Intergeneric Hybrids Research Articles

Correlation analysis of the mRNA and miRNA expression profiles in the nascent synthetic allotetraploid Raphanobrassica.

Raphanobrassica is an allopolyploid species derived from inter-generic hybridization that combines the R genome from R. sativus and the C genome from B. oleracea var. alboglabra. In the present study, we used a high-throughput sequencing method to identify the mRNA and miRNA profiles in Raphanobrassica and its parents. A total of 33,561 mRNAs and 283 miRNAs were detected, 9,209 mRNAs and 134 miRNAs were differentially expressed respectively, 7,633 mRNAs and 39 miRNAs showed ELD expression, 5,219 mRNAs and 57 miRNAs were non-additively expressed in Raphanobrassica. Remarkably, differentially expressed genes (DEGs) were up-regulated and maternal bias was detected in Raphanobrassica. In addition, a miRNA-mRNA interaction network was constructed based on reverse regulated miRNA-mRNAs, which included 75 miRNAs and 178 mRNAs, 31 miRNAs were non-additively expressed target by 13 miRNAs. The related target genes were significantly enriched in the GO term ‘metabolic processes’. Non-additive related target genes regulation is involved in a range of biological pathways, like providing a driving force for variation and adaption in this allopolyploid. The integrative analysis of mRNA and miRNA profiling provides more information to elucidate gene expression mechanism and may supply a comprehensive and corresponding method to study genetic and transcription variation of allopolyploid.

Inter-specific and Inter-generic Hybridization Compatibility of Eriobotrya Species (Loquat) and Related Genera

We studied the cross-compatibility among 91 inter-specific combinations and 21 inter-generic combinations in 7 Eriobotrya plants and 2 related genera (Raphiolepis indica Lindl. and Photinia serrulata Lindl.) using emasculation, bagging, and artificial pollination. Our results showed that 28 of the 91 inter-specific combinations set no fruit, which means nearly 30% of the combinations were incompatible. In the remaining 63 combinations, most showed partial cross-compatibility, and a few showed complete cross compatibility. Eriobotrya plants were incompatible with plants from their related genera (R. indica Lindl. and P. serrulata Lindl.). Backcrossing produced 5 compatible combinations, which could set fruits and produce F1 progeny but only after embryo rescue. Fruit setting ratios varied among various species used as male or female parents. E. prinoides Rehd. & Wils., common loquat (E. japonica) and Eriobotrya × daduheensis, used as female parents resulted in an average fruit-setting ratio of 36.2%–58.2%. E. deflexa Nakai and its two forms, and E. elliptica Lindl. as female parents resulted in 2.9%–16.3% average fruit-setting ratio; however, the fruit set ratio was higher (22.4%–43.1%) if they were used as male parents. Failure of E. deflexa f. koshunensis Nakai × E. prinoides Rehd. & Wils. hybrids to set fruit could be attributed to sporophytic incompatibility.

A Simple and Efficient Approach to Elucidate Genomic Contribution of Transcripts to a Target Gene in Polyploids: The Case of Hexaploid Wheat (Triticum aestivum L.).

Common wheat (Triticum aestivum L.) is one of the most economically important crops in the world, however, gene functional studies in this crop have been lagging mainly due to the complexity of its polyploid genome, which is derived through two rounds of intergeneric hybridization events that led to the presence of six copies for each gene. Elucidating the transcript contribution of each genome to the total expression of a target gene in polyploids such as hexaploid wheat has a paramount significance for direct discovery of genes and the associated molecular mechanisms controlling traits of agronomic importance. A polymerase chain reaction approach that involved primers amplifying DNA fragments unique to each homeolog of a target gene and quantitation of the intensity of the resulting fragment bands were able to successfully determine the genomic transcript contributions as a percentage of target gene's total expression in hexaploid wheat. Our results showed that the genomic contributions of transcripts to a target gene vary with genotype and tissue type, suggesting the distinct role of each homeolog in regulating the trait associated with the target gene. The approach described in this study is an effective and economical method to elucidate the genomic transcript contribution to the total expression of individual target genes in hexaploid wheat. It can also be applied to determine the transcript contribution of each genome towards the collective expression of a target gene in other economically important polypoid crop species.

