Search

Economically, cacao (Theobroma cacao L.) is a major tropical commodity for the Americas; however, severe losses due to Moniliophthora roreri (Cif. and Par.), which causes frosty pod rot (FPR), and Phytophthora spp., which causes black pod rot (BPR), have reduced cacao production in the Americas. The objectives of this study are to (i) re-confirm the QTL using different marker set; (ii) discover new QTL associated with FPR and BPR resistance using SNP markers; and (iii) find genes in the candidate QTL regions. At CATIE in Turrialba, Costa Rica, an F1 mapping population of cacao was obtained by crossing “POUND 7,” a clone moderately susceptible to FPR and resistant to BPR, with “UF 273,” resistant to FPR and highly susceptible to BPR. A total of 179 F1 progeny were fingerprinted with 5149 SNP markers and a dense linkage map composed of 10 linkage groups was developed using 2910 polymorphic SNP markers. Also segregating F1 trees were screened for resistance to FPR and BPR diseases. Seven QTL previously reported on chromosomes 2, 7, and 8 for FPR resistance and on chromosomes 4, 8, and 10 for BPR resistance were confirmed. Additionally, eight QTL were identified for FPR resistance (chromosomes 4, 9, and 10) and BPR resistance (chromosome 2). The expression of genes commonly associated with plant defense and disease resistance that are located within the identified QTL was confirmed.

Avocado (Persea americana Mill.) is a subtropical domesticated fruit tree indigenous to Mesoamerica. It is a member of the Lauraceae family and is separated into three horticultural races (Guatemalan, Mexican, and West Indian) mainly corresponding to their ecological adaptation, botanical, and physiological traits. Main objectives of this study were to characterize the population structure, genetic diversity, and horticultural race of a total of 354 Persea spp. trees whose origin is as follow: 221 trees [P. americana, (218), P. nubigena (2) and P. krugii (1)] from the USDA-ARS-Subtropical Horticultural Research Station, Miami; 105 trees from the Fairchild Farm [P. americana (104) and P. schiedeana (1)], and 28 trees collected in Mexico [P. schiedeana (23) and P. americana (5)]. The complexity of their interracial admixture; as well as mislabeling frequency was also evaluated. Molecular marker analysis utilizing a set of 55 simple sequence repeat (SSR) markers amplified a total of 869 alleles with a mean number of alleles per locus of 15.8 and average polymorphism information content value of 0.71, indicating a high variability in the allele frequency for the collection. Significant deviations from Hardy–Weinberg equilibrium were identified after Bonferroni correction for a large number of loci (48; 87%) due to the presence of null alleles. The main source of variation for this population was found to be within individuals (66.84%), with 19.30% variation among populations, and 13.86% variation among individuals within populations. Moreover, population specific inbreeding indices (F IS ) were calculated for West Indian, Guatemalan, and Mexican [(0.1918; p value 0.0000), (0.1879; p-value 0.0000), (0.0925; p-value 0.0022)], respectively. Bayesian analysis divided the individual genotypes into groups associated with the Guatemalan, Mexican, West Indian races; interracial admixture; complex hybrids and P. schiedeana species. Also, results of the multivariate clustering method (PCA) and genetic distance analyses calculated among all possible individual combinations within the SSR diversity data agreed with Bayesian or Structure analyses results. The 55 SSRs provided complete resolution of all individuals and the estimated mislabeling error was approximately 0.28%.

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.

Thirty-four aroma-active compounds, previously identified with high flavor dilution factors by application of an aroma extract dilution analysis, were quantified in tree-ripened fruits of mango (Mangifera indica L. 'Haden'). From the results, the odor activity value (OAV) was calculated for each compound as the ratio of its concentration in the mangoes to its odor threshold in water. OAVs1 were obtained for 24 compounds, among which ethyl 2-methylbutanoate (fruity; OAV 2100), (3E,5Z)-undeca-1,3,5-triene (pineapple-like; OAV 1900), ethyl 3-methylbutanoate (fruity; OAV 1600), and ethyl butanoate (fruity; OAV 980) were the most potent, followed by (2E,6Z)-nona-2,6-dienal (cucumber-like), ethyl 2-methylpropanoate (fruity), (E)-β-damascenone (cooked apple-like), ethyl hexanoate (fruity), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel-like), 3-methylbut-2-ene-1-thiol (sulfurous), γ-decalactone (peach-like), β-myrcene (terpeny), (3Z)-hex-3-enal (green), 4-methyl-4-sulfanylpentan-2-one (tropical fruit-like), and ethyl octanoate (fruity). Aroma simulation and omission experiments revealed that these 15 compounds, when combined in a model mixture in their natural concentrations, were able to mimic the aroma of the fruits.

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.

