Embryo rescue of interspecific hybrids of Brachiaria spp

Age of explant and six different media were evaluated with the objective of regenerating higher numbers of interspecific hybrids between sexual and apomictic Brachiaria. Immature embryos of 7–8, 9–10 and 11–12 days after pollination (DAP), from artificial hybridization between Brachiaria ruziziensis (R) as female parent, and B. brizantha (B) or B. decumbens (D) as male parent, were cultured in modified MS media (M4) – supplemented with different combinations of growth regulators and vitamins. Embryos cultured 9–12 DAP showed high percentage (85–100%) of germination for all the crosses examined. Germination and survival rates varied according to accessions within crosses. Six different media (all modified MS with different growth regulators and vitamins) were tested with the objective of inducing multiple shoots from 7 to 10 DAP embryos, from crosses between R × B. The media M1, supplemented with Kinetin (13.94 μM) and NAA (5.37 μM), and media M3, supplemented with BA (4.44 μM and IAA 2.85 μM), regenerated adventitious shoots and calli about 30–40 days after inoculation. The highest multiplication rate observed was 2.85 shoots per explant in media M1, 60–70 days after culturing. Two other media, M6, supplemented with 2,4-D (13.57 μM) and M2, with 2,4-D (9.05 μM) and BA (8.87 μM) exclusively induced the formation of calli. The described protocols proved to be efficient in regenerating healthy seedlings from immature embryos of interspecific hybrids in Brachiaria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses

To develop doubled haploid (DH) mapping populations of hexaploid Brassica, 10 F₁ hybrids derived from crosses between allohexaploid Brassica parents were evaluated in this study. The allohexaploid Brassica parents were selfed progenies of unique interspecific crosses between Brassica rapa (genome AA) × B. carinata (BBCC), B. nigra (BB) × B. napus (AACC), and a complex cross between B. juncea (AABB), B. napus and B. carinata, with relatively stable chromosome number (2n = 54). Hexaploid status and chromosome behavior during meiosis I in four promising F₁ hybrids were assessed using microscopy and flow cytometry, and progeny were obtained following microspore culture. Hybrids H11-2 and H16-1 demonstrated higher amenability for embryo generation, plantlet regeneration, and frequency of production of DH microspore-derived progeny of hexaploid DNA content (6x) compared to hybrids H08-1 and H24-1. A total of 370 6x DH progeny were selected out of 693 plantlets from H11-2, 241/436 from H16-1, 23/54 from H08-1, and 21/56 from H24-1. DH progenies of hybrids H11-2 and H16-1 were then designated as promising mapping populations of a new hexaploid Brassica species.     

Application of embryo rescue after interspecific crosses in the genus Rhododendron

Interspecific hybridization between evergreen pot azalea (Tsutsusi) cultivars and genotypes of other Rhododendron subgenera or sections (Rhododendron, Hymenanthes, Pentanthera, Vireya) is significantly hampered by many prezygotic and postzygotic barriers. The objective of our work was to overcome spontaneous abortion and lack of endosperm formation and to increase germination rates by establishing an embryo rescue protocol. The optimal germination medium for immature Rhododendron seeds was a basal medium supplemented with 145 μM GA₃. This medium induced germination of fertilized ovules after several sexual combinations of subgenera. Its use was clearly more efficient than in vivo sowing. The direction of the cross significantly influenced the occurrence of abortion, germination and albinism. The obtained seedlings were multiplied on Woody Plant Medium + 4.5 μM 2iP, and rooted afterwards. Finally, about 9% of the germinated ovules resulted in vigorously growing seedlings that were successfully acclimatized.     

