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Courtney A. Weber and William Boone

The role of plant pigments in human health has been under intense scrutiny recently. Anthocyanin pigments have been shown to be powerful antioxidants and may contribute to other areas of human health. In red and black raspberry, Rubus idaeus and Rubus occidentalis, respectively, no less than eight different anthocyanin pigments have been identified. However, the genetics controlling the presence and ratios of the different pigments is poorly understood. Various researchers have identified four loci that impart fruit pigment deficiencies and three loci that affect the pigment ratios. The underlying gene function of these loci is not known. Efforts are under way to map two pigment deficiency loci in red raspberry using bulked segregant analysis. Screening with 800 random primers has produced two markers with >90% and two with >80% correlation to one loci. For the other loci, 10 markers with >80% correlation have been identified. Mapping is ongoing with the first linkage map for raspberry to be presented. Populations to test allelism between sources of pigment deficiency are being evaluated for further mapping of loci of the anthocyanin production pathway. Data on cloning of genes in the anthocyanin pathway based on database sequences with degenerative primers for further elucidation a anthocyanin production in raspberry will be presented.

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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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Milton E. Tignor Jr. and Courtney A. Weber

Current efforts in the study of citrus freeze hardiness including gene mapping and elucidating early induction processes require large populations of uniform seedlings. Related genera and intergeneric hybrids are often used in these studies and little is known about factors effecting their seedling emergence. We tested a total of 8 genotypes including Poncirus trifoliata `Rubidoux', Citrus grandis, C. sinensis `Pineapple', C. jambhiri `Schaub', C. paradisi `Duncan', C. aurantium (Brazilian), Carrizo citrange (P. trifoliata × C. sinensis), and Troyer citrange. A total of seven pre-planting treatments were used to evaluate seedling emergence rates. Expanding on the work of previous researchers, treatments were seed coat removal, hydrating in water (96 hours) at either 4, 25, or 35°C, acid scarification, or boiling. Generally, seed coat removal resulted in the most uniform emergence as compared to untreated controls. Presoaking at each temperature enhanced emergence in most varieties tested and 25°C was the best hydrating temperature. Acid scarification greatly delayed emergence in all genotypes tested except Troyer citrange and `Pineapple' orange which had enhanced emergence rates as compared to controls. Preplanting treatment with 100°C water was lethal in all varieties. Pretreatment of citrus seeds can enhance uniformity of germination, although optimum treatments for individual genotypes vary.

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Kim S. Lewers and Courtney A. Weber

Researchers developing new cultivars of red raspberry (Rubus idaeus subsp. idaeus L.) and black raspberry (R. occidentalis L.) observe progeny of breeding populations for several seasons to identify those that perform reliably. If a portion of any breeding population could be eliminated based on a qualitative character or molecular marker, resources used for that portion could be used for other progeny. Our objective is to identify such molecular markers for red raspberry and black raspberry. A black raspberry × red raspberry cross was made to develop a map of each parent, and an F2 population was generated to join the maps. Simple sequence repeat (SSR) markers derived from red raspberry and strawberry were used. The level of homozygosity for the red raspberry was 40%, and the level for the black raspberry was 80%. Severe segregation skewing was observed in the F2 generation and indicates problems with transmission. Our findings help quantify the relative levels of homozygosity previously reported for red raspberry and black raspberry. In addition, the severe skewing observed in the F2 generation provides a molecular perspective to the fertility problems previously reported for the black raspberry × red raspberry hybrids (purple raspberry). Since black raspberry is highly homozygous, purple raspberry has transmission and fertility problems, and black raspberry breeders have reported a frustratingly low level of diversity in this subgroup, development of a black raspberry map is expected to require twice the markers as for a red raspberry map, emphasizing the need for a black raspberry sequence from which to develop molecular markers.

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Courtney A. Weber, Gloria A. Moore, Z. Deng, F. Gmitter and Courtney A. Weber

Specific primers were designed for 61 cloned RAPD fragments and from 10 Citrus EST sequences for the production of SCAR, CAPS, and STS markers for a Citrus grandis `DPI 6-4' × Poncirus trifoliata `Rubideaux' F1 pseudo-testcross population. Fifteen SCAR, three CAPS, and one EST/STS markers were developed. An additional 17 SCAR and CAPS primer pairs developed at the Citrus Research and Education Center for a Citrus grandis `Thong Dee' × (Citrus grandis `Thong Dee' × Poncirus trifoliata `Pomeroy') BC1 population were screened in the pseudo-testcross population. A total of 27 markers were identified and scored in the pseudo-testcross population in which 24 were mapped; 13 in the Citrus parental linkage map on seven linkage groups and 11 in the Poncirus parental map on five linkage groups. In the BC1 population, 20 of 27 markers tested were found to be polymorphic and 13 mapped to seven of nine linkage groups. Of these, 11 were mapped in both populations and could be used for aligning presumed homologous regions on the three linkage maps.