Cacao (Theobroma cacao L.) is an important cash crop in tropical growing regions of the world and particularly for small cacao farmers. Over the past two decades, ‘CCN 51’ has become one of the most planted cultivars in Ecuador, mainly as a result of its high productivity and disease resistance. Intermixing of Nacional fine flavor Ecuadorian beans with beans of ‘CCN 51’ has become common practice, reducing overall bean quality and decreasing value. The primary goals of this study were to determine the genetic identity, structure, and allelic richness of ‘CCN 51’, its maternal origin and to compare ‘CCN 51’s’ agronomic characteristics against a composite group of Nacional cultivars. To investigate the complex genetic background of this cultivar, 70 simple sequence repeat loci were used. The high heterozygosity observed (56 of 70 loci) for ‘CCN 51’ is not characteristic of traditional Nacional cultivars. Comparison of agronomic characteristics between ‘CCN 51’ and several Nacional cultivars indicates significant differences in cacao dry bean weight, yield potential, production efficiency, percent healthy pods, and witches' broom [Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora] disease incidence. Additionally, physical, chemical, and organoleptic characteristics suggest that ‘CCN 51’ is different from those of Nacional lineage. Based on population structure analysis, the predominant ancestries for ‘CCN 51’ are Iquitos (45.4%), Criollo (22.2%), and Amelonado (21.5%) genetic groups. A lesser proportion of its genome was accounted for by genetic groups Contamana (3.9%), Purús (2.5%), Marañon (2.1%), and Nacional (1.1%) admixtures. Results of phylogenetic analyses using the unweighted pair group method with arithmetic mean yielding high bootstrap values strongly support the relatedness of ‘CCN 51’ with Iquitos, Criollo, and Amelonado genetic groups. Moreover, seven mitochondrial simple sequence repeat loci revealed that ‘CCN 51’ maternally inherited the ‘IMC 67’ cytotype. ‘CCN 51’ constitutes a valuable cacao genetic resource that is currently used not only in its country of origin, but also in many other national breeding and selection programs worldwide.

The majority of the world's cacao for chocolate manufacture is produced in West Africa. Cocoa breeding programs in West Africa need genetic markers to reduce the time needed for improving cocoa by screening seedlings for the presence of the markers rather than mature plants for the phenotypic traits (i.e., marker-assisted selection [MAS]). For MAS to be successful, the breeder must have both access to markers linked to desired traits and a convenient marker-assay system that can be performed locally. In this study, microsatellite markers that flanked disease resistance quantitative trait loci (QTL) but could not be assayed conveniently in West Africa were converted using a genome walking method into single nucleotide polymorphism (SNP) markers that could be assayed locally. The SNP and microsatellite markers were equally effective in identifying off-types in two different mapping populations of cacao. Also, SNPs cast doubt on whether all microsatellite markers are identical by descent.

Cacao (Theobroma cacao L.) is a significant agricultural commodity in the Dominican Republic, which ranks 11th in the world for cacao exports. To estimate genetic diversity, determine genetic identity, and identify any labeling errors, 14 SSR markers were employed to fingerprint 955 trees among cacao germplasm accessions and local farmer selections (LFS). Comparisons of homonymous plants across plots revealed a significant misidentification rate estimated to be 40.9 % for germplasm accessions and 17.4 % for LFS. The 14 SSRs amplified a total of 117 alleles with a mean allelic richness of 8.36 alleles per locus and average polymorphism information content (PIC) value of 0.67 for the germplasm collection. Similar levels of variation were detected among the LFS where a total of 113 alleles were amplified with a mean of 8.07 alleles per locus and PIC of 0.57. The observed heterozygosity (Hobs) was 0.67 for the germplasm collection and 0.60 for LFS. Based on population structure analysis 43.9 % of the germplasm accessions and 72.1 % of the LFS are predominantly of the Amelonado ancestry. Among these Amelonado, 51.7 % for the germplasm collection and 50.6 % for LFS corresponded to Trinitario hybrid lineage. Criollo ancestry was found in 7.6 and 9.5 % of the germplasm accessions and LFS, respectively. The Contamana, Nacional, and Iquitos backgrounds were also observed in both populations, but the Curaray background was only detected in the germplasm accessions. No Purus or Guiana ancestry was found in either of the populations. Overall, significant genetic diversity, which could be exploited in the Dominican Republic breeding and selection programs, was identified among the germplasm accessions and LFS.

