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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.

Recent enhancement of the pool of known molecular markers for avocado has allowed the construction of the first moderately dense genetic map for this species. Over 300 SSR markers have been characterized and 163 of these were used to construct a map from the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage'. One hundred thirty-five primer pairs amplified 163 usable loci with 20 primer pairs amplifying more than one locus. 'Tonnage' was heterozygous for 152 (93%) loci, whereas 'Simmonds' was heterozygous for 64 (39%). Null alleles were identified at several loci. Linkage maps were produced for both reciprocal crosses and combined to generate a composite linkage map for the F1 population of 715 individuals. The composite map contains 12 linkage groups. Linkage groups ranged in size from 157.3 cM (LG2) to 2.4 cM (LG12) and the number of loci mapped per group ranged from 29 (LG1) to two (LG12). The total map length was 1,087.4 cM. Only seven markers were observed to have segregation distortion (α ≤ 0.05) across both sub-composite (reciprocal) maps. Phenotypic data from traits of horticultural interest are currently being collected on this population with the ultimate goal of identifying useful quantitative trait loci and the development of a marker-assisted selection program.

Phytophthora cinnamomi causes a severe root rot in avocado, Persea americana. Breeding tolerant rootstocks is thought to be the most promising method for phytophthora root rot disease control but breeding avocado is challenging. The avocado flowering syndrome (synchronous protogynous dichogamy), combined with high flowering and low fruit set, render controlled pollination exceedingly difficult. Juxtaposing complementary flowering types of elite parent cultivars (cultivars that produce progeny with tolerance to phytophthora root rot) was performed in an effort to increase the number of full-sib progeny for elite maternal parents and, hypothetically, the number of phytophthora root rot tolerant progeny. Although high outcrossing rates were achieved (estimated ∼93%), the majority of progeny had a non-elite paternal parent (56% of progeny were offtypes) implying maternal trees were pollinated by non-elite distant trees. Among progeny that could be confidently genotyped, a high number of cross types were detected (33). Contrary to our hypothesis, a significant portion of the progeny were the result of crosses between like, and not complementary, flowering types. The spatial distribution of productive trees and grafts helped to explain these data, as productive grafts were directly adjacent to grafts of the same flowering type more often than that of the complementary flowering type. Selfed progeny were significantly less tolerant to phytophthora root rot than outcrossed progeny. Progeny resulting from crosses between an elite maternal parent and non-elite pollen donor (offtypes) were less tolerant than full-sib progeny resulting from crosses between elite parents. Maternal effects may interfere with identifying truly disease tolerant selections. Thus, to reduce maternal effects and non-elite pollen donor contamination, removal of seedling cotyledons before screening for disease tolerance and better isolation of elite parent trees and windbreaks may improve breeding efficacy. This study also demonstrates the usefulness of microsatellite markers in parentage analysis where a high proportion of the putative parents are closely related.

In this paper, we report the construction of the first composite map of cacao from linkage data of one F2 and two F1 mapping populations with a high number of codominant markers in common. The combination of linkage information from all three maps results in the currently most precise estimates of marker locations and distances between markers, especially in densely marked areas. JoinMap®V4 software was used for all marker quality assessment and mapping. Individual (sub-composite) maps and the composite map contained 10 major linkage groups, corresponding to the number of cacao chromosomes. Homogeneity of marker placement was very high among sub-composite maps, the composite map, and the designated “reference” map. Care was exercised in the re-creation of sub-composite maps and the composite map to include only markers with acceptable mapping quality parameters. The composite map places more markers with higher precision than any individual map. This research clearly demonstrates for the first time a very high level of marker homogeneity among commercial cacao clones compared to other species. The observed homogeneity between different maps, including the composite one, is probably due to a narrow genetic base of commercial cacao clones. Markers linked to identified quantitative trait loci (QTLs) are more likely to retain linkage in other commercial clones, rendering the QTLs in cacao potentially more stable than in other species.

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 func- tionally male the next morning. Avocado growers typically interplant cultivars of opposite flowering types to maxi- mize 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 out- crossing, 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, micro- satellite 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. Avocado is an evergreen tree native to Mesoamerica.Persea americana is a polymorphic species consisting of several botanical varieties, or subspecies, adapted to geographically distinct microenvironments. These include wild botanical varieties (var. floccosa Mez, var. steyermarkii Allen, var. nubigena (Williams) Kopp) and three horticulturally important botanical varieties, also known as ''races'' (var. americana Mill. (''West Indian''), var. guatemalensis Williams (''Guate- malan''), and var. drymifolia (Schlecht. and Cham.) Blake (''Mexican''); Scora and Bergh 1990). Because of the adapta- tion to microclimates, hybrids of Guatemalan-Mexican botan- ical varieties are cultivated in the dry Mediterranean-like environments of California, Israel, Chile, South Africa, and the upland areas of Asia. West Indian or hybrids of West Indian-Guatemalan botanical varieties are cultivated in the more humid tropical environments of Florida, Hawaii, South- east Asia, and the Caribbean. Mexico is the primary producer of avocados, centered around the state of Michoacan, and 'Hass' is

