Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.     

Genetic diversity of root anatomy in wild and cultivated Manihot species

An anatomical study of roots was conducted on two wild Manihot species, namely M. glaziovii and M. fortalezensis, and two cassava varieties, M. esculenta Crantz variety UnB 201 and M. esculenta variety UnB 122, to identify taxonomic differences in primary growth. Anatomical characters of cassava roots have been rarely investigated. Their study may help cassava breeders to identify varieties with economically important characters, such as tolerance to drought. We investigated tap and lateral adventitious roots of two specimens of each clone or species. Free-hand cross-sections of roots were drawn; these had been clarified with 20% sodium hypochlorite solution, stained with 1% safranin-alcian blue ethanolic solution, dehydrated in ethanol series and butyl acetate and mounted in synthetic resin. Anatomical differences among Manihot species and varieties were found in the epidermal and exodermal cell shape and wall thickness, content of cortical parenchyma, and number of xylem poles. Wall thickness of the epidermis and exodermis of tap root were similar in all species, while in the lateral root there were differences in cell shape and wall thickness. Epidermal cells with thick walls were found in the tap root of all species and in lateral roots of cassava varieties. This character is apparently associated with tolerance to drought and disease. The variation in the number of xylem poles of cassava varieties was larger (4-8) than in wild species (4-6), and appears to support the hybrid origin of cassava.     

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.     

水稻84-15细胞胚胎学的研究

粳稻品种84-15是非洲长花药野生稻(O．longistaminata)和栽培稻80-6195杂交,在杂种第三代出现的不分离粳稻品系。它们的杂种F10代农艺性状保持整齐—致(赵世绪,1990;陈建三,1995)。它比水稻品种郑粳107增产 22.2％。用粳稻84-15与水稻喜峰杂交,在杂种第二代选育出中新一号比秋光增产11％,已种植F10代农艺性状保持—致。为了证实粳稻84-15固定杂种优势的原因,我们进行了细胞胚胎学的研究。通过研究发现了粳稻84-15珠心胚生殖现象(陈建三,1988;1990;1991)。本文旨在进一步研究此种现象。     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

Microsatellite markers confirm high apomixis level in cassava bred clones

Apomixis genes have successfully been transferred to cassava by hybridization with the wild species, Manihot glaziovii. The interspecific hybrid of cassava and M. glaziovii was left for open pollination during the subsequent three generations. Seven sibs and their maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii itself proved to be identical when examined by the same microsatellite loci. These results lead to the conclusion that apomixis do occur in wild cassava relatives and apparently has played an important role in Manihot speciation.     

Comparative life table statistics of Apoanagyrus lopezi reared on the cassava mealybug Phenacoccus manihoti fed on four host plants

The demographic statistics of Apoanagyrus lopezi De Santis (Hymenoptera: Encyrtidae) were studied in the laboratory on its host, the cassava mealybug Phenacoccus manihoti Matile-Ferrero (Homoptera: Pseudococcidae), reared on four host plants characterized by different levels of antibiotic resistance to the mealybug: two cassava varieties, Incoza and Zanaga (Manihot esculenta Crantz: Euphorbiaceae), the faux-caoutchouc (hybrid of M. esculenta × M. glaziovii Muel. Arg.) and Talinum (Talinum triangulare Jack:Portulacaceae).Total and daily mean fecundities of female parasitoids were strongly influenced by the host plant but there was no link with antibiotic resistance. The mean duration of the oviposition period was also significantly modified by the host plant and was again not correlated to the level of antibiotic resistance. Net reproduction rates of female parasitoids were nearly 2 times higher on hosts fed on Zanaga and Talinum than on Incoza and Faux-caoutchouc, and were not related to the total fecundities. The generation time was significantly longer on Faux-caoutchouc than on the other three plants. The intrinsic rate of increase varied significantly with host plants and was higher on Talinum and Zanaga than on Incoza and Faux-caoutchouc. With the varieties of cassava, Incoza and Zanaga, it was observed that antibiosis had a significant negative effect on the survival of A. lopezi, which would influence the effectiveness of the parasitoid.     