DNA amplification fingerprinting analysis of bermudagrass (Cynodon): genetic relationships between species and interspecific crosses

We have used DNA amplification fingerprinting (DAF) to study the genetic variation of bermudagrass (Cynodon) species and cultivars of interspecific crosses that exhibit leaf-blade textural characteristics ranging from coarse to fine. Arbitrary octamer primers produced complex and reproducible amplification profiles with high levels of polymorphic DNA. Phylogenetic analysis using parsimony (PAUP) and unweighted pair group cluster analysis using arithmetic means (UPGMA) grouped 13 bermudagrass cultivars into several clusters, including one containing the African-type bermudagrasses (C. transvaalensis) and another containing the common-type bermudagrasses (C. dactylon). The latter group included C. magennissii (Sunturf) and a interspecific C. transvaalensisxC. dactylon cross (Midiron), 2 cultivars that exhibited leaf textural characteristics closer to the common-types. All other C. transvaalensisxC. dactylon crosses grouped between the African and common types. An extended screen of 81 octamer primers was needed to separate cultivar Tifway from the irradiation-induced mutant Tifway II. The use of either template endonuclease digestion prior to amplification or arbitrary mini-hairpin primers increased detection of polymorphic DNA and simplified the task of distinguishing these closely related cultivars. Alternatively, the use of capillary electrophoresis (CE) resolved fingerprints adequately and detected products with high sensitivity, thereby promising to increase throughput and the detection of polymorphic DNA. When used to fingerprint samples from commercial sources, DAF identified bermudagrass plant material on the basis of unique reference profiles generated with selected primers. DAF represents an excellent technique for bermudagrass cultivar verification, seed certification, varietal protection, and for the identification of mistakes in plantings, mislabeled plant materials, and contamination or substitutions of sod fields.     

Hybrid crosses and the genetic basis of interspecific divergence in lifespan in Pristionchus nematodes

Characterizing the genetic basis of among‐species variation in lifespan is a major goal of evolutionary gerontology research, but the very feature that defines separate species – the inability to interbreed – makes achieving this goal impractical, if not impossible, for most taxa. Pristionchus nematodes provide an intriguing system for tackling this problem, as female lifespan varies among species that can be crossed to form viable (although infertile) hybrids. By conducting reciprocal crosses among three species – two dioecious (long‐lived Pristionchus exspectatus and short‐lived Pristionchus arcanus) and one androdioecious (short‐lived Pristionchus pacificus) – we found that female lifespan was long for all hybrids, consistent with the hypothesis that the relatively short lifespans seen for P. pacificus hermaphrodites and P. arcanus females are caused by independent, recessive alleles that are masked in hybrid genomes. Cross‐direction had a small effect on survivorship for crosses involving P. exspectatus, indicating that nuclear–mitochondrial interactions may also influence Pristionchus longevity. Our findings suggest that long lifespan in P. exspectatus reflects the realization of an ancestral potential for extended longevity in the P. pacificus species complex. This work demonstrates the utility of interspecific hybrids for ageing research and provides a foundation for future work on the genetic architecture of interspecific lifespan variation.     

Molecular mechanism of manipulating seed coat coloration in oilseed Brassica species

Yellow seed is a desirable characteristic for the breeding of oilseed Brassica crops, but the manifestation of seed coat color is very intricate due to the involvement of various pigments, the main components of which are flavonols, proanthocyanidin (condensed tannin), and maybe some other phenolic relatives, like lignin and melanin. The focus of this review is to examine the genetics mechanism regarding the biosynthesis and regulation of these pigments in the seed coat of oilseed Brassica. This knowledge came largely from recent researches on the molecular mechanism of TRANSPARENT TESTA (tt) and similar mutations in the ancestry model plant of Brassica, Arabidopsis. Some key enzymes in the flavonoid (flavonols and proanthocyanidin) biosynthetic pathway have been characterized in tt mutants. Some orthologs to these TRANSPARENT TESTA genes have also been cloned in Brassica species. However, it is suggested that some alterative metabolism pathways, including lignin and melanin, might also be involved in seed color manifestation. Polyphenol oxidases, such as laccase, tyrosinase, or even peroxidase, participate in the oxidation step in proanthocyanidin, lignin, and melanin biosynthesis. Moreover, some researches also suggested that melanic pigment in black-seeded Brassica was several fold higher than in yellow-seeded Brassica. Although more experiments are required to evaluate the importance of lignin and melanin in seed coat browning, the current results suggest that the flavonols and proanthocyanidin are not the only roles affecting seed color.     