Saccharum species are autopolyploids with ploidy level ranging from 5× to 16x, and are considered the most complex genomes among crop plants. In present study, the genome sizes of 28 Saccharum spontaneum accessions, 15 Saccharum officinarum accessions, 28 Saccharum robustum accessions, and 12 Saccharum hybrids spp. were analyzed using flow cytometry. The estimated genome sizes of S. officinarum accessions ranged from 7.50 to 8.55 Gb with an average size of 7.88 Gb. In S. robustum, the estimated genome sizes ranged from 7.65 to 11.78, reflecting the variation of ploidy level. In S. spontaneum, the estimated genome sizes varied widely, with a range from 3.36 to 12.64 Gb, also due to variation of ploidy level. The average monoploid genome size of S. officinarum was 985 Mb, and that of S. spontaneum was 843 Mb. The results also showed that genome sizes were correlated with chromosome numbers, and based which, that the unknown chromosome numbers of some accessions could be predicted. The estimated genome sizes of Saccharum germplasm also helped identify some mislabeled accessions and yielded information critical for sugarcane breeding and genome sequencing programs.

Avocado sunblotch viroid (ASBVd) causes an important disease of avocado, Persea americana. Symptoms of avocado sunblotch were first observed in the avocado germplasm collection at the National Germplasm Repository in Miami in the early 1980s; however, the extent of infection was unknown. An ASBVd-specific reverse transcription polymerase chain reaction (RT-PCR) protocol was developed in 1996 and used to screen every tree in the collection. Surveys in 1996 and 2000 found that although 23 newly infected trees were detected, the proportion of ASBVd-positive accessions remained unchanged at 19%. However, in a 2009 survey, 50 newly infected trees were detected for an overall infection rate of 21%. Results of spatial analyses indicate that for the older plantings, the effective range of spread increased more than threefold during the 13 year span, while in the newer plantings, the pattern of infection indicates a reintroduction of the viroid rather than natural spread. Despite strict sanitization procedures in field and greenhouse operations, ASBVd infections have increased in the USDA collection. Although genetic diversity in the collection would be reduced, eliminating all ASBVd-positive plants may be necessary to ensure that other accessions in the collection do not become infected.

Theobroma cacao (cacao) is a tree cultivated in the tropics around the world for its seeds that are the source of both chocolate and cocoa butter. Genetic marker development for marker-assisted selection (MAS) is critical for the success of cacao breeding for disease resistance and yield. To develop conserved ortholog set II (COSII) single-nucleotide polymorphism (SNP) markers for MAS in cacao, we have used three strategies and three types of cacao genetic and sequence data to identify and map 98 cacao COSII genes. The resources available at the time these studies were first undertaken dictated the strategy utilized. For the first strategy, SNPs were identified using cacao expressed sequence tags homologous to COSII sequences. Strategy II utilized a leaf transcriptome of cacao genotype “Matina 1–6” and Strategy III the genomic sequence of a 3-Mb region of “Matina 1–6” linkage group 5 associated with an important quantitative trait locus (QTL) for resistance to black pod. We have identified SNP markers for 83 of the 98 mapped COSII genes, and 19 of these SNP markers co-locate with QTLs. These COSII SNP markers, the first identified for cacao, will be used for genotyping and off-typing in cacao breeding programs and employed for genetic mapping and syntenic studies to trace co-location of genes regulating traits of importance between cacao and other species.

Two molecular marker systems, SCoT and ISSR were used for identification and genetic comparison analysis of 23 mango germplasm accessions collected within Guangxi province of China. Using 18 selected SCoT primers 158 bands were generated, of which 104 (65.82%) were polymorphic. Eighteen selected ISSR primers amplified 156 bands with 87 (55.77%) being polymorphic. The cultivars of Xiang Ya Mango type and their progeny have high genetic similarity with each other. The 23 cultivars were clustered into two major groups based on the SCoT analysis and three major groups based on the ISSR analysis with UPGMA. These clusters are in accordance with their known origins and main phenotypic characteristics. Our results indicated that the SCoT analysis better represents the actual relationships than ISSR analysis, although both analyses give similar results. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.

The tropical tree crop Theobroma cacao L. is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. Although the upper Amazon region is the center of origin for cacao, 70% of the world’s supply of cacao beans currently comes from small farms in West Africa. While cacao breeding programs in producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. While DNA extraction can be routinely performed, the equipment needed to analyze simple sequence repeats (SSRs) is seldom available, forcing the outsourcing of genotyping to foreign laboratories and delaying the breeding process. We describe a 5′ nuclease (TaqMan)-based single nucleotide polymorphism (SNP) assay for genotyping cacao plants under conditions similar to those found in most cacao-producing areas. The assay was tested under field conditions by planting open pollinated seeds of seven pods from four different maternal plants. The resulting 171 seedlings were successfully genotyped with 18 SNP markers representing 12 loci. The ability to use temperature-stable reagents and rapid DNA extraction methods is also explored. Additionally, by examining the seedling genotypes for the SNP markers and 14 additional SSR markers, we investigated whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method of genotyping cacao seedlings in the field should allow for more efficient resource management of seed gardens and is currently being implemented in Ghana.

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao’s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches’ broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao’s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.