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Jeremy A. Pattison*, Suren K. Samuelian and Courtney A. Weber

RAPD and AFLP markers were first used to construct a molecular map in a BC1 red raspberry population consisting of 70 individuals that segregated for Phytophthora root rot resistance. RAPD markers linked to root rot resistance were identified by bulk segregant analysis and through QTL anlaysis. Two common genomic regions were identified by both analyses and were estimated to explain ≈50% of the phenotypic variation. RAPD markers flanking the QTL were cloned and made into sequence specific markers for potential use in marker assisted selection. In addition to the linked markers, RAPDs spread throughout the linkage map were also sequenced and developed into either SCARs, CAPs, or codominant SSRs. Attempts were made to locate red raspberry resistance gene analogs using degenerate primers designed on conserved regions encoding known resistance genes. Results on the type and map position of identified RGA's and selection efficiency of linked markers analyzed in red raspberry cultivars of characterized root rot resistance will be discussed.

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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.

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Kim S. Lewers*, Eric T. Stafne, John R. Clark, Courtney A. Weber and Julie Graham

Some raspberry and blackberry breeders are interested in using molecular markers to assist with selection. Simple Sequence Repeat markers (SSRs) have many advantages, and SSRs developed from one species can sometimes be used with related species. Six SSRs derived from the weed R. alceifolius, and 74 SSRs from R. idaeus red raspberry `Glen Moy' were tested on R. idaeus red raspberry selection NY322 from Cornell Univ., R. occidentalis `Jewel' black raspberry, Rubus spp. blackberry `Arapaho', and blackberry selection APF-12 from the Univ. of Arkansas. The two raspberry genotypes are parents of an interspecific mapping population segregating for primocane fruiting and other traits. The two blackberry genotypes are parents of a population segregating for primocane fruiting and thornlessness. Of the six R. alceifolius SSRs, two amplified a product from all genotypes. Of the 74 red raspberry SSRs, 56 (74%) amplified a product from NY322, 39 (53%) amplified a product from `Jewel', and 24 (32%) amplified a product from blackberry. Of the 56 SSRs that amplified a product from NY322, 17 failed to amplify a product from `Jewel' and, therefore, detected polymorphisms between the parents of this mapping population. Twice as many detected polymorphisms of this type between blackberry and red raspberry, since 33 SSRs amplified a product from NY322, but neither of the blackberry genotypes. Differences in PCR product sizes from these genotypes reveal additional polymorphisms. Rubus is among the most diverse genera in the plant kingdom, so it is not surprising that only 19 of the 74 raspberry-derived SSRs amplified a product from all four of the genotypes tested. These SSRs will be useful in interspecific mapping and cultivar development.

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Eric T. Stafne, John R. Clark, Courtney A. Weber, Julie Graham and Kim S. Lewers

Interest in molecular markers and genetic maps is growing among researchers developing new cultivars of Rubus L. (raspberry and blackberry). Several traits of interest fail to express in seedlings or reliably in some environments and are candidates for marker-assisted selection. A growing number of simple sequence repeat (SSR) molecular markers derived from Rubus and Fragaria L. (strawberry) are available for use with Rubus mapping populations. The objectives of this study were to test 142 of these SSR markers to screen raspberry and blackberry parental genotypes for potential use in existing mapping populations that segregate for traits of interest, determine the extent of inter-species and inter-genera transferability with amplification, and determine the level of polymorphism among the parents. Up to 32 of the SSR primer pairs tested may be useful for genetic mapping in both the blackberry population and at least one of the raspberry populations. The maximum number of SSR primer pairs found useable for mapping was 60 for the raspberry population and 45 for the blackberry population. Acquisition of many more nucleotide sequences from red raspberry, black raspberry, and blackberry are required to develop useful molecular markers and genetic maps for these species. Rubus, family Rosaceae, is a highly diverse genus that contains hundreds of heterozygous species. The family is one of the most agronomically important plant families in temperate regions of the world, although they also occur in tropical and arctic regions as well. The most important commercial subgenus of Rubus is Idaeobatus Focke, the raspberries, which are primarily diploids. This subgenus contains the european red raspberry R. idaeus ssp. idaeus L., as well as the american black raspberry R. occidentalis L. and the american red raspberry R. idaeus ssp. strigosus Michx. Interspecific hybridization of these, and other raspberry species, has led to greater genetic diversity and allowed for the introgression of superior traits such as large fruit size, fruit firmness and quality, disease resistance, and winter hardiness.

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Kim S. Lewers, Patricia R. Castro, John M. Enns, Stan C. Hokanson, Gene J. Galletta, David T. Handley, Andrew R. Jamieson, Michael J. Newell, Jayesh B. Samtani, Roy D. Flanagan, Barbara J. Smith, John C. Snyder, John G. Strang, Shawn R. Wright and Courtney A. Weber