Reproduction and cytogenetic characterization of interspecific hybrids derived from crosses between Brassica carinata and B. rapa

The tri-genomic hybrid (ABC, 2n=27) between Brassica carinata (BBCC, 2n=34) and B. rapa (AA, 2n=20) is a unique material for studying genome relationships among Brassica species and a valuable bridge for transferring desirable characteristics from one species to the other within the genus Brassica. The crossability between B. carinata and B. rapa was varied with the cultivar of B. rapa. Hybrid pollen mother cells (PMCs), confirmed by morphological observation and molecular marker assay, could be grouped into 20 classes on the basis of chromosome pairing configurations. More than 30% of the PMCs had nine or more bivalents. Genomic in situ hybridization confirmed that two of the bivalents most likely belonged to the B genome. Nearly one-half of the PMCs had trivalents (0–2) and quadrivalents (0–2), which revealed partial homology among the A, B, and C genomes and suggested that there is a good possibility to transfer genes by means of recombination among the three genomes. The advantages of using the tri-genomic hybrids as bridge material for breeding new types of B. napus are discussed.     

Sources of genetic diversity in feral oilseed rape (Brassica napus) populations

Feral oilseed rape populations on roadsides and along other semi-natural habitats contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified (GM) oilseed rape to wild or weedy populations. Knowledge about the underlying mechanism of establishment, persistence, and the evolutionary potential of feral populations is a prerequisite for a successful risk assessment and of high policy relevance. We analysed the origin, persistence, and genetic variation of feral oilseed rape populations in northwest Germany based on a field survey (2004–2007) and nuclear microsatellite markers. A maximum-likelihood approach was used to allocate individuals of feral populations to their source varieties and to hybrids between these varieties. The majority (72%) of the analysed sites was inhabited by oilseed rape for at least 2 years. The proportion of feral populations setting seeds varied between years (30–48%) and was higher than in comparable studies. Within-population genetic diversity of feral oilseed rape populations was higher than that of the common grown varieties studied. Repeated escapes of different varieties and hybridisation between these varieties were identified as the most important sources of genetic variation in feral oilseed rape populations. Up to four different source varieties were identified in feral populations. The detection of three hybrids between different varieties indicates that feral oilseed rape populations may persist via self-recruitment. Our results highlight the evolutionary potential of feral oilseed rape populations and have consequences for approaches to regulate the coexistence of genetically modified and non-GM oilseed rape varieties in the same region. Feral populations may serve as stepping stones for intraspecific and interspecific gene flow and can promote persistence of transgenes outside cultivation. The likelihood of introgressive interspecific hybridisation depends on the abundance of crossing partners, which may vary strongly from region to region.     

Effect of kinetin on interspecific crosses inBrassica

Application of kinetin to four interspecific crosses in Brassica, either singly or in combination with NAA, resulted in larger length and retention time of ovaries, increased frequency of ovules with embryos and aided embryo development.     

Dominance status of shape of male genitalia in interspecific crosses of some Drosophila virilis group species

The heritability of the shape of the main species-specific morphological trait for the Drosophila virilis group-the male mating organ has been analyzed using the hybrid males D. virilis × D. lummei and D. virilis × D. novamexicana. The results suggest an increase in the share of the characters with a recessive status in the evolutionarily younger species and demonstrate the role of sex chromosomes in the implementation of a dominant or recessive status of the trait. The roles of additive and epistatic components of the total variation in the evolution of the dominance status, shown in several known theoretical models and confirmed by our data, are considered. The published data on sterility of hybrid males in interspecific crosses are discussed from the standpoint of the evolution of dominance.     

