QTL analysis of citrus tristeza virus-citradia interaction

Citrus tristeza virus (CTV) has caused the death of millions of trees grafted on sour orange (Citrus aurantium). However, this rootstock is very well adapted to the Mediterranean, semi-arid conditions. The aim of the present research is to genetically analyze the accumulation of CTV in a progeny derived from the cross between C. aurantium and Poncirus trifoliata, both resistant to CTV isolate T-346. Graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. Three months later, each rootstock was graft inoculated with two patches of infected tissue (isolate T-346). One, 2, and sometimes, 3 and 4 years after inoculation, hybrids and infected patches were tested for CTV by tissue-blot immuno-assay. Additionally, CTV multiplication was evaluated every year as the optical density of double-antibody sandwich enzyme-linked immuno-sorbent assay reactions. Linkage maps for P. trifoliata based on 63 markers, and for C. aurantium based on 157 markers, were used. Most molecular markers were microsatellites and IRAP (inter-retrotransposon amplified polymorphisms). Some analogues of resistance and expressed sequences were also included for candidate gene analysis.Resistance against CTV was analyzed as a quantitative trait (CTV accumulation) by QTL (quantitative trait loci) analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected where the P. trifoliata resistance gene, Ctv-R, had been previously located in other progenies. Up to five minor QTLs were detected (Ctv-A ₁ to Ctv-A ₅ ). A significant epistatic interaction involving Ctv-R ₁ and Ctv-A ₁ was also found. An analogue of a resistance gene is a candidate for Ctv-A ₃ , and two expressed sequences are candidates for Ctv-A ₁ and Ctv-A ₅ . Single-strand conformational polymorphism analysis of CTV genes QTL P20 and P25 (coat protein) in susceptible hybrids, was carried out to test whether or not any QTL accumulation was a defeated resistance gene. Since the same haplotype of the virus was visualized independently on the CTV titer, differences in the amount of virions are not explained through the selection of CTV genotypes by the host, but through differences among citradias in CTV replication and/or movement.Communicated by C. Möllers     

Genetic analysis of citrus leafminer susceptibility

Damage caused by the citrus leafminer (CLM), Phyllocnistis citrella, is highly dependent on the citrus flushing pattern. Chemical control is only required in young trees, both in nurseries and in newly established orchards. However, this situation is completely different in countries where the causal agent of citrus canker, the bacterium Xanthomonas axonopodis pv. citri exists. CLM infestation results in a higher incidence of citrus canker infection. Among preventive control strategies that provide environmentally sound and sustainable solutions, resistant or tolerant varieties remain the most economical means of insect control. The objective of the present study is to genetically analyse the resistance/susceptibility to CLM and two other traits that might be related, the deciduous behaviour and leaf area of the tree, in a progeny of citradias derived from the cross between two species with different CLM susceptibility—C. aurantium L. and Poncirus trifoliata—using linkage maps of each parent that include several resistance gene analogues. We detected two antibiosis and six antixenosis putative quantitative trait loci (QTLs) in a random sample of forty-two of those citradias. An important antibiosis QTL (R²=18.8–26.7%) affecting both percentage of infested leaves and number of pupal casts per leaf has been detected in P. trifoliata linkage group Pa7, which is in agreement with the CLM antibiotic character shown by this species, and independent from any segregating QTL involved in its deciduous behaviour. The maximum value for the Kruskal-Wallis statistic of the other putative antibiosis QTL coincides with marker S2-AS4_800 in sour orange linkage map. Given that the sequence of this marker is highly similar to several nucleotide binding site-leucine-rich repeat (NBS-LRR)-type resistance genes, it might be considered as a candidate gene for insect resistance in citrus.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Molecular markers flanking citrus tristeza virus resistance gene from Poncirus trifoliata (L.) Raf.