Sugarcane (Saccharum hybrid spp.) breeders at the Hawaiian Sugar Planters’ Association used biparental and melting pot (modified polycross) crossing methods concurrently from 1935 to 1985. While the annual effort expended to make biparental crosses exceeded the effort to make melting pot crosses over this 50-year period, annual viable seed yield from biparental crosses was usually less than 15% of that from melting pot crosses and hence, the numbers of seedlings planted to the field from those crosses usually accounted for less than 20% of the total seedling population. In 1985, nine of the 10 sugarcane cultivars listed in Hawaii’s variety census originated from melting pot crosses; only one originated from a biparental cross. In the face of a shrinking sugar industry in Hawaii and a smaller work force in the breeding program, the decision was made in 1985 to rely primarily on melting pot crosses for the production of commercial cultivars. From 1985 to 2005, twelve additional clones that were bred prior to 1985 eventually attained “commercial cultivar” status by exceeding 1% of the total cane growing area. All twelve originated from melting pot crosses. Over the 50-year period that the two crossing methods were used, the melting pot method proved to be more labour efficient and ultimately contributed more than the biparental crossing procedure toward the development of new commercial cultivars for the Hawaiian sugar industry. From 1985 forward, the biparental crossing method was used more for introgressing desired traits from exotic germplasm than for the development of commercial cultivars.

Single nucleotide polymorphism (SNP) markers are increasingly being used in crop breeding programs, slowly replacing simple sequence repeats (SSR) and other markers. SNPs provide many benefits over SSRs, including ease of analysis and unambiguous results across various platforms. We have identified and mapped SNP markers in the tropical tree crop Theobroma cacao, and here we compare SNPs to SSRs for the purpose of determining off-types in clonal collections. Clones are used as parents in breeding programs and the presence of mislabeled clones (off-types) can lead to the propagation of undesired traits and limit genetic gain from selection. Screening was performed on 186 trees representing 19 Theobroma cacao clones from the Institute of Agricultural Research for Development (IRAD) breeding program in Cameroon. Our objectives were to determine the correct clone genotypes and off-types using both SSR and SNP markers. SSR markers that amplify 11 highly polymorphic loci from six linkage groups and 13 SNP markers that amplify eight loci from seven linkage groups were used to genotype the 186 trees and the results from the two different marker types were compared. The SNP assay identified 98% of the off-types found via SSR screening. SNP markers spread across multiple linkage groups may serve as a more cost-effective and reliable method for off-type identification, especially in cacao-producing countries where the equipment necessary for SSR analysis may not be available.

Cacao (Theobroma cacao L.) is an important cash crop in many tropical countries. Cacao accessions must be propagated vegetatively to conserve genetic integrity due to its allogamous nature and its seed recalcitrance (lack of dormancy). Therefore, cacao germplasm is usually maintained as living trees in fi eld collections and has resulted in varying rates of misidentifi cation and duplication. Using a high throughput genotyping system with 15 microsatellite loci, all 924 trees in the USDA-ARS Mayaguez cacao collection were fi ngerprinted. Nineteen accessions (12.3%) were found to have intraplant errors while 14 (9.1%) synonymous sets were identifi ed that included replicates of 49 accessions. The average number of alleles (8.8; SE = 0.56) and gene diversity (H Obs = 0.65; SE = 0.026) indicate a high allelic diversity in this collection. A distance-based cluster analysis and a Bayesian assignment test showed that the cacao accessions can be classifi ed into four distinct clusters, with their geographical origins covering most of the cacao growing regions in the Americas. Assessment of the representative diversity of the collection led to the identifi cation of several genetic gaps, including underrepresented genetic populations and particular traits of economic and agronomic value. The improved understanding of identities and structure in the USDA-ARS cacao collection will contribute to more effi cient use of cacao in conservation and breeding.

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.

The visual appearance of mangos is a primary factor in determining consumer acceptance and sale, similar to other fruit and vegetable commodities. Even if the appeal of visual appearance is based on consumer perception rather than on established quality factors, breeders must usually select within the range of acceptance, at least in some countries. Mango selection using multiyear breeding programs is slowly replacing the former method by which most earlier cultivars were selected, namely from chance seedlings either from planned or unplanned crosses. The knowledge of heritability of traits as they are controlled by genetics and experimental design and the effects and interaction of these two sets of factors on achieved gain have become more critical. The use of portable colorimeters has been shown to give repeatable scores in a quantitative, three-dimensional space for fruits and vegetables. In this experiment, we calculated broad-sense heritability estimates for five color traits, three morphological fruit traits, and one disease resistance trait (anthracnose expressed on the fruit). Estimates were found to be relatively high, indicating good potential for improvement through breeding. For nearly all traits measured, variance within families was greater than that among families, illustrating the likely importance of heterozygosity, dominance, and epistasis in these crosses. The careful estimation of heritability and repeatability will help prioritize and increase the efficiency of trait improvement as breeding methods become more sophisticated and competition for funding increases.

