrid backcrossing. While the greatest advantage for hybrids was in pollen and seed production, hybrid seeds germinated, and seedlings survived and grew as well or better than the native species. As native S. foliosa becomes increasingly rare, hybrid seed floating on the tides will predominate, overwhelming recruitment sites and resulting in further colonization by hybrids. In an evolutionary context, hybrids with exceptional pollen and seed production will be initially favoured by natural selection, leading to the evolution of even more fertile hybrid genotypes.     

Pollination between maize and teosinte: an important determinant of gene flow in Mexico

Gene flow between maize [Zea mays (L.)] and its wild relatives does occur, but at very low frequencies. Experiments were undertaken in Tapachula, Nayarit, Mexico to investigate gene flow between a hybrid maize, landraces of maize and teosinte (Z. mays ssp. mexicana, races Chalco and Central Plateau). Hybridization, flowering synchrony, pollen size and longevity, silk elongation rates, silk and trichome lengths and tassel diameter and morphology were measured. Hybrid and open-pollinated maize ears produced a mean of 8 and 11 seeds per ear, respectively, when hand-pollinated with teosinte pollen, which is approximately 1–2% of the ovules normally produced on a hybrid maize ear. Teosinte ears produced a mean of 0.2–0.3 seeds per ear when pollinated with maize pollen, which is more than one-fold fewer seeds than produced on a maize ear pollinated with teosinte pollen. The pollination rate on a per plant basis was similar in the context of a maize plant with 400–500 seeds and a teosinte plant with 30–40 inflorescences and 9–12 fruitcases per inflorescence. A number of other factors also influenced gene-flow direction: (1) between 90% and 95% of the fruitcases produced on teosinte that was fertilized by maize pollen were sterile; (2) teosinte collections were made in an area where incompatibility systems that limit fertilization are present; (3) silk longevity was much shorter for teosinte than for maize (approx. 4 days vs. approx. 11 days); (4) teosinte produced more pollen on a per plant basis than the landraces and commercial hybrid maize; (5) teosinte frequently produced lateral branches with silks close to a terminal tassel producing pollen. Collectively these factors tend to favor crossing in the direction of teosinte to maize. Our results support the hypothesis that gene flow and the subsequent introgression of maize genes into teosinte populations most probably results from crosses where teosinte first pollinates maize. The resultant hybrids then backcross with teosinte to introgress the maize genes into the teosinte genome. This approach would slow introgression and may help explain why teosinte continues to co-exist as a separate entity even though it normally grows in the vicinity of much larger populations of maize.     

The role of seed traits as segregation factors of hybrids in wild populations of Cistus (Cistaceae)

Hybridization has been postulated as a main speciation mechanism in Cistus, a hard-seeded group of plants widely distributed in fire-prone Mediterranean shrublands. However, ecological evidence about how the segregation of hybrids from parents might occur is nonexistent. The presence of two hybrid populations in Morocco and Spain from Cistus albidus and Cistus heterophyllus led us to investigate their seed trait differences (seed size, mass and coat thickness) with the parentals and their seed germinability. A sowing experiment to compare the seedling emergence ability and seedling fitness of hybrids and their parentals was also carried out. Hybrid seeds germinated in a similar proportion to those of the parents. The hybrid seeds were significantly lighter, smaller and had thinner seed coats in both populations. However, these seed trait differences did not affect the seedling fitness of hybrids. These results were interpreted as a successful shift in the regeneration niche of the hybrids due to a greater proportion of soft-coated seeds, able to emerge from the soil seed bank in absence of fire. Smaller seeds also would favour further and deeper seed dispersal to escape from a fire. Monitoring of the seed yield of the Spanish population showed a competitive ability of hybrids.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.     

Production,morphology, and cytogenetic analysis of Elymus caninus (Agropyron caninum) x Triticum aestivum F1 hybrids and backcross-1 derivatives

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD) and wheatgrass, Etymus caninus (Agropyron caninum) (2n=4x=28, SSHH) — the first successful report of this cross. Reciprocal crosses and genotypes differed for percent seed set, seed development and F₁ hybrid plant production. With E. caninus as the pollen parent, there was no hybrid seed set. In the reciprocal cross, seed set was 23.1–25.4% depending upon wheat genotype used. Hybrid plants were produced only by rescuing embryos 12–13 days post pollination with cv Chinese Spring as the wheat parent. Kinetin in the medium facilitated embryo germination but inhibited root development and seedling growth. The hybrids were vigorous, self sterile, and intermediate between parents. These had expected chromosome number (2n=5x=35, ABDSH), very little chromosome pairing (0.51 II, 0.04 III) and some secondary associations. The hybrids were successfully backcrossed with wheat. Chromosome number in the BC₁ derivatives varied 54–58 with 56 as the modal class. The BC₁ derivatives showed unusually high number of rod bivalents or reduced pairing of wheat homologues. These were sterile and BC₂ seed was produced using wheat pollen.     

