Interspecific Origin Research Articles

Analysis of genome-wide linkage disequilibrium in the highly polyploid sugarcane

Linkage disequilibrium (LD) in crops, established by domestication and early breeding, can be a valuable basis for mapping the genome. We undertook an assessment of LD in sugarcane (Saccharum spp), characterized by one of the most complex crop genomes, with its high ploidy level (>or=8) and chromosome number (>100) as well as its interspecific origin. Using AFLP markers, we surveyed 1,537 polymorphisms among 72 modern sugarcane cultivars. We exploited information from available genetic maps to determine a relevant statistical threshold that discriminates marker associations due to linkage from other associations. LD is very common among closely linked markers and steadily decreases within a 0-30 cM window. Many instances of linked markers cannot be recognized due to the confounding effect of polyploidy. However, LD within a sample of cultivars appears as efficient as linkage analysis within a controlled progeny in terms of assigning markers to cosegregation groups. Saturating the genome coverage remains a challenge, but applying LD-based mapping within breeding programs will considerably speed up the localization of genes controlling important traits by making use of phenotypic information produced in the course of selection.

Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes

Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100microM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45mgCukg(-1) shoot dry weight. Higher levels of copper in solution (250 and 500microM) were lethal. Sugarcane displayed tolerance to 500microM Cd without symptoms of toxicity, accumulating 451mgCdkg(-1) shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.

B chromosomes in Amazonian cichlid species

B chromosomes are reported in three different Amazonian cichlid species. One to three supernumerary microchromosomes were detected in the peacock bass Cichla monoculus (4 out of 28 specimens) and Cichla sp. (4 out of 13 specimens), and pike cichlids Crenicichla reticulata (2 out of 5 specimens), with no similar standard chromosomal morphology. C-banding revealed that B chromosomes are totally heterochromatic. We suggest two scenarios for the origin of these B chromosomes either by chromosomal breakdowns due to mutagenic action of methyl mercury present in the aquatic environment or by interspecific origin due to hybridization events.

Transfer of Powdery Mildew Resistance from Brassica carinata to B. oleracea

Powdery mildew is a fungal disease of crucifers, caused by Erysiphe polygoni D.C. and it can be problematic during seed increase in green-houses. Crosses were made between Brassica carinata (Ethiopian mustard) accession (PI 360883) and B. oleracea cultivars `Titleist' and `Cecile' to transfer resistance to powdery mildew to B. oleracea germplasm. It was not possible to obtain interspecific hybrids between Ethiopian mustard and B. oleracea through natural seed set. However, interspecific hybrids and backcross one (BC1) progenies were produced via embryo rescue following sexual crosses. Four interspecific hybrid plants were produced with the aid of embryo rescue from cultured pistils with B. carinata as the maternal parent, and their interspecific origin was confirmed through plant morphology and analysis of RAPD polymorphisms. No interspecific hybrids were obtained when `Titleist' was used as a maternal parent. Interspecific hybrid plants were male sterile and they were used as maternal parents to produce BC1 plants. Twenty one BC1 plants were obtained through natural seed set and embryo rescue, although embryo rescue was not necessary to produce first backcross generation plants. When tested in greenhouse with powdery mildew, all interspecific hybrids and eight of the BC1 plants were resistant to the disease. Crosses are being made to produce BC2 plants with 2n = 18 chromosomes for introgression of the resistance in B. oleracea.