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.

A prior analysis of eight coconut cultivars with 15 microsatellite (SSR) markers drew unexpected relationships between two of the out-crossing tall cultivars evaluated: ‘Atlantic Tall’ and ‘Panama Tall’. We further investigated the relationships between these eight cultivars by increasing the number of individuals studied (particularly for ‘Atlantic Tall’ and ‘Panama Tall’), by including 28 more molecular markers, and by adding two other cultivars to our analysis. Our results show that five to ten coconut individuals do not represent a dependable sample to withdraw conclusions regarding cultivar/variety relationships, particularly when studying out-crossing genotypes. As suggested in the prior study, a high level of hybridization was observed between the ‘Atlantic Tall’ and ‘Panama Tall’ cultivars. However, at this time we were able to identify distinct groups for each one of these two cultivars. The two clustering methods used (Neighbor Joining, NJ and Unweighted Pair Group Method with Arithmetic mean, UPGMA) produced dendrograms that resolved contrasting cultivar relationships, especially for the ‘Atlantic Tall’ and ‘Panama Tall’ cultivars. We discuss the implications of our results in regard to current scenarios of coconut domestication and future considerations when assessing genetic relationships among different varieties.

Knowledge of genebank and on-farm genetic diversity, particularly in an introduced crop species, is crucial to the management and utilization of the genetic resources available. Microsatellite markers were used to determine genetic diversity in 574 accessions of cacao, Theobroma cacao L., representing eight groups covering parental populations in West Africa, genebank, and farmers’ populations in Nigeria. From the 12 microsatellite markers used, a total of 144 alleles were detected with a mean allelic richness of 4.39 alleles/locus. The largest genetic diversity was found in the Upper Amazon parent population (Hnb = 0.730), followed by the 1944 Posnette’s Introduction (Hnb = 0.704), and was lowest in the Local parent population (Hnb = 0.471). Gene diversity was appreciably high in the farmers’ populations (Hnb = 0.563–0.624); however, the effective number of alleles was lower than that found in the genebank’s Posnette’s population. Fixation index estimates indicated deficiency of heterozygotes in the Upper Amazon and the Local parent populations (Fis = 0.209 and 0.160, respectively), and excess of heterozygotes in the Trinitario parent population (Fis = −0.341). The presence of inbreeding in the Local parent populations and substructure (Wahlund effect) in the Upper Amazon were suggested for the deficiency of heterozygotes observed. Non-significant genetic differentiation observed between the genebank’s and farmers’ populations indicated significant impact of national breeding programs on varieties grown in farmers’ plantations. From this study, we showed that appreciable genetic diversity was present in on-farm and field genebank collections of cacao that can be exploited for crop improvement in West Africa. Suggestions for future conservation of on-farm genetic diversity and local landraces are further discussed.

‘Hass’ is the most popular avocado (Persea americana Mill.) cultivar in the world. It has been characterized as a crop requiring cross-pollination. However, the potential extent of self-pollination and the most effective pollen donors (best cross-pollinizing cultivars) have not been determined. In this study, 56 markers were screened against ‘Hass’ and nine commonly used pollinizing cultivars grown in southern California: ‘Bacon,’ ‘Ettinger,’ ‘Fuerte,’ ‘Harvest,’ ‘Lamb Hass,’ ‘Marvel,’ ‘Nobel,’ ‘Sir Prize,’ and ‘Zutano.’ Seventeen microsatellite, i.e., simple sequence repeat (SSR) markers, were found to be very promising for paternity analysis. Four highly informative SSR markers were selected to accurately and unequivocally identify pollen parents of ‘Hass’ fruit from an orchard interplanted with these pollinizing cultivars. From 2003 to 2006, 7,984 ‘Hass’ fruit were analyzed for their paternity. Overall, the pollen parents of 99.55% of the analyzed fruit could be unequivocally identified with a single multiplex polymerase chain reaction (PCR). Only 36 fruits (

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.

The present study investigates the parentage of farm accessions in Cameroon using data from 12 microsatellite loci. Bayesian analysis suggests that 25.5% of the 400 farm accessions studied is still closely related to the traditional Amelonado variety called 'German Cocoa' by the farmers. Another 46.3% of the farm accessions were found to be direct descendants (20.8% first-generation (F1) hybrids and 25.5% selfed genotypes) from 24 parental clones used in biclonal seed gardens (BSGs) established in the 1970s in southern and western Cameroon. Furthermore, 28.3% of farm accessions appeared to descent from uncontrolled pollination events in cacao farms, which could be related to a common practice of cacao growers to use seeds collected in their own farm for new plantings. All farm accessions descending from BSG could be individually related through parentage analysis to the 24 progenitors of the BSG. Only 25% of progenies distributed from BSG corresponded to F1 hybrids combinations originally planned to be released. Significant biparental inbreeding estimates were observed for all 'traditional' farms and for most 'F1 hybrids' farms due to presence of a high proportion of selfed accessions. Biparental inbreeding occurs when plants receive pollen from genetically related neighbors. High levels of outcrossing observed in 'mixed' farms might be explained by the admixture of traditional varieties and BSG progenies. The implications of our finding for management of seed gardens and for further breeding using farm accessions in Cameroon are discussed.