Localization and overcoming of hybridization barriers in Delosperma and Lampranthus (Aizoaceae)

The large succulent plant family Aizoaceae offers an enormous diversity of growth forms and flower pigmentations as well as prevalent tolerance to drought. Although many family members have potential for a wider use as ornamental plants, directed breeding of midday flowers is still in its infancy and only few cultivars have been launched onto the market so far. Most probably, this fact can be ascribed to the presence of cross-incompatibilities. The present study aimed at localization of intra- and intergeneric hybridization barriers between different genotypes of the genera Delosperma and Lampranthus by pollen germination tests, fluorescence and DIC microscopy, in vitro culture of seeds and ovules, and AFLP analysis. Aside from distinct self-incompatibility, the analyses revealed that pollen tube growth towards the ovules was not inhibited, even after intergeneric pollinations. Cross-combinations within Delosperma seemed to be compatible to a high extent, while hybridizations of the investigated Lampranthus genotypes and intergeneric combinations were largely unsuccessful. Investigations of embryo sacs following wide crosses using ovule clearing and differential interference contrast microscopy revealed fertilization failures as well as abnormal early embryogenesis. Seedlings obtained after wide crossings, mostly exhibited partial or complete loss of chlorophyll causing frequent hybrid breakdown. We therefore conclude that hybridization barriers between the investigated midday flowers occur both pre- and postzygotically.

ULASAN Kajian Filogenetika Molekuler dan Peranannya dalam Menyediakan Informasi Dasar untuk Meningkatkan Kualitas Sumber Genetik Anggrek

<p>Early information<br />resulted from molecular phylogenetic studies of many important<br />ornamental crops is often less attention to many<br />growers and farmers. Phylogenetics is one of the most preferable<br />method in systematics to reconstruct evolutionary<br />relationships of groups of biological organisms in order to<br />understand their biodiversities. This has been revolutionized<br />by DNA sequences data. In this method, a group of organisms<br />that shares many identical characteristics are considered<br />to be closely related; deriving from a common<br />ancestor and is assumed to have similar genetic patterns<br />and biochemical properties. By these basic principles,<br />molecular phylogenetics plays important roles in revealing a<br />basic knowledge on pattern of relationships to which<br />genetic resources can be improved. Over the past decade,<br />botanists have done several thousand phylogenetic analyses<br />based on molecular data of economically and horticulturally<br />important crops. Orchids are the best example for this.<br />There is no doubt that most orchid plants had played roles in<br />horticulture and hybridization. At present, many infrageneric<br />and intergeneric hybrids are available commercially. Successful<br />hybridization can be achieved if two or more individual<br />plants understudy are closely related in respect to their<br />genetics and evolution.</p>

Genomic affinities between Brassica napus and Raphanus raphanistrum as revealed by meiotic GISH

AbstractAn intergeneric hybrid between Brassica napus (AC, n = 19) and Raphanus raphanistrum (Rr, n = 9) was developed using the sequential ovary–ovule culture. Morphological, molecular, cytological studies and fluorescent genomic in situ hybridization were conducted to establish the hybridity and genomic relatedness. F1 hybrid plants were male sterile and morphologically intermediate between the two parents. Cytological analysis of the hybrid plants revealed 10II + 8I as the predominant meiotic configuration with the mean bivalent frequency of 8.83. A maximum of 13II were observed in 8.24% of the pollen mother cells (PMCs). GISH studies facilitated the identification of two allopairs, confirming homoeologous pairing between A/C and Rr genomes. This may have significant implications in terms of transgene flow and possible introgression into R. raphanistrum, which is a common canola weed in Canada and Australia. The intergeneric hybrid B. napus × R. raphanistrum so produced also has the potential to be used as a bridging species for the transfer of desirable genes to amphiploid crop Brassica species carrying A/C genomes.

Embryonic development and inviability phenotype of chicken-Japanese quail F1 hybrids.

Interspecific hybrid incompatibility, including inviability and sterility, is important in speciation; however, its genetic basis remains largely unknown in vertebrates. Crosses between male chickens and female Japanese quails using artificial insemination can generate intergeneric hybrids; however, the hatching rate is low, and hatched hybrids are only sterile males. Hybrid development is arrested frequently during the early embryonic stages, and the sex ratio of living embryos is male-biased. However, the development and sex ratio of hybrid embryos have not been comprehensively analyzed. In the present study, we observed delayed embryonic development of chicken-quail hybrids during the early stage, compared with that of chickens and quails. The survival rate of hybrids decreased markedly during the blastoderm-to-pre-circulation stage and then decreased gradually through the subsequent stages. Hybrid females were observed at more than 10 d of incubation; however, the sex ratio of hybrids became male-biased from 10 d of incubation. Severely malformed embryos were observed frequently in hybrids. These results suggest that developmental arrest occurs at various stages in hybrid embryos, including a sexually non-biased arrest during the early stage and a female-biased arrest during the late stage. We discuss the genetic basis for hybrid inviability and its sex bias.

