Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

Background and Aims

Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.

Methods

Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of ‘Tardivo di Ciaculli’ mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (‘Carrizo’ citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.

Key Results

Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.

Conclusions

Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.     

柑橘愈伤组织生长速度与体细胞胚胎发生的关系

为了探讨柑橘愈伤组织不能再生的原因,试图寻找柑橘愈伤组织生长速度与其体细胞胚胎发生之间的关系,对7种柑橘类型的29种基因型的愈伤组织的生长速度进行了测定,并对愈伤组织生长速度与体细胞胚胎发生之间的相关性进行了统计分析。结果表明,柑橘愈伤组织生长速度与体细胞胚胎发生之间的相关系数为r=-0.3683。由此推断在这两者之间还存在其它影响因素。     

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.     

Specific features of early embryogenesis in apomictic <Emphasis Type="Italic">Poa pratensis</Emphasis> L.

We studied the early stages of embryo formation in apomictic Poa pratensis L. It was shown that during transition to parthenogenesis, at least at the initial stages of embryogenesis, the algorithm of development of the sexual embryo is preserved. This could be due to the system of genetic control of embryogenesis, common for amphimixis and apomixis. We described asynchrony of developmental processes both within the efflorescence (asynchronous maturation of seed-buds) and within the seed-bud and even gametophyte (different timing of induction of apoarchesporic initials and oospores). This feature of pseudogamous apomicts allows them to produce simultaneously both sexual and apomictic progenies.     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Distribution ofGeotrichum candidum citrus race in citrus groves and non-citrus fields in Japan

The presence ofGeotrichum candidum citrus race, the citrus sour rot pathogen, was examined in soils of citrus groves and non-citrus fields of Japan. Soil samples were collected from 223 sites (118 sites in citrus groves, and 105 sites in fields cultivated with 33 species of non-citrus plants and in evergreen broad-leaved forest) in 11 main citrus-growing prefectures, and Hokkaido, a non-citrus-growing area. Of 236 soil samples from citrus groves, 95.76% containedG. candidum citrus race and 0.42% contained the non-citrus race; while of 210 samples from non-citrus fields, 62.85% and 4.76% contained the citrus race and the non-citrus race respectively. All of the citrus race isolates obtained either from citrus groves or non-citrus fields were pathogenic on lemon (Citrus limon) and satsuma mandarin (Citrus unshiu), but some of these isolates failed to infect orange (Citrus sinensis). The non-citrus races were pathogenic on ripe tomato fruit (Lycopersicon esculentum) and ripe muskmelon fruit (Cucumis melo var.reticulatus). Results indicated that citrus sour rot pathogen is widely distributed in citrus groves and non-citrus fields of diverse plant species in Japan.     

花椒球心胚及胚乳的发生和发育

对花椒珠心胚及胚乳的发生和发育过程进行了详细的细胞学及细胞学研究。主要研究结果如下;珠心胚发生前,有性胚囊发育过程中从大孢子发生到胚囊形成的各个阶段均可发生退化,退化频率５０％,未退化的胚囊发育成熟,成熟胚囊仅含卵器和两个极核。卵器最终退化,极核不经受精自发形成胚肥。当胚乳游离核达到１５或３２个时,最早的珠心胚原始细胞由靠近胚囊球孔端的珠心细胞分化形成。随着子房生长,多个原始细胞持续不断地从珠孔端     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.     

Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes

Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1‐BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes – the submetacentric Het′ and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.     

Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula

Apomicts tend to have larger geographical distributional ranges and to occur in ecologically more extreme environments than their sexual progenitors. However, the expression of apomixis is typically linked to polyploidy. Thus, it is a priori not clear whether intrinsic effects related to the change in the reproductive mode or rather in the ploidy drive ecological differentiation. We used sympatric sexual and apomictic populations of Potentilla puberula to test for ecological differentiation. To distinguish the effects of reproductive mode and ploidy on the ecology of cytotypes, we compared the niches (a) of sexuals (tetraploids) and autopolyploid apomicts (penta‐, hepta‐, and octoploids) and (b) of the three apomictic cytotypes. We based comparisons on a ploidy screen of 238 populations along a latitudinal transect through the Eastern European Alps and associated bioclimatic, and soil and topographic data. Sexual tetraploids preferred primary habitats at drier, steeper, more south‐oriented slopes, while apomicts mostly occurred in human‐made habitats with higher water availability. Contrariwise, we found no or only marginal ecological differentiation among the apomictic higher ploids. Based on the pronounced ecological differences found between sexuals and apomicts, in addition to the lack of niche differentiation among cytotypes of the same reproductive mode, we conclude that reproductive mode rather than ploidy is the main driver of the observed differences. Moreover, we compared our system with others from the literature, to stress the importance of identifying alternative confounding effects (such as hybrid origin). Finally, we underline the relevance of studying ecological parthenogenesis in sympatry, to minimize the effects of differential migration abilities.     

激光显微切割技术用于子宫内膜异位组织DNA的提取及其完整性分析

目的：探索一套激光显微切割（LCM）分离子宫内膜异位症腺体细胞后提取微量DNA并进行完整性分析的操作流程。方法：分别对20例石蜡标本及20例冰冻标本进行LCM,收集切割后的腺体细胞;2组标本各取10例提取微量DNA,检测DNA浓度并通过PCR扩增进行验证;余20例标本分别进行全基因组扩增,检测产物浓度并利用8种常见管家基因作为引物通过PCR扩增进行验证,对比分析其结果。结果：石蜡标本与冰冻标本在LCM获取腺体细胞及提取微量DNA两个环节中均可获得满意效果;但经全基因组扩增后,石蜡标本无法保留完整DNA信息。结论：LCM获取子宫内膜异位症腺体细胞提取微量DNA是一种操作简单、结果稳定的方法,可作为日后子宫内膜异位症基因组研究的常规方法;冰冻切片相对石蜡切片,更能保留完整的DNA信息。     

Biotic and abiotic factors associated with citrus progressive decline in Fars Province,Iran

Citrus decline has become a devastating problem in citrus-growing regions of southern Iran. The affected trees show progressive yellowing of leaves, thinning of the canopy, dieback and total collapse. Despite a number of attempts, the aetiology of the decline has remained uncertain. In the present study, we evaluated the potential association of various biotic and abiotic factors with the decline in the Fars Province of Iran. Biotic agents surveyed included Candidatus Liberibacter asiaticus, Spiroplasma citri, phytoplasmas, Tylenchulus semipenetrans and root infecting fungi and oomycetes. Abiotic factors studied were soil and water salinity, changes in prevailing temperature and other environmental conditions. In our surveys, Ca. L. asiaticus, either alone or in combination with other factors, had the highest frequency of association (92%) with the decline, followed by S. citri (75%). Ca. L. asiaticus was not detected in any of the non-decline trees examined. Pythium or Phytophthora species and a fungus of theFusarium solani species complex were also isolated from roots of many declining trees. Phytoplasmas were found only in few cases, and populations of citrus nematode were often below the threshold of economic loss. Soil/water salinity were in the suitable range for citrus cultivation in most cases. It is suggested that the decline is initiated by Ca. L. asiaticus infection, which is known to weaken the root system and make it vulnerable to infection by opportunistic soil fungi and oomycetes. S. citri, summer temperatures, low air humidity and overbearing of the trees seem to be other potential factors contributing to the intensity of the disease.     

A comparison of apomictic reproduction in eastern gamagrass (Tripsacum dactyloides (L.) L.) and maize-Tripsacum hybrids

The expression of gene(s) governing apomictic reproduction inTripsacum provides the best foundation for comparing the effectiveness of apomictic reproduction in a series of maize-Tripsacum hybrids. Several 38-chromosome, apomictic maize-Tripsacum hybrids are available which possess the gene(s) conferring apomictic reproduction fromTripsacum. Without a base line for comparison, studies directed towards discerning the successful transfer or effectiveness of gene expression in a maize background are hampered. The objectives of this study are to compare the reproductive features found in apomicticTripsacum with those in apomictic maize-Tripsacum hybrids. In addition, this study determined the feasibility of utilizing these maize-Tripsacum hybrid materials to continue an attempt to transfer the genes into a pure maize background. The frequency and occurrence of five unique reproductive features found in apomictic accessions ofTripsacum dactyloides were compared to the reproductive behaviours exhibited in the maize-Tripsacum hybrids. Results indicate the genes controlling apomixis in tetraploidTripsacum are fully functional in maize-Tripsacum hybrids with diploid and triploid maize constitutions. The ability of theTripsacum apomictic genes to retain full expression provides evidence to continue their transfer to a diploid or tetraploid maize background.The use of company names in this publication does not imply endorsement by the USDA-ARS, or the product names or criticism of similar ones not mentioned. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Genetic diversity in sexual diploid and apomictic tetraploid populations of Paspalum notatum situated in sympatry or allopatry

