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David N. Kuhn, Giri Narasimhan, Kyoko Nakamura, J. Steven Brown, Raymond J. Schnell and Alan W. Meerow

Identifying genetic markers linked to disease resistance in plants is an important goal in marker-assisted selection. Using a candidate-gene approach, we have previously developed genetic markers in cacao (Theobroma cacao L.) for two families of genes involved in disease resistance: non-TIR-NBS-LRR (Toll/Interleukin-1 Receptor-nucleotide binding site-leucine rich repeat) resistance gene homologues and WRKY transcription factor genes; however, we failed to isolate TIR-NBS-LRR genes. Using a novel algorithm to design degenerate primers, we have now isolated TIR-NBS-LRR loci as determined by DNA sequence comparison. These loci have been developed as genetic markers using capillary array electrophoresis (CAE) and single-strand conformational polymorphism (SSCP) analysis. We have mapped three distinct TIR-NBS-LRR loci in an F2 population of cacao and demonstrated that one is located on linkage group 3 and the other two on linkage group 5.

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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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James W. Borrone, Cecile T. Olano, David N. Kuhn, J. Steven Brown, Raymond J. Schnell and Helen A. Violi

Avocado (Persea americana Mill.) possesses a unique flowering mechanism, thought to promote out-crossing, in which the male and female parts of the perfect flower function at different time periods. Cultivars are classified as Flowering Type A, where flowers are functionally female the morning of one day and functionally male the afternoon of the next day, or Flowering Type B, where flowers are functionally female in the afternoon and functionally male the next morning. Avocado growers typically interplant cultivars of opposite flowering types to maximize yield. Recently, it has been hypothesized that 90% to 95% of avocado flowers are self-pollinated in southern Florida. However, this hypothesis does not address whether mature, marketable avocado fruit in Florida are the result of outcrossing. To determine whether avocado fruit in southern Florida result from self-pollination or outcrossing, fruit were harvested from a commercial orchard in Miami-Dade County, Florida, from a block consisting of two cultivars, Simmonds (Flowering Type A) and Tonnage (Flowering Type B), interplanted in approximately equal numbers. Seeds were germinated and the resulting progeny were genotyped using eight fully informative, microsatellite markers. Seventy-four percent of the ‘Simmonds’ progeny and 96% of the ‘Tonnage’ progeny were judged to be the result of cross-pollination, with an estimated overall outcrossing rate of 63% to 85% within this particular block of the orchard. Seedlings judged to be the result of cross-pollinations between ‘Simmonds’ and ‘Tonnage’ are being maintained at the U.S. Department of Agriculture-Agricultural Research Service, Subtropical Horticulture Research Station and are being evaluated for segregation of important agronomic and horticultural traits.

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Cristina Pisani, Mark A. Ritenour, Ed Stover, Anne Plotto, Rocco Alessandro, David N. Kuhn and Raymond J. Schnell

Avocado (Persea americana Mill.) is a high-value fruit that continues to increase in consumer demand. A population of ‘Hass’–‘Bacon’ hybrids was planted at USDA-ARS, Fort Pierce, as part of a study to find selections with good horticultural and postharvest quality traits for Florida. Extensive phenotypic data on quality were collected over 3 years. Ten selections were identified in 2014 and 2015 with promising fruit quality and postharvest shelf life characteristics and were tested in sensory panels using store-bought ‘Hass’ as the standard. In general, the selections had fruit quality similar to commercial ‘Hass’. Avocados that were most liked were described as creamy in texture with buttery and nutty flavor. Only one selection (R7T54 in 2014) and one store-bought control (‘Hass’ in 2015) were disliked, which was associated with greater firmness at the time of evaluation, likely relating to insufficient postharvest conditioning. Furthermore, CA ‘Hass’ commercial requirements for minimum dry matter (20.8%) were generally achieved by these selections under Florida conditions, ranging from 18.4% to 25.7%. This study identified 10 selections with composition and sensory quality similar to ‘Hass’ that are suitable for further testing and development in Florida.

