Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

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.     

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.     

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.     

Production and molecular characterization of two new Citrus somatic hybrids for scion improvement

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

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.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

QTL mapping of mandarin (<Emphasis Type="Italic">Citrus reticulata</Emphasis>) fruit characters using high-throughput SNP markers

Seedlessness, flavor, and color are top priorities for mandarin (Citrus reticulata Blanco) cultivar improvement. Given long juvenility, large tree size, and high breeding cost, marker-assisted selection (MAS) may be an expeditious and economical approach to these challenges. The objectives of this study were to construct high-density mandarin genetic maps and to identify single nucleotide polymorphism (SNP) markers associated with fruit quality traits. Two parental genetic maps were constructed from an F₁ population derived from ‘Fortune’ × ‘Murcott’, two mandarin cultivars with distinct fruit characters, using a 1536-SNP Illumina GoldenGate assay. The map for ‘Fortune’ (FOR) consisted of 189 SNPs spanning 681.07 cM and for ‘Murcott’ (MUR) consisted of 106 SNPs spanning 395.25 cM. Alignment of the SNP sequences to the Clementine (Citrus clementina) genome showed highly conserved synteny between the genetic maps and the genome. A total of 48 fruit quality quantitative trait loci (QTLs) were identified, and ten of them stable over two or more samplings were considered as major QTLs. A cluster of QTLs for flavedo color space values L, a, b, and a/b and juice color space values a and a/b were detected in a single genomic region on linkage group 4. Two carotenoid biosynthetic pathway genes, pds1 and ccd4, were found within this QTL interval. Several SNPs were potentially useful in MAS for these fruit characteristics. QTLs were validated in 13 citrus selections, which may be useful in further validation and tentative MAS in mandarin fruit quality improvement.     

Linkage maps of the dwarf and Normal lake whitefish (Coregonus clupeaformis) species complex and their hybrids reveal the genetic architecture of population divergence

Elucidating the genetic architecture of population divergence may reveal the evolution of reproductive barriers and the genomic regions implicated in the process. We assembled genetic linkage maps for the dwarf and Normal lake whitefish species complex and their hybrids. A total of 877 AFLP loci and 30 microsatellites were positioned. The homology of mapped loci between families supported the existence of 34 linkage groups (of 40n expected) exhibiting 83% colinearity among linked loci between these two families. Classes of AFLP markers were not randomly distributed among linkage groups. Both AFLP and microsatellites exhibited deviations from Mendelian expectations, with 30.4% exhibiting significant segregation distortion across 28 linkage groups of the four linkage maps in both families (P < 0.00001). Eight loci distributed over seven homologous linkage groups were significantly distorted in both families and the level of distortion, when comparing homologous loci of the same phase between families, was correlated (Spearman R = 0.378, P = 0.0021). These results suggest that substantial divergence incurred during allopatric glacial separation and subsequent sympatric ecological specialization has resulted in several genomic regions that are no longer complementary between dwarf and Normal populations issued from different evolutionary glacial lineages.     

Genetic Map Construction and Detection of Genetic Loci Underlying Segregation Distortion in an Intraspecific Cross of Populus deltoides

Based on a two-way pseudo-testcross strategy, high density and complete coverage linkage maps were constructed for the maternal and paternal parents of an intraspecific F2 pedigree of Populus deltoides. A total of 1,107 testcross markers were obtained, and the mapping population consisted of 376 progeny. Among these markers, 597 were from the mother, and were assigned into 19 linkage groups, spanning a total genetic distance of 1,940.3 cM. The remaining 519 markers were from the father, and were also were mapped into 19 linkage groups, covering 2,496.3 cM. The genome coverage of both maps was estimated as greater than 99.9% at 20 cM per marker, and the numbers of linkage groups of both maps were in accordance with the 19 haploid chromosomes in Populus. Marker segregation distortion was observed in large contiguous blocks on some of the linkage groups. Subsequently, we mapped the segregation distortion loci in this mapping pedigree. Altogether, eight segregation distortion loci with significant logarithm of odds supports were detected. Segregation distortion indicated the uneven transmission of the alternate alleles from the mapping parents. The corresponding genome regions might contain deleterious genes or be associated with hybridization incompatibility. In addition to the detection of segregation distortion loci, the established genetic maps will serve as a basic resource for mapping genetic loci controlling traits of interest in future studies.     

A Study on Glutamate Oxaloacetate Transaminase Isozymes of Citrus Germplasm Resources

The glutamate oxaloacetate transaminase isozymes (GOT) extracted from 125 biotypes of Citrus and its relatives, Fortunella, Poncirus and Microcitrus were analyzed using polyacrylamide gel electrophoresis to investigate the taxonomic relationships among citrus plants. Besides all the isozymes reported before, two new bands were detected and designated their putative alleles B and C in GOT-l. Among pummelo cultivars wide variations were found. Most of the mandarins were identical, having SS for GOI-1 and MM for GOT-2 except for Zou-Pi-Gan and Yao-Gan, which both had FS at GOT-1 and might be hybrids. This finding suggests that all of the mandarins may have originated from a common ancestor. Sour orange biotypes showed a considerable variation in GOT isozymes. Most of the sour oranges in China were assumed to be hybrids between pummelo and mandarin based on GOT isozyme patterns, but two biotypes, Xiao-Hong-Cheng and Zhu-lan, had FS at GOT-1 and MB at GOT-2, which strongly suggests that they be hybrids of pummelo and Ichang papeda because B allele of GOT-2 occurs only in Ichang papeda and its close relatives Yuzu and Ichang lemon. From this study Yuzu is assumed to be derived from hybridizationof Ichan, papeda and mandarin.     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

LTR retrotransposons from the <Emphasis Type="Italic">Citrus x clementina</Emphasis> genome: characterization and application

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis.     

Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance

Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata (Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny.Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange (Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.Communicated by C. Möllers     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.     

Mapping of within-species segregation distortion in Drosophila persimilis and hybrid sterility between D. persimilis and D. pseudoobscura

In contrast to the prevailing dogma in the 1990s, recent studies have suggested that an evolutionary history of segregation distortion within species may contribute to sterility in species hybrids. However, this recent work identified segregation distortion exclusively in species hybrids that may never have had an evolutionary history of segregation distortion in either parent species. We expand on previous work using a strain of Drosophila persimilis exhibiting segregation distortion within species to generate QTL maps for segregation distortion and hybrid sterility in crosses between D. persimilis and D. pseudoobscura. The maps localize regions along the XR contributing to both phenotypes, and they indicate one region of overlap between the two maps. This overlap could provide preliminary evidence for an association between segregation distortion within species and hybrid sterility, but the localizations are currently too broad to have confidence in this conclusion. This work is a first step towards possibly supporting a genetic conflict model of speciation in this system.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

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