Paspalum notatum is a subtropical grass widely distributed in the temperate areas of America. Diploids are sexual while polyploids give rise to clonal seeds through aposporous apomixis. RAPD markers were used to analyze the genetic structure of three natural populations: i) diploids reproducing sexually (R2X); ii) sympatric apomictic tetraploids collected in the vicinity of the diploids (R4X); iii) allopatric apomictic tetraploids growing in isolation (C4X). The apomictic reproduction rate was evaluated by the use of molecular markers in progeny tests, while chromosome-counting allowed the verification of ploidy levels. Data revealed that the R4X group presented a variation considerably higher than that observed for C4X. Jaccards coefficients were used to produce a cluster diagram using the UPGMA method. All but one tetraploid genotypes grouped together and were associated to diploid genotype A21. The possibility of occasional generation of novel tetraploid clones from the interaction between tetraploid and diploid individuals is discussed.     

Population genetic structure of diploid sexual and polyploid apomictic hawthorns (Crataegus; Rosaceae) in the Pacific Northwest

Polyploidy and gametophytic apomixis are two important and associated processes in plants. Many hawthorn species are polyploids and can reproduce both sexually and apomictically. However, the population genetic structure of these species is poorly understood. Crataegus douglasii is represented exclusively by self-compatible tetraploid pseudogamous apomicts across North America, whereas Crataegus suksdorfii found in the Pacific Northwest is known to include self-incompatible diploid sexuals as well as polyploid apomicts. We compare population structure and genetic variability in these two closely related taxa using microsatellite and chloroplast sequence markers. Using 13 microsatellite loci located on four linkage groups, 251 alleles were detected in 239 individuals sampled from 15 localities. Within-population multilocus genotypic variation and molecular diversity are greatest in diploid sexuals and lowest in triploid apomicts. Apart from the isolation of eastern North American populations of C. douglasii , there is little evidence of isolation by distance in this taxon. Genetic diversity in western populations of C. douglasii suggests that gene flow is frequent, and that colonization and establishment are often successful. In contrast, local populations of C. suksdorfii are more markedly differentiated. Gene flow appears to be limited primarily by distance in diploids and by apomixis and self-compatibility in polyploids. We infer that apomixis and reproductive barriers between cytotypes are factors that reduce the frequency of gene flow among populations, and may ultimately lead to allopatric speciation in C. suksdorfii . Our findings shed light on evolution in woody plants that show heterogeneous ploidy levels and reproductive systems.     

橘园中重要天敌具瘤神蕊螨与其主要猎物的生态位

研究了柑橘园中具瘤神蕊螨(Agistemus exsertus)与其主要猎物柑橘皱叶刺瘿螨(Phyllocoptruta oleivora)、柑橘全爪螨(Panonychus citri)、柑橘粉虱(D ialeurodes citri)、黑刺粉虱(Aleurocanthus spiniferus)的生态位宽度、重叠和比例相似性。具瘤神蕊螨与其主要猎物相比,其时间和空间生态位宽度均最大,而其猎物中则以柑橘皱叶刺瘿螨的时间和空间生态位宽度最大,这表明具瘤神蕊螨和柑橘皱叶刺瘿螨的发生期较长,空间分布较广。具瘤神蕊螨与其猎物的生态位重叠在空间上与柑橘全爪螨的重叠值最大,在时间上与柑橘粉虱的重叠值最大。与其猎物相比,具瘤神蕊螨的时—空二维生态位宽度最大,在时空上占有明显的优势。