The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Genetic diversity in the orange subfamily Aurantioideae. II. Genetic relationships among genera and species

Genetic relationships were studied by means of ten isoenzymatic systems, at the genus and species level, using two distances and four methods of aggregation in a germplasm collection of 198 cultivars and accessions of 54 species belonging to Citrus and 13 related genera. The most consistent results were obtained by the chord distance and the neighbor-joining clustering method. Citrus species were distributed in two main groups: the orange-mandarin group and the lime lemon-citron-pummelo group. The species C. halimii and C. tachibana are not included in these groups. Mandarin species fall into three main subgroups: one includes C. sinensis; the second, C. aurantium, the third, small-fruit species. The citron, the pummelo and the ancient lemon subgroups form a cluster to which the species belonging to subgenus Papeda and the cultivated limes, lemons and bergamots are related. Microcitrus spp, to which Severinia buxifolia and Atalantia ceylanica seem to be related, cluster with the lime lemon-citron-pummelo group while Fortunella is close to the orange-mandarin group. Poncirus trifoliata, the most important species for citrus rootstock improvement is located far from Citrus but connected to it through Fortunella spp. A broad distribution of species has been found that should be taken into account to sample new genotypes in the search of desired characters in order to fully and efficiently use genetic resources for citrus improvement.     

Allotetraploid hybrids between citrus and seven related genera produced by somatic hybridization

We have developed an efficient protoplast-fusion method to produce somatic hybrid allopolyploid plants that combine Citrus with seven related genera, including four that are sexually incompatible. In this paper we report the creation of 18 new allotetraploid hybrids of Citrus, including ten among sexually incompatible related genera, that may have direct cultivar potential as improved citrus rootstocks. All hybrids were confirmed by cytological and RAPD analyses. If fertile, the attributes of these hybrids may be amenable to further genetic manipulation by breeding at the tetraploid level. Wide somatic hybridization of Citrus via protoplast fusion bypasses biological barriers to the natural allopolyploidization of Citrus, and creates new evolutionary opportunities that would be difficult or impossible to achieve by natural or conventional hybridization.Florida Agricultural Experiment Station Journal Series No. R-04520     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

基于SSR标记探讨三种金花茶植物的遗传多样性和遗传结构

薄叶金花茶、小花金花茶和小瓣金花茶是三种濒危金花茶植物,为了解珍稀濒危植物遗传多样性和遗传结构,该研究利用微卫星标记对他们的7个种群共184个个体进行了遗传多样性和遗传结构分析。结果表明:11个位点共检测到等位基因92个。在物种水平上,小瓣金花茶平均等位基因数(N_A)为3.9、有效等位基因数(N_E)为2.328、观测杂合度(H_o)为0.520、期望杂合度(H_e)为0.501,高于薄叶金花茶和小花金花茶。在种群水平上,有效等位基因数(N_E)在1.788～2.466之间,期望杂合度(H_e)在0.379～0.543之间;种群间遗传分化系数(FST)在0.143 7～0.453 3之间,种群间基因流(N_m)在0.301 5～1.488 9之间。AMOVA分子变异分析显示65.72%的变异存在于种群内。三种金花茶具有较低水平的遗传多样性和高水平的种群间遗传分化。STRUCTURE和PCoA种群遗传结构分析结果将取样种群分为2组,即薄叶金花茶和小花金花茶大部分个体分为一组,小瓣金花茶大部分个体分为一组。现存所有种群应根据实际情况尽快采取就地保护或迁地保护措施。     

Genetic variability in Lycopersicon species and their genetic relationships

Summary Genetic diversity has to be described and measured in order to establish breeding strategies and manage genetic resources. It is also fundamental to develop a comparative intraspecific study before attempting to discuss and conclude any phylogenetic relationship. The genetic variability of Lycopersicon species was studied using starch gel electrophoresis of 11 enzymatic systems in a hierarchical fashion. The species with the greatest genetic variability are L. chilense, L. peruvianum and L. pennellii, mainly due to the within-line component. L. chmielewskii, L. parviflorum and L. pimpinellifolium show an intermediate total variability and their between-component clearly predominates over the within-component. The least variable species are L. cheesmanii and L. esculentum. Cluster analysis resulted in three main groups: one formed by the cultigen, L. pimpinellifolium, L. cheesmanii and L. peruvianum;another by two species with self-incompatibility systems, L. pennelli and L. chilense; and another by two autogamous species L. chmielewskii and L. parviflorum. With respect to L. esculentum the farthest related species is Solanum rickii and the closest, L. pimpinellifolium.     

Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Study of pollen brush in selected species of Astragalus L. subgenus Pogonophace Bunge (Leguminosae)

Nine species of Lippia (Verbenaceae) were studied by RAPD markers in order to evaluate the degree of genetic diversity. The following species were collected at the Cadeia do Espinhaço Mountains, Southeast Brazil: L. corymbosa, L. diamantinensis, L. filifolia, L. florida, L. hermannioides, L. lupulina, L. rotundifolia, L. rosella and L. sidoides. The analysis was performed using 18 primers that generated 490 fragments and only one primer was found to be monomorphic in all individuals. The average interspecific genetic distances were similar for all species and higher than the intraspecific genetic distances. Species with narrow occurrence did not show low intraspecific diversity. The molecular data were used to generate an UPGMA dendrogram that showed two major groups with a clear distribution among the species. RAPD analysis was efficient to address the genetic diversity of Lippia species and contributed to understand the adaptation to the environment, conservation and taxonomic implications.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Refinement of the Citrus Tristeza Virus Resistance Gene (Ctv) Positional Map in Poncirus trifoliata and Generation of Transgenic Grapefruit (Citrus paradisi) Plant Lines with Candidate Resistance Genes in this Region

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it’s slow spread in R-2 plant lines or an initial appearance of infection and it’s subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it’s 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Factors affecting Citrus tree isozyme-gene expression

Ten enzymatic systems of Citrus species and cultivars have been evaluated for identification purposes and for genetic variability studies. The following factors that could affect their expression were studied: season of sampling, location, rootstock, position of the branch, infection, and age of the tree. Differences involving the presence-absence of the Cu/Zn SOD within the same tree were found. This difference is mainly related to the position of the leaf relative to the sunlight. No change was observed at any of the ten enzymatic systems assayed regarding the location, the rootstock, the growing condition, the season, or the infection with most virus and virus-like pathogens. Viroids induced noticeable changes on 6PG and PRXa zymograms in C. medica. A new peroxidase (not present in healthy plants) was detected that could be related to appearance of symptoms. This may induce errors when trees without sanitary control are characterized by this enzymatic system. On the other hand, it provides a new possibility for studying the plant response to the presence of viroids. An effect of age, from 3 months up to 12 years, was observed on citrange Troyer and mandarin Cleopatra PRX, MDH and 6PG patterns. An important change occurs around the first year, most likely related to the end of the seedling stage. This is followed by a long transition phase, the end of which (around 9 years later) coincides with a change in the PRX pattern. These age-related changes seem to involve post-translational modifications of pre-existing isozymes.     

Development and characterization of ten novel microsatellite markers from olive ridley sea turtle (Lepidochelys olivacea)

Olive ridleys, one of the widely distributed marine turtle species has undergone declines in recent years due to multiple anthropogenic factors warranting conservation efforts for which assessment of genetic variability in existing populations become critical. Here we describe development of ten new microsatellite markers from a short sequence repeat-enriched partial genomic DNA library, which are found to be highly informative for genetic studies. Eight of these markers when tested on 83 olive ridley turtles revealed high allelic diversity (4–27 alleles per marker), and high observed and expected heterozygosity estimates that ranged from 0.29 to 0.82 and 0.62 to 0.94, respectively. Two microsatellites were monomorphic in the tested olive ridley samples, but were found to be informative/polymorphic when tested on related marine turtle species. More importantly, nine of the new markers showed robust cross-species amplifications in three related species Dermochelys coriacea, Chelonia mydas and Eretmochelys imbricata. Thus, this study describes ten new microsatellite markers and also demonstrates their potential as efficient genetic markers in studies related to parentage analysis, population structure, phylogeography and species relationships of olive ridleys and other marine turtle species.     