ROOTSTOCK BREEDING FOR STONE FRUITS

Over the last 20 years stone fruit rootstock development has begun shifting from seedling to clonal types, many of interspecific origin. Publicly funded breeding programs have produced most of these rootstocks due to the time, cost, and risk associated with their development; however, private industry is emerging as a significant contributor of many of the newer rootstocks. Particularly noteworthy among recent releases has been the incorporation of resistance to soilborne diseases, nematodes, waterlogging and vigor control, the last most notably in recent cherry rootstocks. Nevertheless, despite the remarkable progress in the development of clonal stocks, seedling rootstocks still dominate most stone fruit industries around the world, if only because of their relatively low cost, ease of propagation, and proven utility. Many opportunities and challenges remain to be addressed in the areas of disease and insect resistance, adaptation to biotic and abiotic stresses, graft compatibility, and rootstock influence on scion performance and fruit quality. Biotechnology is beginning to show potential in accelerating rootstock development. With the development of markers to assist selection for difficult to evaluate traits, new rootstocks with resistance to multiple diseases are feasible. Future prospects for breeding are presented. INTRODUCTION Several surveys have been undertaken to determine the relative importance of the various “problems” facing stone fruit industries around the world. The peach (Prunus persica) industry has received the most attention, if only because of its large size, hence economic importance, relative to other stone fruit crop industries. Results of two of these surveys are summarized in Table 1. Relative order of importance changed little between 1982 and 1997. At this time it would appear that the need for waterlogging tolerance has been alleviated somewhat by recent releases. This factor was the only one that moved down significantly in importance since the earlier survey. Given the number of rootstock releases that offer some relative promise for most of the problem areas listed, this might seem surprising at first. However, given that orchard life expectations for stone fruits range from 15 to 25 years or longer, adoption is an inherently slow process. This may also be due in part to the fact that many interspecific rootstocks change other “non-target” characteristics of the finished tree, for example, vigor or bloom date which require changes in management that growers may not wish to change. Additionally, many “problem” sites have more than one limitation and require that a new rootstock incorporate resistance to multiple problems for successful adaptation. In many cases, new rootstocks are probably best suited for regional or prescription/niche planting rather than broad use over a large industry. Regional testing is the only way to determine each rootstock’s best adaptation. Priorities vary from one stone fruit crop to another. Rom (1991) has summarized priorities for apricots (P. armeniaca) which generally agreed with those cited for peach. Nematodes were of less importance and one particularly vexing issue with apricot was that of scion/rootstock graft compatibility. However, Rom noted that priorities also varied Proc. XXVI IHC – Genetics and Breeding of Tree Fruits and Nuts

Genome size analysis in the genus Cucurbita and its use for determination of interspecific hybrids obtained using the embryo-rescue technique

The investigation includes the analysis of genome sizes of 11 Cucurbita species (some represented by several botanical varieties). In the majority of species, the genome size was measured for the first time. Values obtained by flow cytometry were based on propidium iodide (PI) staining technique. In contrast to the uniform chromosome number, the genome size differences within this genus appeared to be large, 2C values ranging from 0.686 pg in C. foetidissima to 0.933 in C. ficifolia. PI determined genome sizes of five cultivated species were 0.864 pg ( C. pepo) , 0.887 pg ( C. maxima) , 0.933 pg ( C. ficifolia) , 0.708 pg ( C. moschata) and 0.748 pg ( C. argyrosperma). The investigation also included several interspecific crosses among cultivated species, using embryo rescue technique. F 1 hybrid plants were obtained from crosses: C. maxima× C. pepo (and vice versa), C. pepo× C. moschata, C. ficifolia× C. maxima, (C. maxima×C. moschata)× C. pepo, C. argyrosperma× C. moschata (and vice versa). The formation of plantlets in the last combination, where C. moschata was used as the female parent, had not been previously reported. The interspecific origin of regenerants was confirmed by evaluating relative nuclear DNA content, estimated by DAPI staining. In all hybrid combinations, the relative nuclear DNA contents in interspecific hybrids was intermediate and differed significantly from those determined in parental species. The results show that differences in genome size within the genus Cucurbita are big enough to be used efficiently in determination of interspecific hybrids.

Sugarcane microsatellites for the assessment of genetic diversity in sugarcane germplasm

The ability of microsatellite or simple sequence repeat (SSR) markers to determine the level of genetic diversity between members of the genera Saccharum ( S. officinarum, S. sponfaneum, S. sinense), Old World Erianthus Michx. sect. Ripidium, North American E. giganteus ( S. giganteum), Sorghum and Miscanthus were assessed. Six SSR markers were tested on 66 accessions and produced a total of 187 distinct alleles. Where available, results were compared against published data from other molecular marker systems such as RFLPs, RAPDs, AFLPs and 5S rRNA intergenic spacers. Similarity coefficient calculations and clustering revealed a genetic structure for Saccharum and Erianthus sect. Ripidium that reflected closely the relationship previously identified using other marker systems. The results indicated that SSRs will be an ideal means for the identification of the genetic constitution of modern sugarcane cultivars of interspecific origins.

