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Courtney A. Weber and Gloria A. Moore

A greater saturation of the previously constructed genetic linkage map of Citrus is important in the long term goal of mapping quantitative trait loci (QTL) such as those controlling cold and salt tolerance. Segregation for cold tolerance appears to be greatly enhanced in the intergeneric F1 population of Citrus grandis × Poncirus trifoliata as compared to the BC1 population previously used for mapping due to the higher percentage of P. trifoliata genes present. This is not unexpected since P. trifoliata is the source of cold tolerance in this cross and is a highly heterozygous species. An integration of the maps of the two populations using about 50 random amplified polymorphic DNA (RAPD) markers common to the two populations is possible using the JoinMap computer program. This will allow the placing of approximately 100 new polymorphic RAPD markers from the F1 population identified by screening from 42 random oligonucleotide primers onto the Citrus map. This saturated map will be used to locate QTL following bulk segregation analysis of cold tolerance in the F1 population.

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Peggy Ozias-Akins, Edward L. Lubbers, and Wayne W. Hannna

Apomixis is asexual reproduction through seed. Apomixis in the genus Pennisetum is of the gametophytic (aposporous) type. Genes for apomixis have been transferred from a wild apomictic species (P. squamulatum) to pearl millet (P. glaucum) by conventional breeding to produce an obligately apomictic backcross 3 (BC3) plant (Dujardin and Hanna, 1989, J. Genet. Breed. 43:145). Molecular markers based on restriction fragment length polymorphisms and random amplified polymorphic DNAs were identified in BC3 that were shared only with the apomictic parent. Segregation of these informative markers in a BC4 population indicated that three linkage groups from P. squamulatum were present in BC3 and that minimal recombination between these alien chromosomes and those of the recurrent parent occurred. Transmission of only one of the linkage groups was required for transfer of apomixis. Recombination is essential for genetic mapping, thus we are beginning to map the informative molecular markers in an F, interspecific cross between pearl millet and P. squamulatum, a population that segregates for apomictic and sexual reproduction.

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Norman Weeden and Gail Timmerman-Vaughan

A linkage map for a set of 51 F2-derived recombinant inbred lines has been constructed from the segregation data of ≈850 morphological, isozyme, RFLP, STS, RAPD, and AFLP markers. The final map consists of seven clear linkage groups with a total length of nearly 900 cM. The wide variety of loci placed on this map permits its comparison with partial maps that have been developed in other programs. For the most part, the arrangement of loci agrees with that in previous maps, and no evidence for translocation heterozygosity in this cross is apparent. Although some clustering of markers is observed, for the most part the markers are well-distributed, and few gaps greater than 5 cM are found in the coverage. The availability of this first “complete” and highly saturated map for pea should permit more efficient comparison of the partial maps that have been generated in a number of different crosses, as well as provide a firm basis for future mapping and molecular studies in this species.

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H.M. Ariyarathne, Dermot P. Coyne, and Geunhwa Jung

Halo blight (HB), brown spot (BS), and rust incited by the bacterial pathogens Pseudomonas syringae pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss) and the fungal pathogen Uromyces appendiculatus, respectively, are important diseases of common beans. The objectives were to construct a RAPD linkage map, and to locate HB and BS resistance genes and genes for some other traits. One-hundred-seventy RAPD markers were mapped in 78 RI lines of the cross BelNeb 1 and A 55. Eleven main and nine minor linkage groups were identified. MAPMAKER/QTL, interval mapping, was used to identify genomic regions involved in the genetic control of the traits. One region was found to control HB leaf reactions to strain HB16 while three regions controlled reactions to strain HB 83. These regions accounted for 22% and 18%, 17%, and 17% of phenotypic variation of resistance, respectively. Four putative QTLs were identified for resistance to BS, and accounted for 37%, 26%, 23%, and 19% of the phenotypic variation. Rust resistance was determined by a single major gene to both rust strains US85NP 5-1 and D82vc74fh. However, linked markers were not identified. The V gene controlling flower and stem color was tightly linked with the Operon marker O10.620.

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Sudheer Beedanagari* and Patrick Conner

Pecan, [Carya illinoinensis (Wangenh.) C. Koch], is a member of Juglandaceae family and is one of the most important nut crops produced in the United States. The objective of this study is to generate the first genetic linkage maps for pecan. Maps were constructed for the cultivars `Elliot' and `Pawnee' using the double pseudo-testcross mapping method whereby a separate linkage map is made for each parent using markers heterozygous in that parent. First generation maps consisted primarily of randomly amplified polymorphic DNA (RAPD) markers. We have now used fluorescently labeled amplified fragment length polymorphism (AFLP) markers to produce more complete maps. In the development of the AFLP markers, 64 primer combinations were originally screened to find the most informative combinations. Ten primer combinations were then chosen to produce markers for the maps. The maps currently consist of approximately 100 RAPD and 100 AFLP markers on each cultivar map. `Pawnee' is a high quality commercial pecan cultivar with a very early ripening date. `Elliot' possesses high levels of resistance to pecan scab, caused by the fungus Cladosporium caryigenum. The maps will be used to find markers linked to scab resistance genes and other traits of interest to the breeding program.