New data on the distribution of hybrid necrosis genes in winter bread wheat (Triticum aestivum L.) cultivars

Hybrid necrosis genotypes have been identified in 125 Russian cultivars of winter bread wheat. More than half of them (56%) carry the Ne2 gene (genotype ne1ne1Ne2Ne2); others are free of necrosis genes (genotype ne1ne1ne2ne2). The possible causes of the increase in the Ne2 allele frequency and the loss of the Ne1Ne1ne2ne2 genotype in modem Russian cultivars of winter wheat are discussed. The principal component method has been used to compare the structures of the genetic diversity of cultivars differing in the hybrid necrosis genotype. It has been found that the Ne2 allele in winter wheat cultivars from northern Russia has originated from the cultivar Mironovskaya 808, whereas the cultivar Bezostaya 1 is not a source of this gene. In cultivars from southern Russia, the presence of the Ne2 allele is also mainly accounted for by the use of Mironovskaya 808 wheat in their breeding. The recessive genotype is explained by the presence of descendants of the cultivar Odesskaya 16 in the pedigrees of southern Russian winter wheats. The genetic relationship of cultivars with identical and different necrosis genotypes has been analyzed in nine regions of the Russian Federation.     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae)

Fitness of interspecific hybrids is sometimes high relative to their parents, despite the conventional belief that they are mostly unfit. F(1) hybrids between oilseed rape (Brassica napus) and weedy B. rapa can be significantly more fit than their weedy parents under some conditions; however, under other conditions they are less fit. To understand the reasons, we measured the seed production of B. napus, B. rapa, and different generations of hybrid plants at three different densities and in mixtures of different frequencies (including pure stands). Brassica napus, B. rapa, and backcross plants (F(1) ♀ × B. rapa) produced many more seeds per plant in pure plots than in mixtures and more seeds in plots when each was present at high frequency. The opposite was true for F(1) plants that produced many more seeds than B. rapa in mixtures, but fewer in pure stands. Both vegetative and reproductive interactions may be responsible for these effects. Our results show that the fitness of both parents and hybrids is strongly frequency-dependent and that the likelihood of introgression of genes between the species thus may depend on the numbers and densities of parents and their various hybrid offspring in the population.     

薯蓣属植物人工杂交后代的检测

以湖北武当山的盾叶薯蓣(Dioscorea zingiberensis C.H.Wright)为主要亲本,与重庆金佛山的盾叶薯蓣、小花盾叶薯蓣(D.parviflora C.T.Ting)及黄独(D.bulbifera L.)进行种内和种间杂交,并获得部分杂交组合的F1代植株。将F0代种子与F1代植株进行结实率及种子萌发率比较,并与亲本进行过氧化物酶(POD)、超氧化物歧化酶(SOD)和酯酶(EST)同工酶比较。结果表明,不同亲本及其杂交后代的结实率、种子萌发率和实生苗存活率均存在明显差异;亲本和F1代的同工酶酶谱较丰富,部分杂种与亲本的相似度较高,并出现各自的特征酶带。运用同工酶技术可以鉴别杂种的真伪。     

Reproductive fitness of hybrids between Senecio jacobaea and S. aquaticus (Asteraceae)

Natural hybridization is increasingly recognized as an important process for the ecology and evolution of natural plant populations and species. There is a great need to initiate more studies based on natural populations in order to elucidate the possible role of hybrids in nature. The reproductive success of early generation hybrids can make or break hybrid lineages and may determine the genetic structure of hybrid swarms or the potential for gene flow through future generations, but studies of hybrid reproductive success are lacking. Here we measured components of male and female reproductive success in Senecio jacobaea and S. aquaticus (Asteraceae) species and F(1) hybrids between these species under laboratory conditions, and we measured reproductive output from crosses producing F(1), F(2), and backcross (BC) generation hybrids. F(1) hybrids were readily produced, and on average, the success of crosses producing subsequent generations (F(2), BC) decreased (though remained substantial), but the success of crosses was highly dependent on the genotypes involved. Also, F(1) hybrids were bigger, produced more flowers, and therefore produced more pollen than parental plants. Finally, crosses between parents were asymmetrical, such that S. aquaticus produced more and larger F(1) seeds than did S. jacobaea.