 Two segregating populations for citrus tristeza virus (CTV) resistance derived from Poncirus trifoliata var ‘Flying Dragon’ by self-pollination and pollination to Citrus medica L. var ethrog ‘Arizona’ were inoculated with a common CTV isolate. The presence of virus was checked by the Double Antibody Sandwich Enzyme-Linked Assay and Direct Tissue Blot Inmunoassay at 3, 6, and 12 months after inoculation. Seven RAPDs were found linked to the CTV resistance gene by bulked segregant analysis. The closest linked RAPDs were cloned to obtain linked codominant RFLPs and to increase the precision of the genetic distance estimation. The CTV resistance gene seems to be located between cW18 and cK16. Differences in genetic distances among progenies are large and can be explained by genome-wide reduction in the recombination of progeny derived from male versus female gametes. Received: 5 June 1996 / Accepted: 26 July 1996     

Efficient search for new resistant genotypes to the citrus tristeza closterovirus in the orange subfamily Aurantioideae

 Virulent isolates of the citrus tristeza virus (CTV) are continuously arising and their spread threatens the world citrus industry. Methods for effective utilization of material conserved in germplasm banks are needed in plant improvement. Two objectives are pursued in the present paper: a search for new CTV-resistant genotypes and tests of two strategies for this search. One of these tests is based on a study of genetic relationships among genera and species of the orange subfamily and the other on scores of molecular markers known to be linked to the CTV-resistant locus. Sampled plants were graft-inoculated with a mild CTV isolate (T-346) and two virulent ones (T-388 and T-305). Susceptible plants were those where CTV multiplication was detected beyond 4 months after inoculation. All cultivars of Poncirus trifoliata tested, as well as Severinia buxifolia and Atalantia ceylanica, were resistant to the three CTV isolates; Fortunella crassifolia (Meiwa kumquat) resists two of them. The finding of CTV resistance in this species, closely related to cultivated Citrus species, opens a new arena for CTV-resistance improvement of oranges and mandarines by sexual hybridization. The searching strategy based on phylogenetic data has been successful, whereas the other one may be worthwhile only when the search is restricted to the species where linkage analysis is available. A good documentation system that allows quick sampling of accessions to build up core collections and where the location of new and useful genes could be easily worked out, is suggested to enhance germplasm utilization. Received: 27 April 1997 / Accepted: 5 June 1997     

New gene(s) involved in the resistance of Poncirus trifoliata (L.) Raf. to citrus tristeza virus

 Citrus tristeza virus (CTV) causes important economic losses in the citrus industry worldwide. Resistance to CTV is present in Poncirus trifoliata and is known to be controlled by a dominant gene at the Ctr locus. Short-distance movement of CTV around the inoculum, as well as passive movement through the phloem vessels, were studied in segregant plants derived by self-pollination from P. trifoliata var. “Flying Dragon” in order to genetically analyze the mechanism of CTV resistance. Accumulation of CTV in the vicinity of the inoculum and in new flushes was studied by means of a direct tissue-blot immunoassay (DTBIA). CTV is able to passively move with the phloematic flux from inoculated resistant genotypes Ctr-Rr and Ctr-RR up to a susceptible scion cultivar (Ctr-rr). Differences regarding CTV accumulation around the inoculum were found among Ctr-Rr individuals of the progeny. Bulked segregant analysis identified five RAPD markers linked to a locus (Ctm), or a genomic region, involved in short-distance accumulation of CTV but located in a different linkage group from Ctr. This result indicates that Ctr is not the only locus responsible for resistance to CTV in P. trifoliata, and that at least one other gene is involved. Given that citrus is a perennial crop, breeding for durable disease resistance should take into account selection at both the Ctr and Ctm loci. Received : 13 March 1996 / Accepted : 18 April 1997     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Comparative genome-wide segregation analysis and map construction using a reciprocal cross design to facilitate citrus germplasm utilization

Citrus genetic resources are rich but underutilized in breeding because their complex reproductive biology and the scarceness of inheritance studies on agronomic traits. Here, we investigated the genomic distribution of segregation distortion regions, the inheritance of organelle DNA and colinearity between scion citrus linkage maps by using a reciprocal cross design. The parents were Fortune, a hybrid mandarin from C. clementina, and Chandler, a hybrid pummelo from C. grandis that largely differ in fruit size, taste and colour. The inheritance of organelle DNA was studied in 201 hybrids by using four organelle DNA markers, and the linkage maps were based on 174 of those hybrids. Around ten percent of the seedlings derived from the pummelo as female parent showed the same organelle markers as those of the mandarin, indicating a possible exception to their expected maternal inheritance in citrus. Most segregation distortion affects just the allele frequencies, generally representing differences in pollen fertilization success, as a likely consequence of the presence of gametal factors affecting the functionality of gametes and pollen-pistil interactions. The large extension of colinearity found when comparing the C. grandis and C. clementina linkage maps to those previously reported for rootstock species (C. aurantium and P. trifoliata), will be helpful to infer the position of orthologous genes and QTLs in citrus species and for a more useful genetic characterization of citrus germplasm collections.     