Genetic dissection of a modern sugarcane cultivar (Saccharum spp.).II. Detection of QTLs for yield components

The genetics of current sugarcane cultivars ( Saccharum spp.) is outstandingly complex, due to a high ploidy level and an interspecific origin which leads to the presence of numerous chromosomes belonging to two ancestral genomes. In order to analyse the inheritance of quantitative traits, we have undertaken an extensive Quantitative Trait Allele (QTA) mapping study based on a population of 295 progenies derived from the selfing of cultivar R570, using about 1,000 AFLP markers scattered on about half of the genome. The population was evaluated in a replicated trial for four basic yield components, plant height, stalk number, stalk diameter and brix, in two successive crop-cycles. Forty putative QTAs were found for the four traits at P = 5 x 10(-3), of which five appeared in both years. Their individual size ranged between 3 and 7% of the whole variation. The stability across years was improved when limiting threshold stringency. All these results depict the presence in the genome of numerous QTAs, with little effects, fluctuating slightly across cycles, on the verge to being perceptible given the experimental resolution. Epistatic interactions were also explored and 41 independent di-genic interactions were found at P = (5 x 10(-3))(2). Altogether the putative genetic factors revealed here explain from 30 to 55% of the total phenotypic variance depending on the trait. The tentative assignment of some QTAs to the ancestral genomes showed a small majority of contributions as expected from the ancestral phenotypes. This is the first extensive QTL mapping study performed in cultivated sugarcane.

Application of some biotechnological achievements in sunflower breeding

Basic in vitro techniques, linked with the increase of genetic variability and acceleration of the breeding process, have been mastered and applied in sunflower "breeding programs at Dotoroud-a Agricultural Institute, Bulgaria The method of direct organogenesis has been successfully used for genetic modifications of fertility restorer lines RHA-857, 147 R and Z-8-A and for overcoming the inability for crossing between H annuus (hybrid Albena) and Heltanthas tuberosus, Hehanthus sahcifolius, Hehanthus maximiliam Hehanthus stlphioides, Verbisma hehanthoides and between H molds and H annuus. The method of y-mduced parthenogenesis has been successfully applied to the cultivated sunflower (H annuus L ) for obtaining doubled haploid lines. The parthenogenetic ability of both maternal and paternal parents and the gamma-irradiation effect on parthenogenic embryo development have been studied A number of researches have been focused on the genetic potential of wild species from the genus Heltanthus. Our studies investigated the possibilities of the methods for cultivation of embryos and anthers from interspecific origin. They included the use of the embryo cultivation technique under in vitro conditions until the production and testing of finished lines that could directly participate in the breeding process. Embryos were isolated from interspecific hybrids (cultural x wild and wild x cultural) with the participation of the species H resmosus M-046, H strumosus M-059, H sahcifolius M-045 H ciliaris M-092, H giganteus M-030, H maximiliam M-087 and H glaucophylus M-012. The studies included and investigated 51 new lines which originated from embryos of the above interspecific crosses. A number of lines obtained from embryos of the above interspecific crosses exibited valuable economic indices and high combining ability. They were included as components in highly productive hybrids.

Diversity in sugarcane species and hybrids for peroxidase isozyme

Peroxidase isozyme was surveyed in 28 clones belonging to five species ofSaccharum L.and inter specific hybrid. Presence of more number of isozymes inSacharum reflected genetic complexity of this polyploid genus.Saccharum species could be identified from one another by the presence/ absence of specific isozymes. S.officinarum andS. robustum showed lesser genetic diversity.S. sinense was the most complex species with more number of isozymes and could be distinguished from its close relativeS. barberi. S. officinarum clones link Manjri Red, Baragua, Gungera and 57NG77 had isozymes characteristic of other species, as also reflected in the dendrogram, indicating introgression of genes from other sources in their origin. 2n = 64 cytotypes ofS. spontaneum were more similar, but were divergent from the rest including the other cytotypes of the species. F1 hybridity were intermediate between the parents, proving their hybridity. Commercial hybrids showed less diversity with species accessions in support to their interspecific origin. Peroxidase diversity among commercial hybrids quantified in terms of similarity indices may be useful in identifying diverse cross combinations for deriving heterotic hybrids in sugarcane breeding programmes.

Molecular investigation of the genetic base of sugarcane cultivars

Molecular diversity was analysed among 162 clones of sugarcane using DNA restriction fragment length polymorphism (RFLP). One hundred and nine of them were modern cultivars of interspecific origin; most of them were bred in Barbados or in Mauritius. Fifty three were from Saccharum officinarum species, which is the major source of genes in modern cultivars, prevailing over the part of the genome incorporated from the wild species Saccharum spontaneum. Twelve low-copy nuclear DNA probes scattered over the genome were used in combination with one or two restriction enzymes. A total of 399 fragments was identified, 386 of which were polymorphic. Each sugarcane clone displayed a high number of fragments per probe/enzyme combination, illustrating the polyploid constitution of the genome. Among the S. officinarum clones, those from New Guinea had the largest variability and encompassed that present among clones collected from the Indonesian Islands and those known to have been involved in the parentage of modern cultivars. This is in agreement with the hypothesis that New Guinea is the centre of origin of this species. The clones from New Caledonia formed a separate group and could correspond to S. officinarum clones modified through introgression with other members of the ‘Saccharum complex’. Despite the low number of S. officinarum clones used for breeding cultivars, more than 80% of the markers present in the whole S. officinarum sample were also found in modern cultivars due probably to a high heterozygosity related to polyploidy. Among the cultivars, the two main groups, originating from Barbados and Mauritius, were clearly separated. This appeared essentially due to S. spontaneum alleles present in Mauritian cultivars and absent in Barbadan ones, probably in relation to the regular use of early generation interspecific hybrids in the breeding program employed in Mauritius.