SummaryAvocado (Persea americana Mill.) flowers, with their synchronously dichogamous behaviour, are considered to be pollinated by honeybees, despite the lack of any direct evidence. Results in southern Florida showed that avocado pollen was transferable by wind, and was dispersed over a brief period of time (15 – 60 min) each day. Ten ‘Hass’ avocado orchards in the Santa Clara River Valley, CA, USA, planted far from any known ‘Zutano’ polliniser trees, were selected to investigate the impact of honeybees on pollen transfer. ‘Zutano’ pollen (5 g per insert) was placed at the entry to beehives (approx. eight beehives per orchard) and refreshed four-to-five times during the flowering season. Successful pollinations were determined by parental analysis of harvested ‘Hass’ fruit from trees located at various distances from the beehives, and at three different stages of fruit development, using microsatellite DNA markers. The results showed no significant difference in the proportions of ‘Zutano’-pollinated f...

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.

A heterozygous F 1 mapping population of cacao (Theobroma cacao L.) was created and evaluated for resistance to frosty pod [caused by Moniliophthora roreri (Cif. and Par.) Evans et al.], and black pod [caused by Phytophthora palmivora (Butl.) Butl.] and for fi ve horticultural traits at CATIE in Turrialba, Costa Rica. The population consisted of 256 F 1 progeny from the cross ‘Pound 7’ × ‘UF 273’. Progeny were used to form a linkage map using 180 markers. The linkage map contained 10 linkage groups (LGs), numbered as the LG in the cacao reference map, and was used to locate putative quantitative trait loci (QTL) for resistance to the aforementioned diseases and fi ve horticultural traits. Resistance to frosty pod was measured by internal and external pod resistance. Five QTLs for frosty pod resistance were found on three LGs, 2, 7, and 8, with UF 273 appearing to be the source of resistance. These alleles are being used for scoring progeny in ongoing cooperative marker-assisted selection projects, and constitute the fi rst QTLs identifi ed for frosty pod resistance. Three QTLs for black pod resistance were found on LG 4, 8, and 10, with the most favorable alleles coming from Pound 7. One QTL was found on LG 4 for average trunk growth rate, and two QTLs for height of fi rst jorquette were identifi ed on LGs 4 and 6. One QTL each for average trunk diameter growth and pod color was found on LG 4.

Misidentification is a major constraint to the conservation and utilization of cacao genetic resources. One solution for rapid and accurate identification is to produce a Field Guide for each recognized cacao early generation original population. Each Field Guide in this series of compendia would be comprised of pod digital images with complementary morphological descriptors, molecular fingerprint data with kinship analyses and agronomic data useful to cacao cultivar development. This Field Guide Concept culminated in the production of the first compendium entitled, Field Guide to the ICS Clones of Trinidad. In the current study, this Field Guide was used to verify the identity of 69 trees representing a possible 15 ICS clone genotypes introduced nearly 50 years ago to Costa Rica. Phase one of this study involved identity verification of the trees in Costa Rica using the pod digital image and morphological descriptor data in the Field Guide to the ICS Clones of Trinidad. The error rate was 3.5% for misidentification in the field in Costa Rica. In the second and final phase, SSR fingerprint data were generated for each of the 69 trees and analyzed for verification to the original ICS genotype in the Field Guide. Misidentification was reduced to 0% with the addition of SSR fingerprint analyses. The 69 trees in this study clustered into two groups or sub-populations clearly differentiated by Discriminant analyses with six SSR primers. A 46% cost reduction in SSR fingerprinting of the ICS clones was realized by combining use of the pod image and morphological descriptor data in the Field Guide with genetic diversity estimates derived from these six primers. A Field Guide approach to the identification of reference genotypes for cacao germplasm is discussed.

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.

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.