In vitro pollen germination of species and two interspecific hybrids from the genus Brassica

In vitro pollen germination of five species and two interspecific hybrids from the genus Brassica was tested in four media. Genetically fixed differences in the demands for optimal pollen germination among species were found. The experiments were designed to define optimal content of mineral salts, sugar, and PEG for every investigated species or hybrids. The differences found among species are discussed in relation to the evolutionary trend.     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.     

Intra- and interspecific genetic complexities of two Eothenomys species in Honshu, Japan

Differences in the nuclear ribosomal DNA (rDNA), mitochondrial DNA (mtDNA), cytochrome b (Cytb), and Y chromosomal Sry genes were used to assess intra- and interspecific relationships in two Japanese red-backed voles, Eothenomys andersoni and E. smithii, focusing on areas where the two species might come into contact. In the Kii Peninsula, southwestern Honshu, which contains an allopatric population of E. andersoni isolated from its main range, the rDNA-RFLP data provide robust evidence of past mutual interspecific gene introgression, while the Cytb and Sry sequences were specific to this population. In central Honshu, where E. andersoni and E. smithii inhabit higher and lower altitudes, respectively, with a narrow sympatric zone, the rDNA-RFLP and Sry variation was specific for each species, while introgression of the mtDNA from E. smithii to E. andersoni was seen. These complex patterns in the gene markers are consistent with our previous notions derived from sex chromosome variation. Our previous and present data strongly suggest that the evolution of these vole species, which are morphologically and cytogenetically distinct, involves complex genetic interactions and the resultant combinations of genes are sometimes peculiar, mainly due to the Cytb haplotypes. However, phylogenetic analysis using a combination of maternal, paternal, and biparental markers has proven useful for understanding the evolutionary history given the complex phylogenetic background.     

Molecular genetic analyses of species boundaries in the sea

The tools of molecular genetics have enormous potential for clarifying the nature and age of species boundaries in marine organisms. Below I summarize the genetic implications of various species concepts, and review the results of recent molecular genetic analyses of species boundaries in marine microbes, plants, invertebrates and vertebrates. Excessive lumping, rather than excessive splitting, characterizes the current systematic situation in many groups. Morphologically similar species are often quite distinct genetically, suggesting that conservative systematic traditions or morphological stasis may be involved. Some reproductively isolated taxa exhibit only small levels of genetic differentiation, however. In these cases, large population sizes, slow rates of molecular evolution, and relatively recent origins may contribute to the difficulty in finding fixed genetic markers associated with barriers to gene exchange. The extent to which hybridization blurs species boundaries of marine organisms remains a subject of real disagreement in some groups (e.g. corals). The ages of recently diverged species are largely unknown; many appear to be older than 3 million years, but snails and fishes provide several examples of more recent divergences. Increasingly sophisticated genetic analyses make it easier to distinguish allopatric taxa, but criteria for recognition at the species level are highly inconsistent across studies. Future molecular genetic analyses should help to resolve many of these issues, particularly if coupled with other biological and paleontological approaches.     

Effect of three pollination methods on embryo development and seedset in intra- and interspecific crosses between seven Lilium species

Summary Interspecific crosses were made between seven Lilium species, viz. L. candidum, L. concolor, L. dauricum, L. henryi, L. longiflorum, L. nepalense and L. rubellum. A complete diallel cross was carried out between these seven species, including self- and intraspecific pollinations using three pollination methods: normal pollination on the stigma, pollination on the ovary after cutting the style, and pollination on the stigma with the aid of mentor (non-functional, compatible) pollen. Embryo rescue, starting 35 days after pollination, was applied to all interspecific combinations. The percentage of successful crosses was about 2.8% after normal pollination, 5.4% after cut-style pollination and 3.8% with the mentor pollen technique. Crosses with L. nepalense were exceptional in that embryos died during the embryo culture phase. Seventeen cross combinations (including 4 reciprocals) yielded 62 embryo plantlets from 839 interspecific pollinations.