We previously analyzed genetic diversity and population structure of eight cultivars within Florida coconut (Cocos nucifera L.) germplasm using 15 microsatellite (simple sequence repeat, SSR) markers. Here we report on the analysis of the same genotypes using 13 markers derived from WRKY sequences containing single nucleotide polymorphisms (SNP) and one microsatellite. WRKY transcription factors are unique to plants and are involved in numerous vital processes including disease resistance. Our objective was to determine the value of this type of gene to assess thegenetic diversity of this species. Overall, the WRKY results are similar to thosewith microsatellite markers. Despite the lower number of alleles identified with the WRKY-derived markers (37 versus 67 total alleles identified with the SSR markers), individuals of cultivars ‘Red Malayan Dwarf’, ‘Fiji Dwarf’ (‘Niu Leka’) and ‘Red Spicata’ were clearly clustered, as in the previous analysis. However, individuals of ‘Green Malayan Dwarf’ and ‘Yellow Malayan Dwarf’ cultivars were resolved with other varieties, perhaps due to selective forces operating on these functional genes. Most samples of the ‘Atlantic Tall’ and ‘Panama Tall’ cultivars clustered together as in the previous microsatellite study. We discuss the utility of WRKY-derived markers for assessing the genetic diversity of coconut, and their possible value in the study of other species. Published by Elsevier B.V.

Theobroma cacao L. is an understory tree from the Amazon basin that can be cultivated in a sustainable agro-forestry system, providing income to small farmers while maintaining biodiversity. Four main genetic groups of cacao are traditionally described: Criollo, Trinitario, and lower and upper Amazon Forastero. During the 17 th and 18 th centuries, plants derived from a small number of parents were distributed to many tropical regions of the world, resulting in commercial plantings with a narrow genetic base. Production of cacao in tropical America has been severely affected by two fungal pathogens causing diseases known as witches' broom (WB) and frosty pod (FP). These, along with another pan-tropical fungal disease, black pod (BP), were responsible for over 700 million USD in losses in 2001.[...]

Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for fingerprinting individuals and genetic linkage mapping. Primers were designed to microsatellite-containing ESTs. Twenty-two T. cacao accessions, parents of various mapping populations segregating for disease resistance and crop yield characteristics, were tested. Twenty-seven informative loci were discovered with 26 primer pairs. The number of detected alleles ranged from two to 11 and averaged 4.4 per locus. All 27 markers could be mapped into at least one of the existing F 1 or F 2 populations segregating for agronomically important traits.

Expressed sequence tags for Persea americana Mill. were investigated to expand upon the number of informative microsatellite markers available for avocado. Seventy informative loci were discovered using 24 P. americana var. americana Mill. accessions. The number of alleles detected ranged from two to 17 and averaged 7.1 alleles per locus. These primers successfully amplified products in different varieties of P. americana, hybrids and a related species, Persea schiedeana. These primers will be useful for characterizing germplasm, determining genetic relationships of cultivated accessions, and for marker-assisted development of root rot-tolerant P. americana var. americana rootstock material.

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.

There is currently an international effort in improving disease resistance and crop yield in Theobroma cacao L., an economically important crop of the tropics, using marker-assisted selection for breeding. We are developing molecular genetic markers focusing upon gene families involved with disease resistance. One such family is the WRKY proteins, which are plant-specific transcriptional factors associated with regulating defense responses to both abiotic and biotic stresses. Degenerate PCR primers were designed to the highly conserved DNA-binding domain and other conserved motifs of group I and group II, subgroups a-c, WRKY genes. Sixteen individual WRKY fragments were isolated from a mixture of T. cacao DNA using one pair of primers. Of the 16 WRKY loci investigated, seven contained single nucleotide polymorphisms within the intron as detected by sequence comparison of the PCR products. Four of these were successfully converted into molecular markers and mapped in an F2 population by capillary electrophoresis-single strand conformation polymorphism analysis. This is the first report of a pair of degenerate primers amplifying WRKY loci directly from genomic DNA and demonstrates a simple method for developing useful genetic markers from members of a large gene family.

Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyketides identified to date. The biological activities of these compounds are quite diverse. Compounds having potential therapeutic value as anti-cancer agents as well as deadly neurotoxins, whose production has resulted in severe public health hazards and economic hardships, are represented in this group of secondary metabolites. Stable isotope feeding experiments have firmly established the polyketide origins of representative compounds from each of the three structural classes, the polyether ladders, the macrocycles and the linear polyethers. Yet some unusual labeling patterns have been observed in each class. Pendant methyl groups are most often derived from C-2 of acetate and deletions of C-1 of acetate are common. Studies on the biosynthesis of dinoflagellate derived polyketides at the genomic level have not been reported, in part due to the peculiarities of the dinoflagellate nucleus and the lack of a dinoflagellate transformation system. Nevertheless, a fundamental understanding of the genetics of polyketide biosynthesis by dinoflagellates could be the catalyst for developing several fruitful avenues of research. Dinoflagellate derived polyketides are reviewed with special emphasis on pharmacology and biosynthesis.