Molecular markers (RAPD, AFLP and microsatellites) were used to generate a linkage map and to identify QTLs associated to witches' broom (Crinipellis perniciosa) resistance in cacao (Theobroma cacao), using 82 individuals of an F2 population derived from the clones ICS-1 (susceptible) and Scavina-6 (resistant). Fifteen evaluations of the number of brooms have been carried out in six years (1997–2002). In order to increase the precision and accuracy in the measures of resistance, each F2 plant was cloned in three replications in a randomized block design with single-tree plots and evaluated over 2 years. Three hundred and forty-two markers were obtained, being 33 microsatellites, 77 AFLPs and 232 RAPDs. The distribution of the number of brooms in the F2 population was skewed to resistance, suggesting the involvement of major genes controlling resistance and the repeatability estimated for resistance was 44%. A strong putative QTL was detected as being related to witches' broom resistance. Associated to this QTL, the microsatellite mTcCIR35 explained 35.5% of the phenotypic variation in resistance. This marker is being used for marker-assisted selection in Scavina-6 progenies, including those selected in private plantations, as an auxiliary tool to the phenotypic selection.

Current methods for measures of genetic diversity of populations and germplasm collections are often based on statistics calculated from molecular markers. The objective of this study was to investigate the precision and accuracy of the most common estimators of genetic variability and population structure, as calculated from simple sequence repeat (SSR) marker data from cacao (Theobroma cacao L.). Computer simulated genomes of replicate populations were generated from initial allele frequencies estimated using SSR data from cacao accessions in a collection. The simulated genomes consisted of ten linkage groups of 100 cM in length each. Heterozygosity, gene diversity and the F statistics were studied as a function of number of loci and trees sampled. The results showed that relatively small random samples of trees were needed to achieve consistency in the observed estimations. In contrast, very large random samples of loci per linkage group were required to enable reliable inferences on the whole genome. Precision of estimates was increased by more than 50% with an increase in sample size from one to five loci per linkage group or 50 per genome, and up to 70% with ten loci per linkage group, or equivalently, 100 loci per genome. The use of fewer, highly polymorphic loci to analyze genetic variability led to estimates with substantially smaller variance but with an upward bias. Nevertheless, the relative differences of estimates among populations were generally consistent for the different levels of polymorphism considered.

Knowledge of genetic differences among commonly cultivated cacao clones, as well as the type of gene action involved for disease resistance, yield, quality, and horticultural traits, are essential for cacao breeders to select parental clones efficiently and effectively. This information is also critical for quantitative geneticists in designing and improving quantitative trait loci (QTL) localization strategies using breeding populations, whether they involve analysis of multiple populations crossed to one common parent or association genetic analysis. The objectives of this research were to 1) verify the genetic identity of parental cacao clones used to produce hybrids for field evaluation at the Centro Agrónomico Tropical de Investigación y Enzeñanza (CATIE), Turrialba, Costa Rica, using molecular marker analysis, and 2) estimate general and specific combining ability (GCA and SCA) of the parental clones for resistance to frosty pod (Moniliophthora roreri Cif. and Par.) and black pod [Phytophthora palmivora (Butl.) Butl.] diseases, total number of pods, vigor (as measured by trunk diameter), and measures of maturity (months to first flowering and pod production). Misidentification of cacao clones was found at three levels. Molecular marker analysis revealed that six parental clones differed in identity to supposedly identical accessions from other germplasm collections. Trees of the parental clone UF 273 consisted of two clearly different genotypes, resulting in two types of progeny, requiring separate designation for correct statistical analysis. Out-crossed progeny, presumably from foreign pollen, and selfed progeny were also found. Two of the traits measured, percent healthy pods and percent pods with frosty pod, showed predominantly additive gene action, while the traits total number of pods and trunk diameter, demonstrated regulation by both additive and nonadditive gene action. Number of months to first flowering and first fruit both showed evidence of predominant regulation by nonadditive gene effects. Crosses of two parental clones, UF 712 and UF 273 Type I, were identified as potential candidates for QTL analysis as breeding populations, given their favorable GCA estimates for frosty pod resistance and total pod production, respectively.

Crinipellis perniciosa causes a serious disease of cacao known as witches’ broom (WB). Heritable resistance to witches’ broom has been used in cacao improvement programs. ‘SCA6’ and ‘SCA12’ are highly resistant and are the most commonly used parents in the breeding schemes. However, SCA hybrids are not resistant to witches’ broom in all production areas. Presumably, different populations of C. perniciosa cause these variable responses. Amplified fragment length polymorphism (AFLP) markers were used to assess variation and population structure in this pathogen. We examined 40 isolates of C. perniciosa and one isolate of Melanotus subcuneiformis. Nine of 64 primer pairs produced consistent and informative DNA amplification, and were used to screen all isolates. Fifteen haplotypes (AFLP fingerprints) were detected with 186 polymorphic markers. Cluster analysis grouped isolates of the C biotype (pathogenic on cacao) from Bolivia, Brazil, Ecuador and Trinidad together in a major cluster that was distinct from isolates of the S biotype (pathogenic on solanaceous hosts) and M. subcuneiformis. Isolates of the C biotype were divided further into well supported, country-specific groups. Segregation of AFLP alleles was not observed among basidiospore isolates from the same basidiome, broom, tree or field, supporting previous reports that the fungus did not outcross. The results corroborated prior conclusions that C. perniciosa was probably introduced into the Bahia state of Brazil from the Amazon basin. Representative isolates from the genetically distinct groups that were revealed will be used to examine pathogenic specialization in C. perniciosa and differential responses that have been reported in ‘SCA6’-derived germplasm.