Apomixis and cassava

Apomixis means seed formation without fertilization. In cassava (Manihot esculenta) it is an alternative to reproduction by cuttings, which normally transmits pathogens and leads to an accumulation of viral and bacterial diseases. Apomixis also assures preservation of heterosis and avoids genetic segregation. It occurs in wild relatives of cassava and has been transferred successfully from Manihot glaziovii and M. neusana. It is facultative, and occurs at a low frequency, ranging from 1-2%, and apparently is genetically different from apomixis in other crops. With selection, the frequency can reach 13%. Apomixis in cassava is frequently associated with aneuploidy but it does occur in some diploid types. It is due to the formation of aposporic sacs, which can easily be detected by clearing tissue preparations. Apomixis appears to have played an important role in speciation during the evolution of Manihot, since it leads to the maintenance and perpetuation of sterile interspecific hybridization. The use of apomixis in cassava breeding could lead to a boom in line improvement and commercial production. In addition to preserving superior genotypes, avoiding contamination of new plants, it would enable international programs to export their germplasm to destination countries. This would allow the use of superior genotypes even if apomixis occurs at a low frequency. A scheme to maximize benefits is to use diploid apomictic clones as maternal parents, which can be crossed with pollinators of polyploid interspecific hybrids, followed by selection among the progeny of new apomictic types that combine the heteroses of both interspecific hybridization and polyploidy. In addition, they acquire favored genes that have been transferred from the wild to the commercial crop.     

In vitro production of cattle-water buffalo (Bos taurus--Bubalus bubalis) hybrid embryos

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using inter species gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.     

Chromosome doubling induces apomixis in a cassava x Manihot anomala hybrid

A new species was synthesized artificially by chromosome doubling of an interspecific hybrid between cassava and Manihot anomala. The ensuing polyploid type exhibits an apomictic nature and maintains its morphological characteristics in the progeny. It showed a 29% frequency of multiembryonic sacs in its examined ovules whereas the multiembryonic sacs were absent in the diploid type.     

Efficient Stabilization of Phosphodiester (PO), Phosphorothioate (PS), and 2'-O-Methoxy (2'-OMe) DNA·RNA Hybrid Duplexes by Amino Sugars

Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 16S rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2'OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2'OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2'OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2'OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) M(-1) derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.     

CHROMOSOMES AND CROSSING BEHAVIOR OF SOME SPECIES OF SANSEVIERIA

Menzel , Margaret Y. (Florida State U., Tallahassee), and James B. Pate . Chromosomes and crossing behavior of some species of Sansevieria. Amer. Jour. Bot. 47(3) : 230—238. Illus. 1960.–Approximately 20 species (28 clones) studied were diploids, tetraploids or hexaploids of the basic numbers x = 20; about 40% of the taxa were polyploid. All species had similar karyotypes, except for chromosome number. Five of 12 combinations of diploid species gave fertile F₁ hybrids; 4 studied cytologically showed 20 bivalents at metaphase I. Two triploid interspecific hybrids showed high trivalent frequencies. In contrast, multivalent formation in polyploid species was variable but rather low. Morphological relationships appeared reticulate among and between diploids and polyploids and did not coincide with barriers to crossing or to hybrid fertility. The following tentative hypothesis concerning relationships in the genus is proposed: Sansevieria is monophyletic and speciation has proceeded through genetic variation and hybridization at the diploid level and by allopolyploidy (of the segmental type) ; a low level of chromosome differentiation has accompanied speciation such that complete pairing occurs in diploid hybrids, but considerable preferential pairing occurs in allopolyploids. The occurrence of both polyploid and hybrid vigor, the fertility of hybrids between species differing greatly in morphology and physiology, and the high potential for vegetative propagation make the genus a favorable subject for breeding based on interspecific hybridization.     

Epistatic and cytonuclear interactions govern outbreeding depression in the autotetraploid Campanulastrum americanum

The consequences of combining divergent genomes among populations of a diploid species often involve F1 hybrid vigor followed by hybrid breakdown in later recombinant generations. As many as 70% of plant species are thought to have polyploid origins; yet little is known about the genetic architecture of divergence in polyploids and how it may differ from diploid species. We investigated the genetic architecture of population divergence using controlled crosses among five populations of the autotetraploid herb, Campanulastrum americanum. Plants were reciprocally hybridized to produce F1, F2, and F1-backcross generations that were grown with parental types in a greenhouse and measured for performance. In contrast to diploid expectations, most F1 hybrids lacked heterosis and instead showed strong outbreeding depression for early life traits. Recombinant hybrid generations often showed a recovery of performance to levels approximating, or at times even exceeding, the parental values. This pattern was also evident for an index of cumulative fitness. Analyses of line means indicated nonadditive gene action, especially forms of digenic epistasis, often influenced hybrid performance. However, standard diploid genetic models were not adequate for describing the underlying genetic architecture in a number of cases. Differences between reciprocal hybrids indicated that cytoplasmic and/or cytonuclear interactions also contributed to divergence. An enhanced role of epistasis in population differentiation may be the norm in polyploids, which have more gene copies. This study, the first of its kind on a natural autotetraploid, suggests that gene duplication may cause polyploid populations to diverge in a fundamentally different way than diploids.     

