Although no longer as glamorous as it was a few decades past, the routine application of embryo rescue techniques, leading to plant recovery, is a valuable tool for citrus cultivar improvement. Embryo rescue approaches can be used to generate useful variation or to capture various kinds of spontaneous genetic variation. Embryo rescue, by in vitro culture of undeveloped, and presumably unfertilized, ovules in colchicine-supplemented media is a practical method of producing tetraploid clones, which are used then in crosses with diploids to produce seedless triploid hybrids. This same approach, i.e., in vitro culture of undeveloped ovules, is also used to recover plants from chimeric sectored fruit exhibiting economically important mutations for fruit characteristics, and for producing potentially variant somaclones. Seedlessness is an important objective for fresh citrus fruit cultivar improvement, and triploidy following 2x × 4x hybridizations is one approach being exploited for this objective. When monoembryonic diploid seed parents are crossed with tetraploid pollen parents, however, normal seed development is not usually possible. Embryos must be excised from abortive seeds fairly early in development and cultured appropriately to ensure the recovery of sufficient numbers of 3x offspring from these crosses, to increase the likelihood of identifying superior seedless hybrids. These applications will be described in some detail, and progress toward breeding objectives are highlighted.
Fred G. Gmitter Jr. and Jude W. Grosser
Pan-chi Liou, Fred G. Gmitter Jr. and Gloria A. Moore
Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.
Chunxian Chen, Jude W. Grosser, Milica Ćalović, Patricia Serrano, Gemma Pasquali, Julie Gmitter and Fred G. Gmitter Jr
Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [Citrus grandis (L.) Osbeck] + mandarin (C. reticulata Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (Citrus aurantium L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [C. sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.
Peng Ling, Fred G. Gmitter Jr., Larry W. Duncan and S. Y. Xiao
A family of 63 citrus intergenemic backcross hybrids was used for this study. The parents and hybrids were multiplied by rooted cuttings, with 6 uniform replicates selected per hybrid, and each plant was inoculated with citrus nematodes (Tylenchulus semipenetrans) 5 times over 2 mo. The number of nematode female larvae per gram of fine fresh root was determind 2 mo after the last inoculation. The phenotypic variation of the hybrids was continuous and wide-ranged, from 8.0 females· g-1 of root tissue (resistant parent Swingle citrumelo=15.6) to 620.0 females· g-1 of root tissue (susceptible parent LB 6-2=540.5). Bulked segregant analysis (BSA), using RAPD fragments, was conducted with 2 DNA bulks of individuals from the extremes of the phenotypic distribution. Three hundred twenty primers were screened and 5 were found to generate repeatedly single RAPD fragments specific to the resistant bulk. The segregation of resistance-associated fragments among the individuals was examined, and the linkage between these markers and potential nematode resistance loci was estimated.
Zhan'ao Deng, Fred G. Gmitter Jr., Shunyuan Xiao and Shu Huang
Citrus tristiza virus (CTV) is the most-significant viral pathogen of citrus in the world. Rapid decline of trees on sour orange and stem pitting of grapefruit and sweet orange, two diseases induced by CTV, severely jeopardize citrus production worldwide. It is recognized that all future rootstocks should be resistant to this virus, and scion resistance to stem pitting stains is desirable. To facilitate introgression of the CTV resistance gene from Poncirus trifoliata and development of CTV-resistant varieties in citrus, gene mapping projects have been initiated and more than a dozen RAPD markers have been identified with tight linkage to the resistance gene. As part of our efforts to use marker-assisted selection with a large number of crosses, and ultimately to accomplish map-based cloning of the CTV resistance gene, we have been converting the most tightly linked RAPD markers into SCAR (sequence characterized amplified region) markers by cloning, sequencing the marker fragments, and designing locus-specific primers. One codominant and several dominant SCARs have been developed thus far. The updated progress and utilization of these SCARs in marker-assisted selection and possibly in characterization of a BAC library will be presented and discussed.
Courtney A. Weber, Gloria A. Moore, Zhanao Deng and Fred G. Gmitter Jr.
Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F1 pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.
Chunxian Chen, Qifa Zheng, Xu Xiang, Jaya R. Soneji, Shu Huang, Young A Choi, Madhugiri Nageswara Rao and Fred G. Gmitter Jr.
Eight new green fluorescent protein (GFP) binary vectors were developed by inserting gfp reporter gene cassettes into pGreen vectors. We chose one of them, pG52KF, with the nptII selection and gfp reporter gene and one recombinant construct, pG52KFp, for a preliminary evaluation in citrus using Agrobacterium-mediated transformation. High-transformation efficiency was observed, whereas green fluorescence greatly facilitated the early in vivo screening and categorizing of the transformants. These pGreen-derived GFP binary vectors, freely available on request, provide more and flexible options for genetic transformation in citrus and other woody plants.