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Donald Livingstone III, Barbie Freeman, Cecile L. Tondo, Kathleen A. Cariaga, Nora H. Oleas, Alan W. Meerow, Raymond J. Schnell and David N. Kuhn

The ability to rapidly genotype a large number of individuals is the key to any successful marker-assisted plant breeding program. One of the primary bottlenecks in high-throughput screening is the preparation of DNA samples, particularly the quantification and normalization of samples for downstream processing. A rapid and simple Sybr Green I-based quantification procedure that can be performed in a 96-well format is outlined. In this procedure, a dual standard curve method is used to allow better resolution of dilute samples and to reduce fluorescence value variation between samplings. A method to quickly normalize samples, and the importance of normalization, is also explored. We demonstrate that successful fragment amplification of a Theobroma grandiflorum (Willd ex Spreng) Schum. population is increased from 70% to 98% when each DNA extract is quantified and normalized as opposed to quantifying only a subset and normalizing all the samples based on the average of that subset. Improved microsatellite amplification was also observed among individuals in the monocot genus Phaedranassa Herb. ssp. Additionally, when our normalization method is applied to a Persea americana Mill. population, 97% of the samples normalized to 4 ng·μL−1 amplify at least three of six microsatellite regions, whereas only 30% of the samples below 4 ng·μL−1 (i.e., samples that could not be normalized) amplify at least three regions. We describe an undemanding method to quantify and normalize a large number of samples, which can be done manually or can be automated.

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Raymond J. Schnell, Cecile L. Tondo, J. Steven Brown, David N. Kuhn, Tomás Ayala-Silva, James W. Borrone and Thomas L. Davenport

Avocado (Persea americana Mill.) has an unusual flowering mechanism, diurnally synchronous protogynous dichogamy, that promotes crosspollination among avocado genotypes. In commercial groves, which usually contain pollinizer rows adjacent to the more desirable commercial cultivars, the rate of outcrossing has been measured with variable results. Using microsatellite markers, we estimated outcrossing in a commercial California ‘Hass’ avocado orchard with adjacent ‘Bacon’ pollinizers. Seedlings grown from mature harvested fruit of both cultivars were genotyped with five fully informative microsatellite markers and their parentage determined. Among the 919 seedlings of ‘Hass’, 688 (75%) were hybrids with ‘Bacon’; the remaining 231 (25%) seedlings were selfs of ‘Hass’. Among the 850 seedlings of ‘Bacon’, 382 (45%) were hybrids with ‘Hass’ and the remaining 468 (55%) seedlings were selfs of ‘Bacon’. The high outcrossing rate observed in the ‘Hass’ seedlings was expected, because adjacent rows of opposite flowering types (A versus B) are expected to outcross. However, the high selfing rate in ‘Bacon’ was unexpected. A previous study in Florida using the cultivars ‘Simmonds’ and ‘Tonnage’ demonstrated differences in outcrossing rates between complementary flowering type cultivars. In both Florida and California, the A type parents (‘Hass’ and ‘Simmonds’) had similar outcrossing rates (≈75%); however, the B type parents (‘Bacon’ and ‘Tonnage”) had highly skewed outcrossing rates of 45% and 96%, respectively. Two new avocado lethal mutants were discovered among the selfed seedlings of ‘Hass’ and ‘Bacon’. These were labeled “spindly” and “gnarly” and are similar in phenotype to mutants described in Arabidopsis and other crop species.

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Vanessa E.T.M. Ashworth, Haofeng Chen, Carlos L. Calderón-Vázquez, Mary Lu Arpaia, David N. Kuhn, Mary L. Durbin, Livia Tommasini, Elizabeth Deyett, Zhenyu Jia, Michael T. Clegg and Philippe E. Rolshausen

The glossy, green-fleshed fruit of the avocado (Persea americana) has been the object of human selection for thousands of years. Recent interest in healthy nutrition has singled out the avocado as an excellent source of several phytonutrients. Yet as a sizeable, slow-maturing tree crop, it has been largely neglected by genetic studies, owing to a long breeding cycle and costly field trials. We use a small, replicated experimental population of 50 progeny, grown at two locations in two successive years, to explore the feasibility of developing a dense genetic linkage map and to implement quantitative trait locus (QTL) analysis for seven phenotypic traits. Additionally, we test the utility of candidate-gene single-nucleotide polymorphisms developed to genes from biosynthetic pathways of phytonutrients beneficial to human health. The resulting linkage map consisted of 1346 markers (1044.7 cM) distributed across 12 linkage groups. Numerous markers on Linkage Group 10 were associated with a QTL for flowering type. One marker on Linkage Group 1 tracked a QTL for β-sitosterol content of the fruit. A region on Linkage Group 3 tracked vitamin E (α-tocopherol) content of the fruit, and several markers were stable across both locations and study years. We argue that the pursuit of linkage mapping and QTL analysis is worthwhile, even when population size is small.