Early embryo and larval development of inviable intergeneric hybrids derived from Crassostrea angulata and Saccostrea cucullata

To detect the intergeneric hybridization between the oyster Crassostrea angulata and Saccostrea cucullata coexisting along the southern coast of China, reciprocal crosses were conducted between the two species. Barriers for sperm recognizing, binding, penetrating the egg, and forming the pronucleus were detected by fluorescence staining. From the results, although fertilization success was observed in hybrid crosses, the overall fertilization rate was lower than that of intraspecific crosses. A large number of hybrid larvae died at 6–8 d after hatching, and those survived could not complete metamorphosis. C. angulata ♀× S. cucullata ♂ larvae had a growth rate similar to that of the maternal species, whereas S. cucullata ♀ × C. angulata ♂ larvae grew the slowest among all crosses. Molecular genetics analysis revealed that hybrid progeny were amphimixis hybrids. This study demonstrated that hybrid embryos generated by crossing C. angulata and S. cucullata could develop normally to the larval state, but could not complete metamorphosis and then develop to the spat stage. Thus, there is a post-reproductive isolation between C. angulata and S. cucullata.

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH, PCR-RFLP and RFLP.

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.

Interspecific and Intergeneric Hybridization in Dissotis and Tibouchina

Four species of Dissotis and three species of Tibouchina, two genera of the Melastomataceae family, were crossed in an attempt to create interspecific and intergeneric hybrids. Intergeneric crosses set seed at a rate of 18.1% and interspecific crosses had a 32.3% rate of seed set. Germination was extremely poor, with only four crosses having germinated seed. Crosses produced 31 seedlings. Three of the seedlings were from intergeneric crosses between Dissotis canescens and Tibouchina lepidota. Interspecific crosses produced 25 seedlings from crosses between Dissotis princeps and Dissotis rotundifolia and three seedlings from crosses between D. canescens and D. princeps. The prognosis for conventional breeding for species in Dissotis and Tibouchina is poor due to low seed set, poor germination, and slow growth of progeny.

Physiological and antioxidant enzyme gene expression analysis reveals the improved tolerance to drought stress of the somatic hybrid offspring of Brassica napus and Sinapis alba at vegetative stage

Drought is a major constraint of agriculture development. The intergeneric somatic hybrids between Brassica napus and Sinapis alba were created by electrofusion to obtain materials with enhanced drought tolerance. The drought tolerance of B. napus cv. Yangyou 6 (Y6) and one offspring line (W146) of the somatic hybrids was evaluated by morphological observation. Physiological parameters were determined in this study. Moreover, the activities of a few antioxidant enzymes and the transcript level of the antioxidant enzyme encoding genes were analyzed by qPCR. W146 and Y6 showed apparent wilting after drought stress for 7 days. However, Y6 wilted more severely than W146. The result of the physiological analysis showed that the relative electronic conductivity and malondialdehyde content of Y6 were higher than that of W146. The relative water content, net photosynthesis rate, proline content, and the activities of superoxide dismutase (SOD) and peroxidase in W146 were higher than that in Y6 after drought stress for 12 days. The DNA and nitrotetrazolium blue chloride staining analysis revealed less accumulation of O2 ·− and H2O2 in W146 than that in Y6 after drought stress. Moreover, the transcript level of some antioxidant enzyme encoding genes, such as Cu/ZnSOD, MnSOD, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase in W146, was higher than that in Y6 under drought stress. Results revealed that line W146 showed more drought stress tolerance than Y6 because line W146 could reduce oxidative damage by efficient antioxidant systems.

Nonhomologous Chromosome Pairing in Aegilops-Secale Hybrids

Intergeneric hybrids and amphidiploid hybrids from crosses of Aegilopstauschii and Secale cereale were produced using young embryo rescue. The hybrids showed complete sets of both parental chromosomes. The dihaploid plants showed an average meiotic pairing configuration of 10.84 I + 1.57 II + 0.01 III. Genomic in situ staining revealed 3 types of bivalent associations, i.e. D-D, R-R and D-R at frequencies of 8.6, 8.2 and 83.3%, respectively. Trivalents consisted of D-R-D or R-D-R associations. These results suggested that both intra- and intergenomic chromosome homology were contributed to chromosome pairing. Derived amphidiploids with 2n = 28 paired at metaphase I of meiosis as 4.51 I + 11.70 II + 0.03 III. Chromosome pairing of amphidiploids appeared more or less regular, i.e. bivalent-like with some trivalent configurations.

