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Rebecca Nelson Brown and James R. Myers

A molecular and morphological marker map would improve our knowledge of Cucurbita genetics, and would facilitate efforts to breed improved summer and winter squash cultivars. Random amplified polymorphic DNA (RAPD) markers were used to construct a partial map of the Cucurbita genome. The mapping population was the BC1 progeny of the Cucurbita pepo L. yellow straightneck inbred A0449 and the tropical Cucurbita moschata Duchesne ex Lam. landrace `Nigerian Local'. A0449 was the recurrent parent. This cross was chosen because of the relatively greater economic importance of summer squash, traits of value to be introgressed from the C. moschata parent, and maximized genetic variation from the interspecific cross. The map contains 148 RAPD markers in 28 linkage groups. Loci controlling five morphological traits were placed on the map. The map covers 1,954 cM, which is estimated to be 75% of the Cucurbita genome. The qualitative traits placed on the map include the B gene for fruit which turn yellow before anthesis, the M gene for silver mottling of leaves, and a locus controlling the intensity of rind color on mature fruit. Quantitative trait loci (QTL) associated with fruit shape and the depth of the indentations between primary leaf veins were identified.

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Maureen C. O'Leary and Thomas H. Boyle

Polyacrylamide gel electrophoresis was used to study inheritance and linkage of isozymes in Easter cactus (Hatiora species and interspecific hybrids). Five isozyme systems were analyzed: aspartate aminotransferase (AAT), glucose-6-phosphate isomerase (GPI), malate dehydrogenase (MDH), phosphoglucomutase (PGM), and triosephosphate isomerase (TPI). F1, F2, BC1, and S1 progeny were used for inheritance studies. Six polymorphic loci (Aat-1, Gpi-1, Mdh-1, Pgm-1, Pgm-2, and Tpi-2) were identified. Aat-1 and Pgm-1 were linked (recombination frequency = 26% ± 7%), but the other isozyme loci assorted independently. Aberrant segregation ratios were observed in at least one segregating family for all six isozyme loci. We hypothesize that segregation distortion was due to linkage between isozyme loci and other genes subject to pre- or postzygotic selection. The existence of five additional isozyme loci (Aat-2, Gpi-2, Mdh-2, Mdh-3, and Tpi-1) was inferred from segregation patterns and by comparison of isozyme profiles from phylloclades and pollen. These isozyme loci may prove useful for confirming hybridity in intra- and interspecific crosses, determining parentage of cultivars, and assessing genetic diversity in germplasm collections.

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Ying Wang, Gregory L. Reighard, Desmond R. Layne, Albert G. Abbott and Hongwen Huang

Pawpaw (Asimina triloba) produces the largest fruit native to the United States. Six linkage groups were identified for A. triloba using the interspecific cross [PPF1-5 (A. triloba) × RET (A. reticulata Shuttlw. ex Chapman)], covering 206 centimorgans (cM). A total of 134 dominant amplification fragment length polymorphism (AFLP) markers (37 polymorphic and 97 monomorphic) were employed for estimating the genetic diversity of eight wild populations and 31 cultivars and advanced selections. For the wild populations, the percentage of polymorphic loci over all populations was 28.1% for dominant markers and Nei's genetic diversity (He) were 0.077 estimated by 134 dominant markers. Genetic diversity and the percentage of polymorphic loci estimated using only polymorphic dominant AFLPs were 0.245 and 79%, respectively, which are comparable with other plant species having the same characteristics. Estimated genetic diversity within populations accounted for 81.3% of the total genetic diversity. For cultivars and advanced selections, genetic diversity estimated by 134 dominant markers was similar to that of wild pawpaw populations (He = 0.071). Thirty-one cultivars and advanced selections were delineated by as few as nine polymorphic AFLP dominant loci. Genetic relationships among wild populations, cultivars and advanced selections were further examined by unweighted pair group method with arithmetic mean (UPGMA) of Nei's unbiased genetic distance. The genetic diversity estimated for wild populations using the clustered polymorphic markers was lower than the result estimated using the nonclustered polymorphic markers. Therefore, this study indicates that the number of sampled genomic regions, instead of the number of markers, plays an important role for the genetic diversity estimates.

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R.L. Fery and J.A. Thies

The peanut root-knot nematode (Meloidogyne arenaria race 1) is potentially a major pest of pepper cultivars belonging to the species Capsicum chinense. Greenhouse tests were conducted to: 1) compare the level of resistance to the peanut root-knot nematode exhibited by the recently released C. chinense germplasm line PA-353 to that exhibited by the C. annuum cv. Carolina Cayenne; 2) to determine the inheritance of the resistance in the C. chinense germplasm line PA-353; and 3) to determine the genetic relationship between the resistance exhibited by the C. chinense germplasm line PA-353 and that exhibited by the C. annuum cv. Carolina Cayenne. The level of resistance exhibited by the C. chinense germplasm line PA-353 was equal to the high level of resistance of the C. annuum cv. Carolina Cayenne. Evaluation of parental, F1, F2, and backcross populations of the cross between the resistant C. chinense germplasm line PA-353 and the susceptible C. chinense accession PA-350 indicated that the resistance in C. chinense is conditioned by a single dominant gene. The F2 population of the interspecific cross between the resistant C. chinense germplasm line PA-353 and the resistant C. annuum cv. Carolina Cayenne did not segregate for resistance, indicating that the dominant resistance gene in C. chinense is likely allelic to or closely linked to a gene conditioning resistance in C. annuum. The availability of a simply inherited source of outstanding resistance makes breeding for peanut root-knot nematode resistance a viable objective in C. chinense breeding programs.