Chemically Induced Polyploidization in Spathiphyllum wallisii Regel through Somatic Embryogenesis

Polyploid Spathiphyllum wallisii Regel 'Speedy' plants were obtained from somatic embryos induced on anther filaments exposed to mitosis inhibitors. Primary embryos yielded less polyploid plantlets than secondary embryos. Colchicine could be efficiently replaced by oryzalin or trifluralin (10 μM), resulting in an average yield of 5% polyploids. The mitosis inhibitors were directly added to the induction medium. Morphological differences between diploid and tetraploid S. wallisii hybrids were observed. Throughout our experiments flow cytometry was applied as an unambiguous screening tool. Ploidy breeding within the genus Spathiphyllum holds promises towards the development of tetraploid hybrids with an altered morphology, or triploids with a reduced fertility.     

Molecular analysis of an additional case of hybrid sterility in rice (Oryza sativa L.)

Hybrid sterility hinders the exploitation of the heterosis displayed by japonica?×?indica rice hybrids. The variation in pollen semi-sterility observed among hybrids between the japonica recipient cultivar and each of two sets of chromosome segment substitution lines involving introgression from an indica cultivar was due to a factor on chromosome 5 known to harbor the gene S24. S24 was fine mapped to a 42?kb segment by analyzing a large F(2) population bred from the cross S24-NIL?×?Asominori, while the semi-sterility shown by the F(1) hybrid was ascribable to mitotic failure at the early bicellular pollen stage. Interestingly, two other pollen sterility genes (f5-Du and Sb) map to the same region (Li et al. in Chin Sci Bull 51:675-680, 2006; Wang et al. in Theor Appl Genet 112:382-387, 2006), allowing a search for candidate genes in the 6.4?kb overlap between the three genes. By sequencing the overlapped fragment in wild rice, indica cultivars and japonica cultivars, a protein ankyrin-3 encoded by the ORF2 was identified as the molecular base for S24. A cultivar Dular was found to have a hybrid-sterility-neutral allele, S24-n, in which an insertion of 30?bp was confirmed. Thus, it was possible to add one more case of molecular bases for the hybrid sterility. No gamete abortion is caused on heterozygous maternal genotype with an impaired sequence from the hybrid-sterility-neutral genotype. This result will be useful in understanding of wide compatibility in rice breeding.     

马铃薯杂种F1的SSR鉴定

为选育抗黑痣病、高产优质的马铃薯新品种,选用引进品种‘大西洋’分别与‘陇薯6号’、‘陇薯7号’杂交,获得了杂种F1代,利用SSR标记技术对‘大西洋’与‘陇薯6号’的42个杂种F1、‘大西洋’与‘陇薯7号’的9个杂种F1单株进行了鉴定。从59对SSR引物中筛选出2对在亲本间存在差异、扩增稳定、条带清晰的引物S184和STM1049,用于‘大西洋’ב陇薯6号’杂种F1、‘大西洋’ב陇薯7号’杂种F1及其亲本的基因组DNA扩增。SSR带型分析显示,杂种F1的SSR带型呈双亲互补型、缺失型、父本型和母本型4类,依据带型特征鉴定出供试的51个马铃薯杂种F1单株均为真杂种,表明SSR分子标记技术用于马铃薯杂种真实性鉴定是可行的。该研究可为进一步开展马铃薯杂交后代目标性状优异株系选育提供依据。     

Metabolic fingerprinting reveals differences between shoots of wild and cultivated carrot (Daucus carota L.) and suggests maternal inheritance or wild trait dominance in hybrids