Sex beyond species: the first genetically analyzed case of intergeneric fertile hybridization in pinnipeds.

A species, according to the biological concept, is a natural group of potentially interbreeding individuals isolated by diverse mechanisms. Hybridization is considered the production of offspring resulting from the interbreeding of two genetically distinct taxa. It has been documented in over 10% of wild animals, and at least in 34 cases for Artic marine mammals. In Otariids, intergeneric hybridization has been reported though neither confirming it through genetic analyses nor presenting evidence of fertile offspring. In this study, we report the finding of a hybrid adult female between a South American fur seal (Arctocephalus australis) and a South American sea lion (Otaria byronia), and its offspring, a male pup, in Uruguay. Further based on morphological constraints and breeding seasons, sex-biased hybridization between the two species is hypothesized. Morphological and genetic (nuclear and mitochondrial) results confirm de hybrid nature of the female-pup pair. Here we discuss a genetic dilution effect, considering other hybridization events must be occurring, and how isolation mechanisms could be circumvented. Moreover, the results obtained from stable isotope analysis suggest feeding habits may be a trait transmitted maternally, leading to consider broader issues regarding hybridization as an evolutionary innovation phenomenon.

Targeted genotyping-by-sequencing permits cost-effective identification and discrimination of pasture grass species and cultivars.

A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis.

In this study, we confirmed that Vasconcellea cundinamarcensis resists Papaya leaf distortion mosaic virus (PLDMV), and used it to produce intergeneric hybrids with Carica papaya. From the cross between C. papaya and V. cundinamarcensis, we obtained 147 seeds with embryos. Though C. papaya is a monoembryonic plant, multiple embryos were observed in all 147 seeds. We produced 218 plants from 28 seeds by means of embryo-rescue culture. All plants had pubescence on their petioles and stems characteristic of V. cundinamarcensis. Flow cytometry and PCR of 28 plants confirmed they were intergeneric hybrids. To evaluate virus resistance, mechanical inoculation of PLDMV was carried out. The test showed that 41 of 134 intergeneric hybrid plants showed no symptoms and were resistant. The remaining 93 hybrids showed necrotic lesions on the younger leaves than the inoculated leaves. In most of the 93 hybrids, the necrotic lesions enclosed the virus and prevented further spread. These results suggest that the intergeneric hybrids will be valuable material for PLDMV-resistant papaya breeding.

An Investigation on Identification of The Ideal Age of Fruit For Extraction of Viable Embryo in Hot Pepper

Production of interspecific and intergeneric hybrids is often attempted for transfer of desirable genes from wild species into cultivated species. However, several times, progeny from wide crosses is difficult to produce owing to several barriers. Post fertilization barriers such as endosperm abortion and, at later stages, embryo degeneration are of common occurrence, leading to low fertility; but successful hybrids could be regenerated in these crosses using embryo rescue techniques which necessitates a reproducible embryos culture protocol as well as knowledge of ideal stage and age of fruit for extraction of viable embryos. In the present investigation seeds from fruits of 3 days to 18 days old (days after pollination) were cultured on MS medium supplemented with 5 mg/l BAP and 1mg/l NAA in different seasons. It was observed that growing season has marked influence on the germination potential of seeds. In the month of March-April, when the fruits were taken from net house, germination was observed within 4 days in seeds of 7–18 days old fruits, whereas in the months of July-August when the fruits were taken from field even 20 days fruits took about two weeks to germinate in the same media.

Complete mitochondrial genome of the parrotfish Calotomus japonicus (Osteichthyes: Scaridae) with implications based on the phylogenetic position

The complete mitochondrial genome sequence was determined for a specimen of Calotomus japonicus, a temperate parrotfish endemic to coastal East Asia. It was compared phylogenetically with previously published partial sequences from this species and other parrotfishes. The obtained tree indicated that the three cytb sequences of C. japonicus from a recent molecular study (LC068806-8) probably resulted from introgression through intergeneric hybridization, or possibly from sample confusion. Taking the presently obtained mitogenome as representative of C. japonicus, the species most closely related to this one among congeners is C. zonarchus, which is endemic to the Hawaiian islands.