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Michael E. Compton, Brenda L. Fuchs and Jack E. Staub

Cucumis hystrix Chakr. is a rare cucurbit species native to Asia. The species is valued by breeders because of its multiple branching habit and has been used in interspecific crosses with Cucumis sativus. However, individual C. hystrix plants have not been identified in the wild since 1990. Therefore, it was our objective to develop a micropropagation protocol that would allow us to clonally propagate plants in cultivation. Shoots tips (2 cm) were excised from a single C. hystrix plant grown in the greenhouse. All tendrils and leaves were removed before surface-sterilization in 1.25% NaOCl for 5 or 10 min and rinsed six times with sterile distilled water. Shoot tips were trimmed to 1 cm (meristem with two to three young leaf primordia) and placed into 25 × 125-mm test tubes containing 25 ml of initiation medium [MS plus (per liter) 100 mg inositol, 30 g sucrose and 5 g Agargel; pH 5.7-5.8]. PGR combinations tested were initiation medium with 1 μM BA, and initiation medium with 1.7 μM IBA, 0.5 μM kinetin and 0.3 μM GA3 (IKG). Explant survival was greater when shoot tips were surface-sterilized for 5 min (75%) compared to 10 min (33%). More axillary shoots formed when shoot tips were cultured in IKG medium (10.8) than in medium with BA (5.5). Shoots were considerably longer (10 mm) when cultured in medium with IKG compared to BA (1.5 mm). About 64% of shoots place in medium containing 8 μM NAA formed roots and were acclimatized to greenhouse conditions.

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Robert H. Bors and J. Alan Sullivan

Interspecific crosses with Fragaria moschata (6x) have been hampered by ploidy level differences, poor seed set, and extremely poor seed germination. Modification of pollination practices, embryo rescue, and use of several genotypes has allowed over 80 synthetic tetraploids to be created from 14 cross combinations. Germplasm for the experiment consisted of eight selections of F. moschata (6x), two of F. nubicola (2x), and two of F. viridis (2x). Both 2x × 6x and 6x × 2x crosses were performed. Initially, negligible seed set occurred on F. nubicola and F. viridis when multiple flowers per truss were pollinated. When only one cross was performed per truss, with other flowers removed, seed set was greatly enhanced. F. moschata was much more tolerant of multiple crosses per truss. The crossing combination of F. moschata × F. nubicola gave the worst seed production. Other species combinations were capable of producing good seed set with noticeable differences between individual selections. When achenes were halved, only 1% appeared normal, 2% were underdeveloped or shrunken, the remainder were empty. Many of the malformed and most of the normal embryos germinated using the cut achene method. Achenes were surface-sterilized, cut in half, and placed on MS media with activated charcoal (3g·L–1), sucrose (30g·L–1), and no hormones. Germination occurred only from achenes from fully ripened fruit. Viable hybrids were obtained from 2x × 6x as well as 6x × 2x crosses. Fragaria viridis–F. moschata hybrids closely resembled F. moschata while F. nubicola–F. moschata hybrids were more intermediate in leaf morphology.

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Mary Ann Start, James Luby, Robert Guthrie and Debby Filler

The hardy Actinidia species represent a source of genetic diversity for improving A. deliciosa (kiwifruit) as well as for creating new economically important cultivars through intra- and interspecific crosses. Attempts at breeding in Actinidia have been complicated by the existence of intraspecific as well as interspecific variation in ploidy. The haploid chromosome number in Actinidia is 29 and diploid (2n=2x=58), tetraploid (2n=4x=116), and hexaploid (2n=6x=174) levels have been identified. Because of the problems encountered when crossing parents differing in ploidy level, it is desirable to know the ploidy levels of plants to be used in breeding. We determined the ploidy levels of 61 Actinidia accessions currently available in the U.S., including primarily accessions of relatively winter-hardy species. The 61 accessions, representing eight species and three interspecific hybrids, were screened for ploidy using flow cytometry. Mitotic root tip cells from one plant from each putative ploidy level were examined microscopically to confirm the ploidy level derived from flow cytometry. There were 17 diploids, 40 tetraploids, and 4 hexaploids. Intraspecific variation was not found among accessions of the species arguta, callosa, deliciosa, kolomikta, melanandra, polygama, or purpurea. All kolomikta and polygama accessions were diploid. All arguta, callosa, melanandra, and purpurea accessions were tetraploid. Actinidia deliciosa was hexaploid. One chinensis accession was tetraploid. Two accessions (NGPR 0021.14 and 0021.3), acquired as chinensis, were hexaploid and may, in fact, be A. deliciosa based on their morphology. `Issai' (arguta × polygama) was hexaploid and `Ken's Red' and `Red Princess' (both melanandra × arguta) were tetraploid.