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.

cDNA microarray spot variability arises from many sources, and different systems have varying requirements for achieving the desired level of precision. We determined relative contributions to variance and investigated sequence discrimination using a multiple-array experimental design, with arrays subdivided to determine position and pin effect. Related fragments of 67 resistance gene homologs (RGHs) isolated from Theobroma cacao L. and grouped by sequence similarity were spotted onto arrays, using two of the same RGHs in the fluorescent dye channels (Cy™3, Cy5) of the hybridization solution in a “dye-flip” design. A comprehensive statistical model accounted for variability well, giving a coefficient of variation (CV) based on experimental error of 2.12%. Although we were able to separate 85% of RGH group means clearly, some groups more similar to the target were indistinguishable due to nonspecific hybridization. Genetic factors together contributed 72.2% of the total variation, while position and pin effects and their interactions contributed 9.8%. Replication effect was statistically significant. Otherwise, no tests for position effects were significant. The results of the analysis indicate that our Genetic Microsystems 417™ arrayer and Affymetrix 428™ scanner are performing with sufficient precision, and we produced useful information for planning efficient future experiments.

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIII site. After cloning of the ≈5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruitflies.

The aroma-active compounds present in tree-ripened fruits of the five mango (Mangifera indica L.) cultivars Haden, White Alfonso, Praya Sowoy, Royal Special, and Malindi were isolated by solvent extraction followed by solvent-assisted flavor evaporation (SAFE) and analyzed by gas chromatography-olfactometery (GC-O). Application of a comparative aroma extract dilution analysis (cAEDA) afforded 54 aroma-active compounds in the flavor dilution (FD) factor range from 4 to ≥2048, 16 of which are reported for the first time in mango. The results of the identification experiments in combination with the FD factors revealed 4-hydroxy-2,5-dimethyl-3(2H)-furanone as an important aroma compound in all cultivars analyzed. Twenty-seven aroma-active compounds were present in at least one mango cultivar at an FD factor ≥128. Clear differences in the FD factors of these odorants between each of the mango cultivars suggested that they contributed to the unique sensory profiles of the individual cultivars.

Twenty-six jackfruit accessions, one interspecific hybrid, champedak, and one breadfruit accession were analyzed using amplified fragment length polymorphic (AFLP) markers to determine the degree of genetic diversity within the Fairchild Tropical Garden (FTG) germplasm collection. Of the 30 primer pairs evaluated, 12 were identified for collection screening based on number and quality of polymorphic fragments. A total of 187 AFLP markers were scored using the 12 primer pairs, 92 (49.2%) being polymorphic. All accessions could be uniquely identified using the 12 primer pairs. Among the jackfruit accessions, similarity coefficients ranged from 0.567 to 0.950; the accessions also shared a large number of monomorphic fragments (54.9%). Cluster analysis and principal component analysis (PCA) grouped all of the jackfruit accessions with south-east Asian origins into one major cluster with little bootstrap support for groupings within the cluster. The Indian accessions were grouped in a different cluster, as did the hybrid and the breadfruit accession. The AFLP marker based analysis indicates that limited genetic diversity exists within this collection. These observations are in agreement with the phenotypic evaluation and suggest that new accessions be obtained from the center of origin for the species.

In previous studies we developed an avocado sunblotch viroid (ASBVd) specific reverse transcription-polymerase chain reaction (RT-PCR) protocol and tested all avocado germplasm accessions in the U.S. National Germplasm Repository (NGR) for ASBVd. It was found that 19% of the accessions were infected with the viroid. To characterize the population of sequence variants, single trees of seven accessions with a history of infection were selected. RT-PCR products were cloned and sequenced from these trees and a total of 60 sequence variants were identified from 122 clones. Most of the variants were found to be unique to the individual tree from which they were isolated. The fraction of unique variants was 81% in ‘Lima Late’, 81% in ‘Aycock Red’, 67% in ‘Hawaii’, and 60% in ‘Young Special’. Analysis of the sequence data suggests that variants evolved within a tree rather than arising from multiple infection events, supporting the quasispecies concept for ASBVd. Changes in the right terminal loop (Rtl), previously associated with leaf variegation and bleaching, were found in all variants isolated from bleached tissue; however, 16 clones isolated in non-symptomatic tissue also had changes in the Rtl. The addition of a U between bases 115 and 118 was found in 21 of the 23 clones isolated from the bleached tissue but not found in the 99 clones isolated from green tissue. These data suggest that the insertion of a U between bases 115 and 118 is responsible for the bleaching condition.