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Tsuyoshi Habu, Fumio Kishida, Miki Morikita, Akira Kitajima, Toshiaki Yamada, and Ryutaro Tao

Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits S-RNase-based gametophytic self-incompatibility as do other Prunus species. Both self-incompatible and self-compatible Japanese apricot cultivars are grown commercially in Japan. These self-compatible cultivars are shown to have a common S-haplotype called S f that contains S f-RNase and SFB f (S-haplotype-specific F-box protein). This study describes a simple and rapid detection of SFB f, in Japanese apricot, based on loop-mediated isothermal amplification (LAMP) method. A set of 4 primers, F3, B3, FIP, and BIP primer, were designed from the exon and the putative inserted sequence of SFB f. Optimal reaction time at 63 C was determined to be 90 minutes. It appeared that the LAMP method combined with the ultrasimple DNA extraction efficiently detected SFB f. The advantage of the marker-assisted selection of self-compatibility based on the LAMP method was discussed.

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Hongrun Yu and Thomas M. Davis

Agriculture plant genome program grant #92-27300-7442. All crosses were made by Scott Williamson. Kathy Cruz and Lynn Boyden assisted with the isozyme analysis. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal

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Steven R. Triano and Dina A. St. Clair

30 POSTER SESSION 4 (Abstr. 460-484) Breeding/Genetics/Molecular Markers

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R.J. Schnell, C.T. Olano, J.S. Brown, A.W. Meerow, C. Cervantes-Martinez, C. Nagai, and J.C. Motamayor

The authors wish to thank Tom Menezes of Hawaii Gold Cacao Tree Inc.for the selection of the productive and unproductive seedlings.

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Geunhwa Jung, Paul Skroch, James Nienhuis, and Dermot Coyne

One of the highest levels of common bacterial blight (CBB) resistance identified in Phaseolus vulgaris is found in XAN-159, which was developed for leaf resistance to CBB through six generations of pedigree selection of progenies derived from the interspecific cross [(`Pinto UI 114' × PI 319441) × P. acutifolius PI 319443] × `Masterpiece'. A RAPD genetic linkage map was previously constructed in a recombinant inbred population derived from the common bean cross PC-50 × XAN-159 for identification of genomic regions associated with bacterial disease resistance in XAN-159. To confirm that chromosomal regions associated with CBB resistance in XAN-159 were introgressed from tepary bean, we investigated the parentage of each genomic interval in XAN-159 by studying the genomic constitutions of the four different parents involved in the pedigree. The results indicate that all genomic regions associated with CBB resistance contain intervals derived exclusively from tepary bean. The uniqueness of marker polymorphisms associated with resistance to CBB in XAN-159 will allow the application of marker assisted selection for these resistance genes in most populations of common bean.

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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*, 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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Frederick J. Ryan and David W. Ramming

The development of grapevines with berries with small seed traces, so-called seedless grapes, is a costly process. Marker assisted selection would save time and money. Adam-Blondin et al. (Vitis 40:147. 2001) demonstrated that a sequence characterized amplified region, SCC8, could identify seedless grapevine cultivars in European accessions of Vitisvinifera L. We have applied this marker to two populations of grapevines in a breeding program in California. One population consisted of 100 individuals while the second had 109. The two crosses had a common female parent, derived from `Flame Seedless'. Fruit were evaluated over several seasons for parameters including total weight of seeds or traces. DNA was isolated from leaves during the spring. Amplification was carried out with SCC8 primers, followed by digestion with Bgl II, and agarose gel electrophoresis. Individuals were scored as homozygous SCC8+ (small seeded), heterozygous SCC8+/scc8-(intermediate sized seeds), or homozygous scc8-(large seeded) and mean total seed weight per berry was calculated for each genetic class. In the first population, the number of individuals in the inferred genotypes fit an expected 1:2:1 distribution (χ2 = 0.480, P> 0.787) and seed weights for each genetic class were reasonable. For the second population, it was necessary to postulate a null allele in one parent, with a 1:1:1:1 expected distribution for genotypes SCC8+/SCC8+, SCC8+/null, SCC8+/scc8-, and scc8-/null. The actual distribution was in agreement with this model (χ2 = 4.379, P> 0.223). The genotype SCC8+/null had the SCC8+ marker and total seed weight >10 mg per berry. Large seeded individuals and heterozygotes could be reliably identified with this marker.

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Reddy R. Chinthakuntla, Frank Matta, Rao S. Mentreddy, Umesh Reddy, Padmavathi Nimmakayala, Daniel Peterson, and Om Prakash Vadhwa

Chilepepper (Capsicum spp.) is the third most important vegetable crop in the United States. The market value of chile peppers for spices and condiments exceeds $650 million per year. With a growing Hispanic population across the United States, the demand for high yielding, good quality cayenne pepper continues to increase. In order to fulfill this niche market, a study has been initiated to develop pepper varieties that combine high yield potential with superior agronomic traits, including insect and disease resistance, and fruit characteristics, using molecular marker assisted breeding/selection. In preliminary trials, several F1 generations were created through inter- and intra-specific crosses among 220 germplasm lines belonging to six Capsicumsp. in the greenhouse. Selected F1 progeny, parent lines, and selected accessions were planted in single-row field plots the following summer. The crossing success was higher within species than between. The genotypic variation was significant for all parameters examined. The average percent germination (81.1) of F1 progeny was 32% and 45% higher than that of the parent lines and selected accessions, respectively. The F1 progeny were shorter in height; more vigorous in growth, flowered early, and with fewer, but heavier, fruits per plant out-yielded the parent lines and accessions by 50% and 120%, respectively. The study showed a marked heterosis in F1 progeny compared to the parent lines and accessions. Microsatellite genotyping to estimate genetic diversity and validation of markers that are linked to various traits is in progress and will be discussed in the presentation.

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Josefina Alcalá, Leonard M. Pike, and James J. Giovannoni

The relatively low evolution rate of the chloroplast DNA has made it an ideal tool to study evolutionary processes in plants above the species levels. However, recent studies have demonstrated that intraspecific variation in the chloroplast DNA is also common. This variation has provided useful insights into population level evolutionary processes. The polymerase chain reaction and sequencing of a noncoding chloroplast region used to classify onion lines for cytoplasmic type facilitated the identification of one sterile and two normal plastome variants in onion (Allium cepa L.). Sequence comparison revealed that differences between plastome variants included the presence of single-nucleotide polymorphisms associated with cytoplasmic type and variable numbers of tandem repeats, possibly resulting from slipped-strand mispairing. Our observations support the use of chloroplast-specific markers to assist in the selection of specific cytoplasmic types, suggest the potential to facilitate genotype determination, and demonstrate the presence of additional variation within cytoplasm type which gives insight into plastome evolution and may facilitate more accurate genotyping and selection.