Advances in papaya biotechnology

Abstract Papaya ( Carica papaya L.) is an important tropical fruit crop. The fruit is consumed fresh and used in the pharmaceutical, rayon and food industries. Papaya improvement for stress tolerance and qualitative traits using conventional breeding has been difficult due to the narrow germplasm pool in the Carica genus and sexual incompatibility problems encountered during intergeneric hybridization with other genera in the Caricaceae family. Genetic engineering is an important tool in papaya improvement for modifying one or more traits in elite cultivars without altering existing characteristics. Advances in genetic engineering have been facilitated by concerted efforts for genome sequencing of papaya, development of papaya regeneration systems and efficient gene insertion techniques for transfer of desirable traits. Papaya regeneration via organogenesis and somatic embryogenesis has been refined during the past 3 decades. Early efforts to optimize gene insertion protocols utilized a number of reporter and selectable marker genes, viral- and bacterial-derived regulatory sequences and functional genes for biotic and abiotic stress tolerance. Transgenic plants were routinely produced with several cultivars. One of the best success stories in the commercialization of a genetically modified fruit crop has involved the development of transgenic papaya ring spot virus (PRSV) resistant Rainbow and SunUp cultivars, which saved the Hawaiian papaya industry. Additionally, genetically modified papayas with traits for disease resistance and extended shelf life have been extensively screened in field tests. The papaya genome sequence was published in 2008 and has opened new avenues for papaya improvement by precision breeding, which involves the use of regulatory and functional gene sequences from related genera of the Caricaceae family, and is a logical extension of conventional breeding and genetic transformation. The application of precision breeding technology for papaya can pave the way for the development of consumer and eco-friendly cultivars that would be developed in ways similar to conventional breeding while causing fewer GMO-related concerns.

Whole genome expression and hydraulic architecture of diploid and doubled diploid citrus seedlings in intra and interspecific contexts

Genome expression in polyploid is usually considered to be quite different depending on the genetic context. While it is admitted that autotetraploids resulting from polyploidization events within species do not present major changes of genes expression, allotetraploid which result from hybridization between divergent species or lines were showed to present very important changes of expression compared to the diploid (2x) parental lines (genomic shock). In this work, we investigated the root citrus genome expression using custom Agilent Citrus microarray (4x44K format) of different couples of 2x and doubled diploid (4x) in absence of stress: Poncirus, Cleopatra Mandarin as well in the Cleopatra Mandarin × Poncirus intergeneric hybrid. We also evaluated the plant hydraulic architecture of this plant material. Our goal was to evaluate if any change in root genome expression related to polyploidy could lead to plant hydraulic architecture changes. (Resume d'auteur)

Development of male sterile Eruca sativa carrying a Raphanus sativus/Brassica oleracea cybrid cytoplasm.

Alloplasmic male sterile breeding lines of Eruca sativa were developed by intergeneric hybridization with CMS- Brassica oleracea, followed by recurrent backcrosses and determination of the breeding value. Male sterile breeding lines of rocket salad (Eruca sativa) were developed by intergeneric hybridization with cytoplasmic male sterile (CMS) cauliflower (Brassica oleracea) followed by recurrent backcrosses. Five amphidiploid F1 plants (2n = 2x = 20, CE), achieved by manual crosses and embryo rescue, showed an intermediate habit. The plants were completely male sterile and lacked seed set after pollination with the Eruca parent. Allotetraploid F1-hybrid plants (4n = 4x = 40, CCEE) obtained after colchicine treatment were backcrossed six times with pollen of the Eruca parent to select alloplasmic diploid E. sativa lines. The hybrid status and the nucleo-cytoplasmic constellation were continuously controlled by RAPD and Southern analysis during subsequent backcrosses. The ploidy level was investigated by flow cytometry and chromosome analysis. Premeiotic (sporophytic) and postmeiotic (pollen abortive) defects during the anther development were observed in the alloplasmic E. sativus plants in comparison to the CMS-cauliflower donor. No further incompatibilities were noticed between the CMS-inducing cybrid cytoplasm and the E. sativa nuclear genome. The final alloplasmic E. sativa lines were diploid with 2n = 2x = 22 chromosomes and revealed complete male sterility and restored female fertility. Plant vigor and yield potential of the CMS-E. sativa BC5 lines were comparable to the parental E. sativus line. In conclusion, the employed cybrid-cytoplasm has been proven as a vital source of CMS for E. sativa. The developed lines are directly applicable for hybrid breeding of rocket salad.