The genetic improvement of tropical fruit trees is limited when compared to progress achieved in temperate fruit trees and annual crops. Tropical fruit tree breeding programs require significant resources to develop new cultivars that are adapted to modern shipping and storage requirements. The use of molecular markers in tropical fruit tree breeding is greatly assisting in solving a number of difficult challenges for breeders such as the development of complex family structures for recombination mapping and for recurrent selection. A review of the literature on molecular markers development and new techniques for increasing single-nucleotide polymorphic markers is discussed. The development of marker-assisted breeding for these tropical tree crops is also discussed.

The WRKY gene family of transcription factors is involved in several diverse pathways and includes components of plant-specific, ancient regulatory networks. WRKY genes contain one or two highly conserved DNA binding domains interrupted by an intron. We used partial sequences of five independent WRKY loci to assess their potential for phylogeny reconstruction. Loci were originally isolated from Theobroma cacao L. by PCR with a single pair of degenerate primers; loci-specific primers were subsequently designed. We tested those loci across the sister genera Herrania Goudot and Theobroma L., with Guazuma ulmifolia Lam. as the outgroup. Overall, the combined WRKY matrices performed as well or better than other genes in resolving the intrageneric phylogeny of Herrania and Theobroma. The ease of isolating numerous, independent WRKY loci from diverse plant species with a single pair of degenerate primers designed to the highly conserved WRKY domain, renders them extremely useful tools for generating multiple, single or low copy nuclear loci for molecular phylogenetic studies at lower taxonomic levels. This is the first demonstration of the potential for members of the WRKY gene family for phylogenetic reconstruction.

We investigated the reliability of capillary array electrophoresis-single strand conformation polymorphism (CAE-SSCP) to determine if it can be used to identify novel alleles of candidate genes in a germplasm collection. Both strands of three different size fragments (160, 245 and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temperatures (18 degrees C, 25 degrees C, 30 degrees C, and 35 degrees C). Mixtures of amplified fragments of either the intron interrupting the C-terminal WRKY domain of the Tc10 locus or the NBS domain of the TcRGH1 locus of Theobroma cacao were electroinjected into all 16 capillaries of an ABI 3100 Genetic Analyzer and analyzed three times at each temperature. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments. A statistical analysis of the means of the fragment mobilities demonstrated that single-stranded conformers of the fragments could be reliably identified by their mobility at all temperatures and size classes. The order of elution of fragments was not consistent over strands or temperatures for the intermediate and large fragments. If samples are only run once at a single temperature, small fragments could be identified from a single strand at a single temperature. A combination of data from both strands of a single run was needed to identify correctly all four of the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large fragments that differed by only a single single-nucleotide polymorphism (SNP) from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be < or =250 bp, samples should be analyzed at two different temperatures between 18 degrees C and 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures, and undenatured double-stranded (ds)DNA molecular weight standards, such as ROX 2500, should be included as internal standards.

Background The Cocoseae is one of 13 tribes of Arecaceae subfam. Arecoideae, and contains a number of palms with significant economic importance, including the monotypic and pantropical Cocos nucifera L., the coconut, the origins of which have been one of the “abominable mysteries” of palm systematics for decades. Previous studies with predominantly plastid genes weakly supported American ancestry for the coconut but ambiguous sister relationships. In this paper, we use multiple single copy nuclear loci to address the phylogeny of the Cocoseae subtribe Attaleinae, and resolve the closest extant relative of the coconut. Methodology/Principal Findings We present the results of combined analysis of DNA sequences of seven WRKY transcription factor loci across 72 samples of Arecaceae tribe Cocoseae subtribe Attaleinae, representing all genera classified within the subtribe, and three outgroup taxa with maximum parsimony, maximum likelihood, and Bayesian approaches, producing highly congruent and well-resolved trees that robustly identify the genus Syagrus as sister to Cocos and resolve novel and well-supported relationships among the other genera of the Attaleinae. We also address incongruence among the gene trees with gene tree reconciliation analysis, and assign estimated ages to the nodes of our tree. Conclusions/Significance This study represents the as yet most extensive phylogenetic analyses of Cocoseae subtribe Attaleinae. We present a well-resolved and supported phylogeny of the subtribe that robustly indicates a sister relationship between Cocos and Syagrus. This is not only of biogeographic interest, but will also open fruitful avenues of inquiry regarding evolution of functional genes useful for crop improvement. Establishment of two major clades of American Attaleinae occurred in the Oligocene (ca. 37 MYBP) in Eastern Brazil. The divergence of Cocos from Syagrus is estimated at 35 MYBP. The biogeographic and morphological congruence that we see for clades resolved in the Attaleinae suggests that WRKY loci are informative markers for investigating the phylogenetic relationships of the palm family.