Nicotiana tabacum L. as a source of cytoplasmic male sterility in interspecific cross with N. alata Link & Otto

A new source of cytoplasmic male sterility (CMS) in tobacco with interspecific origin is reported. In traditional selection wild tobacco species have been used as donors of cytoplasm. In the present study the cultivated species Nicotiana tabacum L. (n = 24) is a source of CMS. It was used as female parent and N. alata (n = 9) was involved as a pollinator. The F1 hybrid of this cross was completely sterile. Tissue culture method was applied to restore the female fertility. Regenerants obtained from the 5th passage were successfully pollinated with N. tabacum and seed-containing capsules were formed. All BC1P1 plants were male sterile. They possessed normally developed corollas, three-loculed or deformed pistils, and 1–2 stamens modified into secondary pistils. In some plants stamenless flowers were observed. Male sterility of BC1P1 was preserved in BC2P1–BC7P1 progenies confirming its cytoplasmic nature.

Cytoplasmic male sterility in Nicotiana tabacum L. obtained through interspecific hybridization

Three new sources of cytoplasmic male sterility in tobacco with interspecific origin are reported. The wild species Nicotiana excelcior Black (n = 19), N. amplexicaulis Burbidge (n = 18) and N. rustica L. (n = 24) are used as donor of cytoplasm. Nuclear donor is N. tabacum L. (n = 24). The genetic effects of cytoplasmic-nuclear interactions of the studied species are discussed. Our experiments prove that N. excelcior, N. amplexicaulis and N. rustica are carriers of cytoplasmic genes for male sterility that express in combination with nuclear genes of N. tabacum.

On the Use of Essential Oil Profiles for the Determination of Parentage of Cultivars of Unknown or Suspected Interspecific Origin

On the Use of Essential Oil Profiles for the Determination of Parentage of Cultivars of Unknown or Suspected Interspecific Origin

Evidência isoenzímica sobre a origem interespecífica do café Piatã

Encontrou-se, anos atrás, em uma lavoura de Coffea arabica (2n = 44), uma forma de café bastante distinta das demais. Inicialmente, julgou-se tratar de um cafeeiro autotetraplóide de C. liberica (2n = 22) ou de C. dewevrei (2n = 22). Estudos morfológicos e citológicos de tal cafeeiro, denominado Piatã (prefixo C387 da Seção de Genética do IAC), assim como de seus descendentes, indicaram tratar-se, provavelmente, de um híbrido natural derivado da união de um gameta normal de C. arabica e de um não reduzido de C. dewevrei. No presente trabalho, os estudos dos padrões eletroforéticos das enzimas PGI, PGM e ADH do endosperma de sementes confirmaram a origem interespecífica do café Piatã. A combinação de alelos dessas enzimas é distinta e específica para C. dewevrei e C. arabica e tais alelos segregam nas sementes do cafeeiro Piatã. O estudo das isoenzimas das sementes mostram também que, ao contrário do que se pensava, o café Piatã não é auto-incompatível, sendo freqüente a autofecundação. Análises semelhantes, além de úteis na identificação de outros híbridos naturais entre espécies de Coffea, serão de grande valia nos trabalhos básicos de genética, evolução e melhoramento do cafeeiro, nos quais importa conhecer a origem, constituição genética e biologia da reprodução das plantas.

