Frederick G. Gmitter

Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus.

Abstract Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.

A localized linkage map of the citrus tristeza virus resistance gene region.

A localized genetic linkage map was developed of the region surrounding the citrus tristeza virus (CTV) resistance gene (designated Ctv) from Poncirus trifoliate L., a sexually compatible Citrus relative. Bulked segregant analysis (BSA) was used to identify potential resistance-associated RAPD fragment markers in four intergeneric backcross families that were segregating for CTV resistance. Eight RAPD fragments were found that were consistently linked to Ctv in the four families. Map distances and locus order were determined with MAPMAKER 3.0, using the results obtained from 59 individuals in the largest family. Also, a consensus map was constructed with JOINMAP 1.3, using pooled results from the four backcross families. Marker orders were identical, except for 1 marker, on these independently developed maps. Family-specific resistance-associated markers were also identified, as were numerous susceptibility-associated markers. The identification of markers tightly linked to Ctv will enable citrus breeders to identify plants likely to be CTV-resistant by indirect, marker-assisted selection, rather than by labor-intensive direct challenge with the pathogen. These markers also provide a basis for future efforts to isolate Ctv for subsequent genetic manipulation.

Intergeneric somatic hybrid plants from sexually incompatible woody species: Citrus sinensis and Severinia disticha

SummaryThe fusion of Citrus sinensis cv. Hamlin (sweet orange) protoplasts isolated from an embryogenic suspension culture with Severinia disticha (Philippine box orange) protoplasts isolated from epicotyl-derived callus with organogenic potential, resulted in the regeneration of allotetraploid somatic hybrid plants. Plant regeneration was a function of complementation, combining the capacity for somatic embryogenesis of C. sinensis with the organogenic ability of S. disticha. Confirmation of somatic hybrid identity was based on leaf morphology, chromosome number, and analyses of phosphoglucose mutase (PGM) and malate dehydrogenase (MDH) zymograms. Hybrid plants were multiplied organogenically and exhibited morphology intermediate to that of the parents. This is the first example of somatic hybrid plants produced between sexually incompatible woody genera.

Linkage of restriction fragment length polymorphisms and isozymes in Citrus

SummaryGenetic linkage analysis was performed using two segregating populations of citrus. One population arose from an intergeneric backcross of Citrus grandis (L.) Osb. cv ‘Thong Dee’ and Poncirus trifoliata (L.) Raf. cv ‘Pomeroy’, using the former as the recurrent (female) parent. The other population came from an interspecific backcross of C. reticulata Blanco cv ‘Clementine’ and C. x paradisi Macf. cv ‘Duncan’, using the former as the recurrent (male) parent. A total of 11 isozyme and 58 restriction fragment length polymorphisms were found to segregate in a monogenic fashion in one or both populations. Linkage analysis revealed that 62 of the loci examined mapped to 11 linkage groups, while 7 loci segregated independently from all other markers. Gene order was highly conserved between the maps generated from the two divergent segregating populations. Possible applications of the use of such maps in tree fruit breeding are discussed.

Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus

Protoplast fusion technology has been utilized in many crops to generate allotetraploid somatic hybrids, and sometimes autotetraploids as a byproduct of the process. A brief history of this technology development is provided, along with a simple protocol developed for citrus, which can be easily adapted to other plants. Protoplast fusion has become a significant tool in ploidy manipulation that can be applied in various cultivar improvement schemes. In rare cases, a new somatic hybrid may have direct utility as an improved cultivar; however, the most important application of somatic hybridization is the building of novel germplasm as a source of elite breeding parents for various types of conventional crosses for both scion and rootstock improvement. Somatic hybridization is generating superior allotetraploid breeding parents for use in interploid crosses to generate seedless triploids. Seedlessness is a primary breeding objective for new fresh fruit citrus varieties, and several thousand triploid hybrids have been produced using somatic hybrids as the tetraploid parent. Protoplast fusion is also being utilized to produce somatic hybrids that combine complementary diploid rootstocks, which have shown good potential for tree size control. Tree size control has gained importance as a means of reducing harvesting costs, maximizing the efficiency of modern cold protection methodology, and facilitating the adaptation of new fruit production systems. Successful somatic hybridization in citrus rootstock improvement has enabled rootstock breeding at the tetraploid level via sexual hybridization, which can yield maximum genetic diversity in zygotic progeny upon which to impose selection for the many traits required in improved rootstock cultivars, including disease and insect resistance, broad adaptation, tree size control, and the ability to consistently produce high yields of quality fruit. Recent progress and successful examples of these applications are discussed. Finally, a discussion of the genetic potential of somatic hybrids as breeding parents, including meiotic behavior and inheritance is provided.

The possible role of Yunnan, China, in the origin of contemporary Citrus species (Rutaceae).

The primitive center of origin of Citrus species has been a subject of speculation and discussion for some time. Japanese Citrus taxonomist Tanaka concluded that the center of origin was northeastern India and northern Burma. He ascribed a secondary role in the origin and distribution of Citrus species to Yunnan and surrounding areas of China on the basis of his belief that there were no important native species. These conclusions were based on incomplete information that was available over 30 yr ago, but important new evidence from Yunnan challenges Tanaka’s ideas on the origin of Citrus species. This report describes the diversity of indigenous primitive and advanced Citrus species in Yunnan and the interaction of topography and climate with the geographic distribution of these species. Numerous river systems arise in or traverse Yunnan and flow to Burma, Indo-China, and southern and central China, thus providing a natural plant dispersal mechanism. The authors suggest that the substantial portion of the Citrus gene pool represented in the rich diversity of indigenous species reported in recent Chinese surveys and the available natural dispersal mechanism provide strong evidence that Yunnan and nearby areas of China played a critical role in the origin and distribution of modern Citrus species.ResumenEl centro de origen de las especies del géneroCitrus ha sido tema de especulación y discusión por mucho tiempo. El taxónomo japonés Tanaka concluyó que el centro de origen se situó en el noreste de la India y el norte de Burma. El atribuyó un papel secundario a Yunnan y áreas circundantes en base a su creencia que no había especies nativas importantes en esa region. Tales conclusiones se basaron en informatión incompleta de hace mas de 30 años; sin embargo, existen evidencias que revierten las ideas de Tanaka acerca del origen del género Citrus. El presente trabajo describe la diversidad de Citrus spp. que existen en Yunnan, tanto primitivas como avanzadas, asícomo la interactión entre la topografía y el clima con la distributión geográfica de esas especies. En Yunnan existen numerosos ríos que fluyen hacia Burma, Indo-China, y centro y sur de China, proporcionando así un mecanismo natural de dispersión vegetal. Los autores del presente trabajo sugieren que la parte primordial del pool genético de las especies del género Citrus está representada en la amplia diversidad de especies nativas encontradas en evaluaciones y recorridos recientes. Lo anterior junto con los mecanismos naturales de dispersión proporcionan firmes evidencias que Yunnan y áreas circundantes en China jugaron un papel determinante en el origen y distributión de las especies modernas de Citrus.

Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

SummarySomatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + ‘Keen’ sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) ‘Valencia’ sweet orange (C. sinensis (L.) Osbeck) + ‘Femminello’ lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.

Induction of triploid Citrus plants from endosperm calli in vitro

SummaryTriploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping

BackgroundMost modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine.ResultsFive parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome.ConclusionsA reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon.

Intergeneric somatic hybrid plants between ‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Flying Dragon’ trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. ‘Hamlin’ protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with ‘Flying Dragon’ protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the ‘Hamlin’ protoplasts with the subsequent expression of the trifoliate leaf character of ‘Flying Dragon.’ Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.