Differences between the metabolic content of cultivars and their related wild species not only have implications for breeding and food quality, but also for the increasingly studied area of crop to wild introgression. Wild and cultivated western carrots belong to the same outcrossing species and hybridize under natural conditions. The metabolic fingerprinting of Dutch wild carrot and of western orange carrot cultivar shoots using (1)H NMR showed only quantitative differences in chemical content, indicating relatively low divergence after domestication. Main differences reside in the primary metabolite content and in the concentrations of chlorogenic acid and feruloyl quinic acid in the shoots of the different carrot types. Wild×cultivar hybrids cannot be distinguished from wild plants based on the metabolome, suggesting maternal, maternal environment, or dominance effects, and indicating high hybrid fitness in wild conditions. Considering these similarities, introgression is a real possibility in carrots, but understanding its consequences would require further studies using backcrosses in a multiple environments.     

Agronomic Trait and Protein Component of F2 Hybrid Originated from Intergeneric Somatic Hybridization Between Triticum aestivumand Agropyron elongatum

Protoplasts derived from common wheat ( Triticum aestivum L． cv. Jinan 177) were fused with UV-treated protoplasts of Agropyron elongatum (Host) Nevski by PEG method, and fertile asymmetric somatic hybrid plants resembling wheat morphology were obtained. The F2 hybrid plants could be divided into 3 types according to their morphology. Type Ⅰ hybrids had high and loosely standing stalks with big spikes and grains. TypeⅡ hybrids were dwarf and compact in shape with high tillering ability and smaller spikes. Type Ⅲ hybrids were similar to typeⅠas a whole but had more compact and erect spikes. All the F2 hybrid lines were superior to wheat in seed protein content, although some difference existed between themselves. Protein analysis of immature embryos and flag leaves from hybrids by two-dimensional electrophoresis showed that they possessed characteristic proteins of both parents and some new proteins. There existed also some different kinds of proteins in different lines.     

Heterogeneity of three molecular data partition phylogenies of mints related to M. x piperita (Mentha; Lamiaceae)

Phylogenetic reconstructions with molecular tools are now widely used, thanks to advances in PCR and sequencing technologies. The choice of the molecular target still remains a problem because too few comparative data are available. This is particularly true for hybrid taxa, where differential introgression of genome parts leads to incongruity between data sets. We have studied the potential of three data partitions to reconstruct the phylogeny of mints related to M. x piperita. These included nuclear DNA (ITS), chloroplast DNA (non-coding regions trnL intron, intergenic spacers trnL-trnF, and psbA-trnH), and AFLP and ISSR, markers. The taxonomic sampling was composed of hybrids, diploid and polyploid genomes. Since the genealogy of cultivated mint hybrids is known, they represent a model group to compare the usefulness of various molecular markers for phylogeny inference. Incongruities between ITS, chloroplast DNA, and AFLP-ISSR phylogenetic trees were recorded, although DNA fingerprinting data were congruent with morphological classification. Evidence of chloroplast capture events was obtained for M. x piperita. Direct sequencing of ITS led to biased results because of the existence of pseudogenes. Sequencing of cloned ITS further failed to provide evidence of the existence of the two parental copy types for M. x piperita, a sterile hybrid that has had no opportunity for concerted evolution of ITS copies. AFLP-ISSR data clustered M. x piperita with the parent that had the largest genome. This study sheds light on differential of introgression of different genome regions in mint hybrids.     

远缘杂交形成的二倍体鱼和多倍体鱼生殖细胞染色体研究

本文采用性腺染色体制片及组织学切片方法,系统地研究了不同发育时期的鲫鲤杂交第二代(F2) (2n=100)、异源四倍体鲫鲤(4n=200)、三倍体鲫鱼(3n=150))、雌核发育二倍体鲫鲤第二代(G2)(2n=100)及鲤鱼(Cypninus carpio L)(2n=100)(对照组)生殖细胞的染色体特征.研究结果表明,对照组中鲤鱼精原细胞染色体数与体细胞染色体数一致,为二倍体精原细胞(2n=100),而远缘杂交形成的二倍体鱼和多倍体鱼的生殖细胞中则观察到明显的染色体数加倍现象,其中,鲫鲤杂交第二代(F2)精巢生殖细胞染色体数加倍现象特别丰富,占检测的染色体分裂相的21.6%,为其产生不减半的二倍体配子提供了直接的细胞学证据,同时也说明远缘杂交是导致生殖细胞染色体数加倍的一个重要因素.该研究在探讨多倍体鱼的发生及鱼类遗传育种方面具有重要意义.