Relationships among ancestral species of sugarcane revealed with RFLP using single copy maize nuclear probes

DNA restriction fragment length polymorphism (RFLP) analysis was performed on 50 wild and old cultivated sugarcane accessions. Ninety-four maize low copy nuclear DNA sequences of known chromosomal position were screened for hybridization to digested sugarcane genomic DNA blots. Seventy-five (80%) gave very strong hybridization signals and usually yielded many bands and detected profuse polymorphism. Twenty-nine probes and 36 probe/enzyme combinations were selected on the basis of the scorability of the banding profiles. A total of 1110 fragments were separately identified among the 50 genotypes. Multivariate analyses of the data allowed the separation of the three basic species, Saccharum spontaneum, S. robustum and S. officinarum, showed that S. spontaneum had structure which could be related to the geographic origin of the clones and supported current hypotheses on the origin of secondary species S. barberi and S. sinense. The use of more probes did not improve the resolution between the various species examined but identified a few key polymorphisms which were not accounted for by current phylogenetic hypotheses and can guide future analyses. RFLPs in sugarcane will be useful essentially for depicting the genomic constitution of modern varieties of interspecific origin.

Chromosome Identification in Potato Trisomics(2n=2x+1=25) by Conventional Staining, Giemsa C-banding and Non-radioactive in situ Hybridization.

Identification of the potato chromosomes 1 (the longest one), 2 (the carrier of the nucleolar organizer, NOR) and 12 (the shortest one) is possible at mitosis using a combined Feulgen-haematoxylin staining or a Giemsa C-banding technique. In two aneuploids containing 27 chromosomes, three specimens of chromosome 1 were found through conventional staining. Variation of the length of the NORs and the size of the satellite of homologues or homoeologues of chromosome 2 was observed in various cytotypes. Pachytene analysis identified trisomics of chromosome 2 of interspecific origin, involving S. tuberosum L. ssp. tuberosum Hawkes and S. phureja Juz. et Buk., but only two specimens of chromosome 2 could be identified using the Giemsa C-banding technique. However, in six different interspecific chromosome 2-trisomics, three chromosomes per cell were found to show a hybridization signal by non-radioactive in situ hybridization with heterologous rDNA from pea. The high polymorphism of chromosomes observed after Giemsa C-banding made it impossible to bring the identity of the mitotic chromosomes in accordance with the results from pachytene identification in cells containing one triplicate chromosome.

BRIDGE SPECIES FOR INTROGRESSION OF WILD GERMPLASM IN SWEET POTATO BREEDING.

The species of section Batatas represent the primary potential source of wild germplasm for sweet potato breeding. Their wide distribution and adaptation make their use desirable, but their direct use is constrained by their ploidy level (2x, and 4x, in contrast with cultivated I. batatas which is 6x) and lack of storage root production.We investigated 4x clones of interspecific origin (6x I. batatas 2x I. trifida) as bridges to introgress genes from several wild species of the section Batatas to I. batatas. Six 2x wild species from different origins were reciprocally crossed to the 4x clones. The crosses were successful only when the diploid wild species were used as females (2x × 4x) indicating unilateral interspecific incompatibility. The unsuccessful types of crosses (4x × 2x) were repeated using wild hybrid males containing I. trifida in their parentage. This time only those crosses involving a hybrid having I. trifida as its female parent produced seeds. Progenies from the successful 2x × 4x crosses were diploid instead of the expected triploid. Triploid progenies were obtained only in the 4x × (2x hybrid) crosses. Some triploid progenies produced 2n pollen in varying frequencies. These results show different levels of crossing barriers among Ipomoea species and give us alternative pathways for overcoming them. I. trifida was identified as a possible bridge species.

Molecular Characterization of the Interspecific Origin of Viviparous Onion

Molecular Characterization of the Interspecific Origin of Viviparous Onion

Mitochondrial involvement in petal and stamen development studied by sexual and somatic hybridization of Nicotiana species

The interspecific origin, frequency of occurrence, and variation in phenotype of cytoplasmic male-sterile cultivars of Nicotiana tabacum are reviewed in order to provide a framework for understanding cell biological investigations of the interaction between mitochondria and the nucleus in regulating development of floral organs in these cultivars. Analyses of mitochondrial protein synthesis mitochondrial DNA, and RNA transcripts have shown that nuclear effects on mitochondrial activities are very common. Cybridization of cytoplasmic male-sterile cultivars has produced heritable alterations in phenotype which can be correlated with changes in mitochondrial DNA and RNA transcripts. A hypothesis is presented that the different morphologies of cytoplasmic male-sterile cultivars are indicative of variation in activity of several different mitochondrial genes, not yet identified. According to this hypothesis, transcripts from these genes differ in each cultivar, in response to nuclear genes of N. tabacum. The particular pattern of differential expression accounts for the variations in phenotype that characterize each different interspecific combination of N. tabacum nucleus and Nicotiana sp. mitochondria. Evidence in support of this hypothesis has been assembled by comparisons of male-sterile cultivars with cultivars restored to fertility either by nuclear restorers or by somatic cybridization.