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Geunhwa Jung, Dermot P. Coyne, James Bokosi, James R. Steadman, and James Nienhuis

Dry bean (Phaseolus vulgaris L.) production is limited by bean rust [Uromyces appendiculatus (Pers.) Unger var. appendiculatus]. An effective control strategy for this disease is to breed cultivars with durable resistance. Information on the inheritance, genetic relationships, and mapping of genes with molecular markers for specific resistance (SR), adult plant resistance (APR), and abaxial leaf pubescence (ALP) is needed to pyramid the desired genes for durable resistance. ALP was found to be associated previously with APR in Andean germplasm. The objective here was to identify and map RAPD markers for the genes controlling SR, APR, and ALP and to examine their relationships. Five rust pathotypes were inoculated on the unifoliate leaves of 68 recombinant inbred (RI) lines derived from `PC-50' (presence of SR, APR, and ALP) × XAN-159 (absence of SR, APR, and ALP). SR was determined by a single major gene (Ur-9) to the five rust pathotypes with no detection of recombinants. The fourth trifoliolate leaves were inoculated with one pathotype (A88T1-4b). A single major gene Ur-12 controlled APR to that pathotype. The Ur-9 gene (SR) was independent of and epistatic to the Ur-12 gene (APR). Because of the low number of APR lines in the RI population resulting from the elimination of RI lines with SR, an F2 population was developed from a cross of two homozygous RI lines selected for unifoliate susceptibility to pathotype A88T1-4b and for resistance and susceptibility of the fourth trifoliolate leaves to tag RAPD markers linked to the Ur-12 gene (APR). The single major gene Pu-a determinated ALP and was not linked to Ur-9 (SR) and Ur-12 (ALP). The gene Ur-9 (SR) was linked to RAPD marker J13-1100 at 5 cM and was not assigned to any linkage group or other markers. The gene Pu-a (ALP) was mapped at 20.2 cM from 116.500 and 3.9 cM from marker G3.1150 in linkage group 3. The Ur-12 gene (APR) was mapped at 34.6 cM from marker O13.1350 in linkage group 4b. This is the first report of mapping a gene for APR in common bean.

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Mikel R. Stevens, Shawn A. Chrisensen, Ammon B. Marshall, JoLynn J. Stevens, Peter Wenzl, Eric Hunter, Jason Carling, and Andrzej Killian

Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.

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Kanta Kobira, Khalid Ibrahim, Elizabeth Jefferey, and John Juvik

Considerable epidemiological evidence exists on the association between consumption of antioxidant-rich vegetables and incidence of chronic diseases, including cancer and cardiovascular disease. Broccoli (Brassica oleracea L. sp. italica) florets are relatively abundant sources of antioxidants, and potentially amenable to genetic manipulation to enhance this vegetable's health-promoting properties. This investigation focuses on the identification of chromosomal segments in the nuclear genome of broccoli associated with antioxidant carotenoid and tocopherol variability. A broccoli F2:3 population consisting of 163 families derived from a cross between two parents (VI-158 and BNC) and previously mapped with 62 polymorphic SSR and SRAP marker loci was evaluated for carotenoid and tocopherol concentration in floret tissue over two growing seasons. Significant differences were observed among F2:3 family means for concentrations of lutein (10-fold difference between the lowest and highest family), beta-carotene 17-fold), alpha-tocopherol (8-fold) and gamma-tocopherol (6-fold). On a concentration basis, beta-carotene, lutein, alpha-tocopherol, and gamma-tocopherol were the most abundant antioxidant forms in broccoli. Heritability estimates of primary phytochemicals ranged from 0.35 to 0.38, 0.40, and 0.44 for beta-carotene, alpha-tocopherol, gamma-tocopherol, and lutein, respectively. Composite interval mapping (CIM) identified two quantitative trait loci (QTL) associated with carotenoid variability on two linkage groups and five QTL associated with tocopherol variability on four linkage groups. The QTL identified in this study have potential for use in marker-assisted crop improvement programs to develop elite germplasm designed to promote health among the consuming public.

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J. Steven Brown, R.J. Schnell, J.C. Motamayor, Uilson Lopes, David N. Kuhn, and James W. Borrone

A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F2 population produced by selfing an F1 progeny of the cross Sca6 and ICS1. Simple sequence repeat (SSR) markers (170) were used principally for this map, with 12 candidate genes [eight resistance gene homologues (RGH) and four stress related WRKY genes], for a total of 182 markers. Joinmap software was used to create the map, and 10 linkage groups were clearly obtained, corresponding to the 10 known chromosomes of cacao. Our map encompassed 671.9 cM, approximately 100 cM less than most previously reported cacao maps, and 213.5 cM less than the one reported high-density map. Approximately 27% of the markers showed significant segregation distortion, mapping together in six genomic areas, four of which also showed distortion in other cacao maps. Two quantitative trait loci (QTL) for resistance to witches' broom disease were found, one producing a major effect and one a minor effect, both showing important dominance effects. One QTL for trunk diameter was found at a point 10.2 cM away from the stronger resistance gene. One RGH flanked the minor QTL for witches' broom resistance, implying possible association. QTLs mapped in F2 populations produce estimates of additive and dominance effects, not obtainable in F1 crosses. As dominance was clearly shown in the QTL found in this study, this population merits further study for evaluation of dominance effects for other traits. This F2 cacao population constitutes a useful link for genomic studies between cacao and cotton, its only widely grown agronomic relative.

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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.