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R.W. Robinson

Cucurbita ecuadorensis is a valuable source of multiple virus resistance. It is resistant to zucchini yellow mosaic virus (ZYMV), papaya ringspot virus (PRSV), watermelon mosaic virus, tobacco ringspot virus, squash mosaic virus, and cucumber mosaic virus (CMV). Its virus resistance can be transferred to squash and pumpkin, but sterility barriers must be overcome. The cross Cucurbita maxima× C. ecuadorensis can readily be made, and there is no need for embryo culture. Pollen fertility of the hybrid is somewhat reduced, but sufficient for producing F2 seed. Segregation for sterility occurs in the F2, but selection can be made for fertile plants that are homozygous for virus resistance. Cucurbita ecuadorensis is much more distantly related to C. pepo than to C. maxima, and there are more formidable barriers in this interspecific cross. The cross is very difficult to make with some C. pepo cultivars, but other cultivars are more compatible. Viable seed were not produced, but hybrid plants were obtained by embryo culture. Although both parents were monoecious, the hybrid was gynoecious. Male flower formation was induced by treating the hybrid with Ag or GA, but they were male-sterile. F2 seed was not obtained, but backcross seed was easily produced by using the interspecific hybrid as the maternal parent in crosses with C. pepo. The most refractory barrier was achieving homozygosity for ZYMV resistance. Disturbed segregation occurred in succeeding generations and the progeny of most resistant plants segregated and were not uniform for resistance. This and other barriers to interspecific gene exchange were overcome and a summer squash variety homozygous for resistance to ZYMV, PRSV, and CMV is being released this year.

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Majid R. Foolad

In tomato, Lycopersi conesculentum Mill., currently there are >285 known morphological, physiological and disease resistance markers, 36 isozymes, and >1000 RFLPs, which have been mapped onto the 12 tomato chromosomes. In addition, currently there are >162,000 ESTs, of which ∼3.2% have been mapped. Several tomato genetic maps have been developed, mainly based on interspecific crosses between the cultivated tomato and its related wild species. The markers and maps have been used to locate and tag genes or QTLs for disease resistance and other horticultural characteristics. Such information can be used for various purposes, including marker-assisted selection (MAS) and map-based cloning of desirable genes or QTLs. Many seed companies have adopted using MAS for manipulating genes for a few simple morphological characteristics and several vertical disease resistance traits in tomato. However, MAS is not yet a routine procedure in seed companies for manipulating QTLs although it has been tried for a few complex disease resistance and fruit quality characteristics. In comparison, the use of MAS is less common in public tomato breeding programs, although attempts have been made to transfer QTLs for resistances to a few complex diseases. The potential benefits of marker deployment to plant breeding are undisputed, in particular for pyramiding disease resistance genes. It is expected that in the near future MAS will be routine in many breeding programs, taking advantage of high-resolution markers such as SNPs. For quantitative traits, QTLs must be sought for components of genetic variation before they are applicable to marker-assisted breeding. However, MAS will not be a “silver bullet” solution to every breeding problem or for every crop species.

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Hongwen Huang, Desmond R. Layne and Thomas L. Kubisiak

Twelve, 10-base primers amplified a total of 20 intense and easily scorable polymorphic bands in an interspecific cross of PPF1-5 pawpaw [Asimina triloba (L.) Dunal.] × RET (Asimina reticulata Shuttlew.). In this cross, all bands scored were present in, and inherited from, the A. triloba parent PPF1-5. Nineteen of the 20 bands were found to segregate as expected (1:1 or 3:1) based on chi-square goodness-of-fit tests, and were subsequently used to evaluate genetic diversity in populations of A. triloba collected from six states (Georgia, Illinois, Indiana, Maryland, New York, and West Virginia) within its natural range. Analysis of genetic diversity of the populations revealed that the mean number of alleles per locus was A = 1.64, percent polymorphic loci was P = 64, and expected heterozygosity was He = 0.25. No significant differences were found among populations for any of the polymorphic indices. Partitioning of the population genetic diversity showed that the average genetic diversity within populations was Hs = 0.26, accounting for 72% of the total genetic diversity. Genetic diversity among populations was Dst = 0.10, accounting for 28% of the total genetic diversity. Nei's genetic identity and distance showed a high mean identity of 0.86 between populations. Genetic relationships among the populations examined by unweighted pair-group mean clustering analysis separated the six populations into two primary clusters: one composed of Georgia, Maryland, and New York, and the other composed of Illinois, Indiana, and West Virginia. The Georgia and Indiana populations were further separated from the other populations within each group. This study provides additional evidence that marginal populations within the natural range of A. triloba should be included in future collection efforts to capture most of the rare and local alleles responsible for this differentiation.