Partial hybridization in crosses between cultivated sunflower and the perennial Helianthus mollis: effect of in vitro culture compared to natural crosses

. Eighty-four plants obtained from the pollination of the annual diploid cultivated sunflower, Helianthus annuus, by the perennial diploid species H. mollis were obtained naturally from mature seeds (62 plants) or following embryo rescue procedures (22 plants) and subsequently studied for phenotype traits and DNA markers. Twenty-two plants were obtained from the reverse cross as a result of natural seed development. The level of hybridization was determined using random amplified polymorphic DNA and restriction fragment length polymorphism markers. All of the resulting plants were found to be diploid 2n=34. Reciprocal crosses led to different progenies, with phenotypes that were predominantly similar to that of the female sunflower or the H. mollis type. The embryo rescue procedure enhanced the level of hybridization, whereas natural seed development led to fewer hybrids. Molecular markers unique to the female or the male parent indicated mechanisms leading to partial hybridization.     

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Partial hybridization in wide crosses between cultivated sunflower and the perennial Helianthus species H. mollis and H. orgyalis

To obtain introgressed sunflower lines with improved disease resistance, interspecific crosses were performed with foreign perennial species. We report on several unusual features displayed by these hybrid plants. The methods used to produce the kernels affected yield and genotypes of progeny. Phenotypic traits and DNA markers were investigated in 97 plants derived from cross-pollination between annual diploid cultivated sunflower (Helianthus annuus) and the perennial diploid species H. mollis or H. orgyalis, and the reverse reciprocal crosses. The level of hybridization in progeny was determined using RAPD and RFLP markers. Hybridization was performed by leaving embryos to develop normally on the head (classical crossing) or using embryo rescue. All observed plants derived from H. mollis were diploid (2n = 34). Phenotypes were predominantly similar to the female when cultivated sunflower was the female parent. Progeny from crosses using a wild species as the female parent resembled that parent. Thus, reciprocal crosses led to different progeny. F1 sister progeny shared different sets of molecular markers representing a few of those of the wild species used as the pollen donor. Our results indicate mechanisms leading to the unusual event of partial hybridization. Possible mechanisms behind these unusual events and their possible impact on evolution are discussed.     

Genetic diversity and population structure in the rare Algodones sunflower (Helianthus niveus ssp. tephrodes)

Assessing levels and patterns of population genetic variation is an important step for evaluating rare or endangered species and determining appropriate conservation strategies. This is particularly important for ensuring the preservation of novel genetic variation in wild relatives of crops, which could provide beneficial alleles for plant breeding and improvement. In this study, we evaluate the population genetics of Helianthus niveus ssp. tephrodes (the Algodones sunflower), which is an endangered, wild relative of cultivated sunflower (H. annuus L.). This rare sunflower species is native to the sand dunes of the Sonoran Desert in southern California, southwestern Arizona, and northern Mexico and is thought to harbor beneficial alleles for traits related to drought tolerance. We genotyped nine populations of this species with a set of simple-sequence repeat markers derived from expressed sequence tags (EST-SSRs) and investigated levels of genetic diversity and population structure, in H. niveus ssp. tephrodes. We also compared our results to findings from five related sunflower species that have been analyzed with these same markers, including annuals and perennials that range from rare to widespread. The Algodones sunflower harbors lower levels of standing genetic variation, but similar levels of population structure as compared to other sunflower species. We also discovered that a disjunct population from northern Mexico was genetically distinct from populations elsewhere in the range. Given the occurrence of such a genetically unique population, our recommendations include population surveys of the southern portion of the range in hopes of bolstering the existing germplasm collection.     

Novel proteinase inhibitors in seeds of sunflower (Helianthus annuus L.): polymorphism, inheritance and properties

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M _r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M _r from about 1,500 to 6,000. Received: 7 March 1999 / Accepted: 18 March 1999     

High genetic diversity in a rare and endangered sunflower as compared to a common congener