The response of citrus plants to the broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae)

1. Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a common polyphagous mite in tropical and subtropical areas and is considered as an important citrus pest.
2. To understand the response of citrus to P. latus infestation, we have characterized the volatile profile and the molecular defence mechanisms of two citrus genotypes, namely sour orange (Citrus aurantium) and Cleopatra mandarin (Citrus reshni), to P. latus infestation. These two species are important rootstocks for the citrus industry and display differential resistance to Tetranychus urticae Koch (Acari: Tetranychidae), with sour orange showing elevated levels of constitutive and induced resistance associated with the jasmonic acid (JA) pathway compared with Cleopatra mandarin.
3. P. latus infestation activated both the JA- and the salicylic acid-dependent pathways in sour orange but not in Cleopatra mandarin. However, this differential activation resulted in the production of similar volatile blends (a mixture of green leaf volatiles and aromatic compounds).
4. Contrary to T. urticae infestation, sour orange supported larger densities of P. latus than Cleopatra mandarin with similar injury levels.
5. Therefore, sour orange may be more tolerant to P. latus than Cleopatra mandarin.

     

GISH,AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange [Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2n=4x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.The first two authors contributed equally to this paper.     

Inheritance of citrus nematode resistance and its linkage with molecular markers

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds. Received: 4 May 1999 / Accepted: 21 September 1999     

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline‐tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus‐based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non‐silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV‐infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.     

The Cytogenetic Map of the Poncirus trifoliata (L.) Raf.��A Nomenclature System for Chromosomes of All Citric Species

A cytogenetic map was established for the trifoliate orange, Poncirus trifoliata. Chromosome size, centromere position and CMA⁺ heterochromatin content were determined for each chromosome pair, together with the position of BAC clones previously described as chromosome-specific markers. Although P. trifoliata is a true biological species, heteromorphisms for the size of the heterochromatic bands in two chromosome pairs were observed. A minimum set of four BACs was proposed for chromosome identification in P. trifoliata, as well as a chromosome nomenclature that can be applied to all citrus species. The Ctv locus was assigned to chromosome 9 and the remaining linkage groups can now be integrated to this physical map. This standard nomenclature for trifoliate orange will be valuable for genome-sequencing projects in all citrus species.     

Intra-farm diversity and evidence of genetic recombination of Citrus tristeza virus in Delhi region of India

Infection of Citrus tristeza virus (CTV) in different citrus orchards of New Delhi was detected by direct antigen coated-ELISA and RT-PCR. Sweet orange (Citrus sinensis) orchards were found to be susceptible to CTV with estimated disease incidence up to 39%. Kagzi kalan (C. lemon), Pumello (C. paradisi) and Kinnow mandarin (C. reticulata) orchards did not show CTV infection. Three CTV isolates, D1, D7 and D15 randomly selected from infected sweet orange orchards were considered for biological and molecular characterization. In the host range study, all the Delhi isolates infected Darjeeling mandarin (C. reticulata), Kagzi lime (C. aurantifolia), sour orange (C. aurantium) and sweet orange but not Kinnow mandarin. A fragment of 5??ORF1a and complete coat protein (CP) gene of these three isolates were cloned, sequenced and compared with other Indian and international CTV isolates. Delhi isolates shared 85?C92% sequence identity for 5??ORF1a fragment and 89?C91% for CP gene among them. Phylogenetic analysis segregated three Delhi isolates into three genogroups for each of 5??ORF1a fragment and CP gene, however phylogenetic relationships for both the genomic regions was incongruent. Recombination detecting program RDP3 detected CTV isolate D7 as recombinant, indicating genetic variability in CTV isolates might be the outcome of recombination events between divergent CTV sequences. An attempt was made in present study to characterize CTV isolates biologically and at genetic level, and to determine genetic diversity at farm level and study the recombination of CTV isolates in Delhi region.     

Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers

A series of crosses designed for introgression of mandarin (Citrus reticulata Blanco) and pummelo (C. maxima Merr.) germplasm, to develop an alternative rootstock to sour orange (C. aurantium L.), were carried out. It is necessary to identify those hybrids that yield nucellar seedlings for rootstock propagation. Rootstocks can be developed through traditional plant breeding methods; however, the ability to screen and select for economically important traits (such as production of true nucellar seedlings) in an efficient fashion is limited by the difficulties of screening techniques based on whole plant performance. To address these problems, we have used randomly amplified polymorphic DNA (RAPD) and fluorescently labeled expressed sequence tag simple sequence repeat (EST-SSR) molecular markers. A total of 204 individual seedlings obtained from 34 hybrid parental plants were successfully characterized using five RAPD primers. Ten hybrid parents and their progenies, found to be genetically similar among themselves, were selected for more scrutiny using eight EST-SSR primer pairs. The degrees of genetic similarity (nucellars) among progeny seedlings were determined and compared with that of their parents. The mean genetic similarity varied from 67–99% among the selected rootstock candidates screened. The genetic similarity relationship identified using RAPD and EST-SSR molecular markers was highly concordant (p = 0.001). Two elite rootstock candidates (B6R5T56; B6R11T129) that seem to be ideal for future mandarin and pummelo derived rootstock breeding programs have been identified. Our results indicate that either RAPD or EST-SSR analyses could be equally successful in identifying true nucellars among the progenies obtained from introgression crosses of mandarin and pummelo, thus improving the accuracy of early selection in a citrus rootstock breeding program.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Segregation and linkage analyses in two complex populations derived from the citrus rootstock Cleopatra mandarin. Inheritance of seed reproductive traits

Two complex populations derived from the salt-tolerant citrus rootstock Cleopatra mandarin were used to investigate (1) the genomic regions affected by segregation distortion and (2) gene segregation heterogeneity and their causes and to obtain (3) a Citrus reshni linkage map to genetically analyze (4) the duration of the juvenility period and the seed embryony type. Both populations differed in the extent and origin of segregation distortion. The population derived from the cross between C. reshni and Poncirus trifoliata (R?×?Pr) showed 75?% of codominant markers with distorted segregation. The origin of this distortion was prezygotic in most cases. Meanwhile, 100?% of codominant markers in the self-pollinated population [F₂(R?×?Pr)] showed genotypic distortion, and the origin of such distortion was mostly postzygotic, with the heterozygote being the most frequent genotype in all cases. In the R?×?Pr population, where two pollinator varieties were used, allele segregation was significantly heterogeneous not only in P. trifoliata (28.6?% of markers) but also in C. reshni (19.5?%). The results on segregation heterogeneity in the F₂(R?×?Pr) suggest the presence at linkage group 4c of a postfertilization system of balanced lethal factors that reduces homozygosis in self-compatible hybrids. Four low to medium contributing quantitative trait loci (QTLs) were detected for the duration of juvenility period by both Kruskal?CWallis and interval mapping methodologies. For seed embryony type, three QTLs were detected by both methodologies, with the previously reported Apo2 being the QTL contributing the most. CR14,290 and TAA15 are good markers for early selection of polyembryonic rootstocks in progenies derived from C. reshni, Citrus aurantium, and Citrus volkameriana.     

The Dry Storage of Citrus Seeds

The survival of seeds of lemon (Citrus limon L.), lime [C. arantifolia(Christm.) Swing.] and sour orange (C. aurantium L.) was examinedunder a wide range of constant moisture contents and temperatures.Seed longevity was increased by decreasing the moisture contentand temperature of the storage environment. Maximum viabilitywas maintained in the combination of storage conditions includingthe lowest moisture content (5 per cent) and lowest temperature(–20 °C) investigated. The practicality of dry storageof citrus seed for genetic conservation is discussed. Citrus limon L., lemon, Citrus aurantifolia (Christm.), Swing, lime, Citrus aurantium L., sour orange, dry storage, moisture content, temperature, seed viability, seed longevity