利用DNA片段测序方法探究枳属和富民枳的分类地位

富民枳(Poncirus polyandra)属于芸香科(Rutaceae)枳属(Poncirus Raf.).自发表以来,分类地位一直备受争议,其中在Flora of China中认为富民枳为柑橘杂交种(Poncirus polyandra),把枳属归并于柑橘属(Citrus).该研究选取枳属的富民枳、枳(Ponci...     

Genetic variation in Simocephalus (Anomopoda: Daphniidae) in North America: patterns and consequences

The presumption of intercontinental distributions of many species of Anomopoda has been reinforced by their considerable morphological uniformity and ease of passive dispersal via ephippial eggs. To test the validity of this dogma, genetic variation among taxa in the cladoceran genus Simocephalus was examined on a continental scale. Genetic variability (percentage polymorphic loci = 8.8%, individual heterozygosity = 3.4%) was comparable to that determined on a local scale in Simocephalus and somewhat less than for other zooplankton groups. Four species complexes were distinguished allozymically with unique allelic substitutions found for at least two loci between these taxa. Eight species in North America were differentiated within these complexes (Nei s genetic distance, D>0.30), all of which were clearly separated from two European species. Both S. cf. vetulus and S. cf. serrulatus are broadly distributed in North America, and intraspecific divergence is relatively low. In contrast, S. cf. congener and S. cf. exspinosus represent species complexes, both consisting of several species, differentiated allozymically and morphologically. The concept of generalist species with moderate genetic variation throughout a broad range and specialist species, genetically depauperate, with restricted distributions, is explored.     

Examining morphological and molecular diagnostic character states of Cichorium intybus L. (Asteraceae) and C. spinosum L.

The inter- and intraspecific variability of Cichorium intybus L. was examined to evaluate potential morphological and molecular diagnostic character states. Two diagnostic and one overlapping morphological character clearly delimit the two species C. intybus and C. spinosum. All applied molecular methods (ITS, AFLP, Microsatellites) failed to significantly discriminate between these accepted species. As the morphological traits are fixed and heritable, criteria for species delimitation are fulfilled. These traits, however, are apparently due to mutations of a few crucial loci affecting the morphological diagnostic character states. Intraspecific variability within C. intybus revealed to be highly influenced by plastic response to local environmental factors and subspecific delimitation cannot be supported.     

Spatial genetic patterns in lagoonal, reef-slope and island populations of the coral Platygyra daedalea in Kenya and Tanzania

Considering the rapid degradation of coral reefs, it is becoming increasingly important to assess factors such as levels of intraspecific genetic diversity and degree of connectivity between populations and reefs. In this study, five DNA microsatellite markers were used to infer migration patterns and levels of genetic diversity in ten populations of the faviid coral Platygyra daedalea along the coast of East Africa. Populations from reef-slopes and offshore islands had significantly greater genetic diversity, measured as expected heterozygosity and allelic richness than those of inshore lagoonal reefs. A combination of F-statistics and individual assignment tests indicated moderate to high levels of gene flow among lagoonal populations, and less migration between lagoonal sites and the reef-slope and island sites. These results suggest that reef-slope and island reefs could be important reservoirs of genetic diversity for this coral species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees

Summary Iodoacetate-treated Citrus protoplasts from embryogenic nucellar calli of Sour orange (C. aurantium) or from Rough lemon (C. jambhiri) were fused with -irradiated protoplasts from a related genus, Microcitrus. The fused protoplasts were cultured to obtain colonies and micro-calli. Micro-calli derived from these two fusion combinations were isolated, propagated and differentiated into embryos, which subsequently regenerated trees having the morphology of Sour orange or Rough lemon. These intergeneric fusions resulted in mitochondria with novel DNA, indicating recombination between the chondriomes of Citrus and Microcitrus. Chloroplast DNA analyses of fusion-derived embryos indicated that they contained the chloroplasts of either fusion-partner or a mix of these chloroplasts. Later plastome analyses of leaves from fully differentiated plants showed that cybrids having Rough lemon morphology had either Rough lemon or Microcitrus chloroplast DNA, indicating complete sorting out of chloroplasts. Likewise, sorting out of Microcitrus chloroplasts was detected in a cybrid plant having Sour orange morphology.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel. No. 2663-E, 1989 series     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Genetics of esterase isoenzymes in Malus

Summary Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species.