Determining the genetic structure of isolated or fragmented species is of critical importance when planning a suitable conservation strategy. In this study, we use nuclear and chloroplast SSRs (simple sequence repeats) to investigate the population genetics of an extremely rare sunflower, Helianthus verticillatus Small, which is known from only three locations in North America. We investigated levels of genetic diversity and population structure compared to a more common congener, Helianthus angustifolius L., using both nuclear and chloroplast SSRs. We also investigated its proposed hybrid origin from Helianthus grosseserratus Martens and H. angustifolius. Twenty-two nuclear SSRs originating from the cultivated sunflower (Helianthus annuus L.) expressed sequence tag (EST) database, and known to be transferable to H. verticillatus and its putative parental taxa, were used in this study thereby allowing for statistical control of locus-specific effects in population genetic analyses. Despite its rarity, H. verticillatus possessed significantly higher levels of genetic diversity than H. angustifolius at nuclear loci and equivalent levels of chloroplast diversity. Significant levels of population subdivision were observed in H. verticillatus but of a magnitude comparable to that of H. angustifolius. Inspection of multilocus genotypes also revealed that clonal spread is highly localized. Finally, we conclude that H. verticillatus is not of hybrid origin as it does not exhibit a mixture of parental alleles at nuclear loci, and it does not share a chloroplast DNA haplotype with either of its putative parents.     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Regulation of the paternal inheritance of centrosomes in starfish zygotes

In most animals, fertilized eggs inherit one centrosome from a meiosis-II spindle of oocytes and another centrosome from the sperm. However, since first proposed by Boveri [Sitzungsber. Ges. Morph. Phys. Münch. 3 (1887) 151-164] at the turn of the last century, it has been believed that only the paternal (sperm) centrosome provides the division poles for mitosis in animal zygotes. This uniparental (paternal) inheritance of centrosomes is logically based on the premise that the maternal (egg) centrosome is lost before the onset of the first mitosis. For the processes of the selective loss of the maternal centrosome, three models have been proposed: One stresses the intrinsic factors within the centrosome itself; the other two emphasize external factors such as cytoplasmic conditions or the sperm centrosome. In the present study, we have examined the validity of one of the models in which the sperm centrosome overwhelms the maternal centrosomes. Because centrosomes cast off into both the first and the second polar bodies (PB) are known to retain the capacity for reproduction and cell-division pole formation, we observed the behavior of those PB centrosomes with reproductive capacity and the sperm centrosome in the same zygotic cytoplasm. We prepared two kinds of fertilized eggs that contain reproductive maternal centrosomes, (1) by micromanipulative transplantation of the PB centrosomes into fertilized eggs, and (2) by suppression of the PB extrusions of fertilized eggs with cytochalasin B. In both types of eggs, the PB centrosomes could double and form cell-division poles, indicating that they are not suppressed by the sperm centrosome, which in turn indicates that selective loss of the maternal centrosome is due to intrinsic factors within the centrosomes themselves.     

Occurrence of partial hybrids in wide crosses between sunflower ( Helianthus annuus) and perennial species H. mollisand H. orgyalis

Hybridisation between the annual diploid sunflower (Helianthus annuus) and the perennial diploid species Helianthus mollis and Helianthus orgyalis was obtained by means of a normal crossing procedure or embryo rescue. Hybridisation success was low. All plants examined cytologically appeared to be diploid. However, the phenotypes of these diploids were not intermediate between the parents and, despite great variation, they resembled the female parent-type predominantly. Thirty five percent of plants issued from sunflower pollinated with perennial Helianthus had a phenotype resembling the female sunflower parent. On average, only 5% of the minimum number of expected RAPD and RFLP bands from male parents were recovered in plants produced from mature seeds after pollination of sunflower by H. mollis. More hybrids were found among plants obtained from embryo rescue, with an average of 25% of the male parent bands recovered per plant. Analysis of individual plants indicated the occurrence of various levels of hybridisation. There was a significant positive correlation between the number of phenotype traits related to hybrid status and the number of bands derived from the male parent. A single hybrid plant might possibly represent the product of a ’normal’ hybridisation event. The mechanisms behind these unusual events and the consequences for the breeder are discussed. Received: 23 March 2001 / Accepted: 28 June 2001     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

Photosynthesis,dry matter accumulation and distribution in the wild sunflower Helianthus petiolaris and the cultivated sunflower Helianthus annuus as influenced by water deficits

Summary This study reports on the effects of water deficits on photosynthesis, plant growth and carbon allocation in the wild sunflower Helianthus petiolaris and in the cultivated sunflower Helianthus annuus grown under controlled conditions in the glasshouse. Water deficits reduced the rate of net photosynthesis and the dry weight of leaves, stems, roots and reproductive parts in both species. The root-to-shoot ratio of about 0.05 in H. petiolaris was lower than the root-to-shoot ratio of about 0.15 in H. annuus. Water stress did not affect the root-to-shoot ratio, but increased the percentage of roots at depth in H. annuus. The decrease in growth induced by water deficits was a consequence of a reduction in both leaf area production and net photosynthesis. Flowering occurred earlier in H. petiolaris than in H. annuus with a consequent earlier allocation of carbon to reproductive parts in the wild compared to the cultivated sunflower. The time to budding and flowering of either species was not altered by mild water stress, but was delayed by severe water deficits. During mild water stress carbon allocation to stems decreased, but that to reproductive parts increased. When plants were severely stressed and then rewatered the proportion af carbon allocated to leaves increased and the proportion allocated to stems decreased when compared to unstressed plants. The adaptative role of these features is discussed.     

Polymorphism and inheritance of seed storage protein in sunflower

The data on polymorphism and inheritance of the seed storage protein helianthinin are presented. The results of hybrid analysis indicate that in the annual sunflower Helianthus annuus, helianthinin synthesis is controlled by at least three loci: HelA, HelB, HelB, and HelC. Codominant alleles controlling different electrophoretic variants of polypeptides were identified at each of the loci. The HelA locus was inherited independently of HelB and HelC in a series of dihybrid crosses. The frequencies of recombination between loci HelB and HelC estimated in F2 and BC of two crossing combinations were respectively 21.8 and 19.0%. Segregation of the Hel-C-controlled variants in the progenies from the crosses of cultured sunflower with annual wild species and forms corresponded to that theoretically expected for Mendelian inheritance. The maternal type of helianthinin inheritance was observed in the progenies from the crosses of inbred H. annuus lines with perennial diploid and polyploid Helianthus species. Altered expression of the HelC locus was detected in some hybrid combinations. These alterations appeared in early (F1, F2) hybrid generations and were similar in different hybrid combinations. They did not depend on the perennial paternal species being more influenced by the maternal genotype and by the mode of obtaining hybrids (in an embryo culture or in the field). These results are explained by "genomic shock" generated by hybridization of genetically incompatible species.     

Adaptive introgression of herbivore resistance traits in the weedy sunflower Helianthus annuus

The role of hybridization in adaptive evolution is contentious. While many cases of adaptive trait introgression have been proposed, the relevant traits have rarely been identified, resulting in a lack of clear examples of this process. Here, we examine a purported case of adaptive introgression in which the annual sunflower Helianthus annuus annuus has captured alleles from a congener (Helianthus debilis) to form a stabilized hybrid, Helianthus annuus texanus. We tested the hypotheses that herbivore resistance traits have introgressed from H. debilis to H. annuus and have increased adaptation in the latter. In two common gardens, fitness (estimated by seed production) was on average 55% higher in H. a. texanus than in H. a. annuus. For H. a. texanus, three damage traits (of seven tested) differed significantly from the H. a. annuus parent in one or both sites and were shifted in the direction of the more resistant H. debilis. Natural selection favored H. a. annuusxH. debilis BC(1) hybrids (synthesized to mimic the ancestors of H. a. texanus) with H. debilis-like resistance to seed midges Neolasioptera helianthis and to receptacle/seed feeding Lepidoptera at one or both sites. Assuming similar herbivore pressures in the past, these results suggest that introgression of biotic resistance traits was important in the adaptation of H. annuus to central and southern Texas.     

Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in the genus Actinidia

PCR amplification of four chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) regions followed by restriction of the amplified products was used to identify restriction fragment length polymorphisms in 21 Actinidia taxa. Subsequently, the mode of organelle inheritance was investigated in both interspecific and intraspecific controlled crosses made between genotypes showing different cpDNA and/or mtDNA haplotypes. Fifty-six seedlings produced from three interspecific crosses, including in one case the pseudo reciprocal (different genotypes of the same species used as opposite parents), were checked for cpDNA inheritance, and 102 seedlings from the same interspecific crosses and 32 seedlings from two intraspecific crosses within the species A. deliciosa were checked for mtDNA inheritance. In all cases, cpDNA was inherited from the father and mtDNA was inherited from the mother. Maternal inheritance of mtDNA was expected, being the rule in plants, but A. deliciosa is the first genus in angiosperms for which a widespread and strictly paternal inheritance of cpDNA has been reported. Transmission of chloroplastic and mitochondrial genomes through opposite parents provides an exceptional opportunity for studying the paternal and maternal genetic lineages of species in the genus Actinidia.     

Persistence of sunflower crop traits and fitness in Helianthus petiolaris populations

Transgenic plants have increased interest in the study of crop gene introgression in wild populations. Genes (or transgenes) conferring adaptive advantages persist in introgressed populations, enhancing competitiveness of wild or weedy plants. This represents an ecological risk that could increase problems of weed control. Introgression of cultivar alleles into wild plant populations via crop–wild hybridisations is primarily governed by their fitness effect. To evaluate this, we studied the second generation of seven wild–crop interspecific hybrids between weedy Helianthus petiolaris and cultivated sunflower, H. annuus var. macrocarpus. The second generation comprised open‐pollinated progeny and backcrosses to the wild parent, mimicking crosses that occur in natural situations. We compared a number of morphological, life history and fitness traits. Multivariate analysis showed that the parental species H. annuus and H. petiolaris differed in a number of morphological traits, while the second hybrid generation between them was intermediate. Sunflower crop introgression lowered fitness of interspecific hybrids, but fitness parameters tended to recover in the following generation. Relative frequency of wild/weedy and introgressed plants was estimated through four generations, based on male and female parent fitness. In spite of several negative selection coefficients observed in the second generation, introgressed plants could be detected in stands of <100 weedy H. petiolaris populations. The rapid recovery of fecundity parameters leads to prediction that any trait conferring an ecological advantage will diffuse into the wild or weedy population, even if F1 hybrids have low fitness.     

Occurrence of Verticillium dahliae on sunflower (Helianthus annus) in the UK

In August 1994 verticilliosis-like symptoms were noticed in a maturing crop of sunflower at IACR-Rothamsted. Wilting plants with chlorotic areas on some leaves, and dark areas of microsclerotia at the base of stems, were first observed on 11 August. As the plants matured, dark stripes were also observed on the stems. Many stems became brittle and later collapsed. Infected xylem showed a brown discolouration, later turning to black, and microsclerotia developed. Verticillium dahliae was isolated from all parts of the plant.
In general, once symptoms were present, the incidence of verticilliosis-affected plants increased, and the disease developed, more rapidly in early maturing varieties than in the later maturing types. At harvest, incidence ranged between 24.5% and 89.8%, depending on variety. Early maturing varieties appeared to be more susceptible than later maturing types.
Isolates of V. dahliae were cultured in the laboratory. Sunflower plants were artificially inoculated with these isolates and developed verticilliosis symptoms when grown under controlled environmental conditions.     

Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in loblolly pine

Summary The inheritance of organelle DNAs in loblolly pine was studied by using restriction fragment length polymorphisms. Chloroplast DNA from loblolly pine is paternally inherited in pitch pine x loblolly pine hybrids. Mitochondrial DNA is maternally inherited in loblolly pine crosses. The uniparental inheritance of organelle genomes from opposite sexes within the same plant appears to be unique among those higher plants that have been tested and indicates that loblolly pine, and possibly other conifers, must have special mechanisms for organelle exclusion or degradation or both. This genetic system creates an exceptional opportunity for the study of maternal and paternal genetic lineages within a single species.     

Propensity for paternal inheritance of de novo mutations in Alexander disease

De novo dominant mutations in the GFAP gene have recently been associated with nearly all cases of Alexander disease, a rare but devastating neurological disorder. These heterozygous mutations must occur very early in development and be present in nearly all cells in order to be detected by the sequencing methods used. To investigate whether the mutations may have arisen in the parental germ lines, we determined the parental chromosome bearing the mutations for 28 independent Alexander disease cases. These cases included 17 different missense mutations and one insertion mutation. To enable assignment of the chromosomal origin of the mutations, six new single nucleotide polymorphisms in the GFAP gene were identified, bringing the known total to 26. In 24 of the 28 cases analyzed, the paternal chromosome carried the GFAP mutation (P<0.001), suggesting that they predominantly arose in the parental germ line, with most occurring during spermatogenesis. No effect of paternal age was observed. There has been considerable debate about the magnitude of the male to female germ line mutation rate; our ratio of 6:1 is consistent with indirect estimates based on the rate of evolution of the sex chromosome relative to the autosomic chromosomes.