Two large mapping populations of avocado consisting of 1582 trees were genotyped with 5050 SNP ma... more Two large mapping populations of avocado consisting of 1582 trees were genotyped with 5050 SNP markers from transcribed genes using an Illumina Infinium SNP chip. A Florida mapping population consisted of 527 progeny from 'Tonnage' x 'Simmonds' and 249 from 'Simmonds' x 'Tonnage'. A California mapping population consisted of 576 progeny from 'Hass' x 'Bacon' and 230 progeny from 'Bacon' x 'Hass'. Microsatellite marker data from the Florida mapping populations that had been used to produce a moderately resolved genetic recombination map (Borrone et al. 2009) were included with the SNP data to anchor the new maps to the old. Individual maps were created for each population and joined using JoinMap4.1. The same twelve linkage groups identified in the previous map (Borrone et al. 2009) were found. The genetic map for avocado has been significantly expanded. The expanded, saturated map will be useful for the assembly of the ...
A saturated genetic linkage map can aid greatly in efforts to improve avocado (Persea americana M... more A saturated genetic linkage map can aid greatly in efforts to improve avocado (Persea americana Mill.) via breeding using a Marker Assisted Selection (MAS) approach. A genetic linkage map for avocado can be generated using molecular genetic markers and a known family structure. Currently, approximately 300 microsatellite markers have been developed for avocado. This number of microsatellite markers should give suitable coverage for the avocado genome.
Avocado (Persea americana Mill.) farmers in South Florida have traditionally grown West Indian an... more Avocado (Persea americana Mill.) farmers in South Florida have traditionally grown West Indian and Guatemalan x West Indian hybrid cultivars because they are more suitable to prevalent growing conditions. Currently, there is a growing interest in expanding the avocado production to other areas of the state as an alternative crop to citrus. However, the West Indian and Guatemalan x West Indian hybrids are sensitive to cold damage. The objectives of this research were to a) evaluate cold tolerance in a F1 avocado mapping population of the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage' b) identify DNA markers associated with cold tolerance and flower type, and place them in their respective linkage groups. A total of 800 trees were used to collect phenotypic data for traits of horticultural interest during three years. A single QTL has been identified for cold tolerance using composite interval mapping. The cold tolerance QTL is located on LG1 and ac...
Supplementary material from: Borrone JW, Brown JS, Tondo CT, Mauro-Herrera M, Kuhn DN, Violi HA, ... more Supplementary material from: Borrone JW, Brown JS, Tondo CT, Mauro-Herrera M, Kuhn DN, Violi HA, Sautter RT, Schnell RJ (2009). An EST-SSR based linkage map of Persea americana Mill. (avocado). Tree Genetics & Genomes. 5: 553-560. doi: 10.1007/s11295-009-0208-y
The genetic improvement of tropical fruit trees is limited when compared to progress achieved in ... more 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 signi fi cant 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 dif fi cult 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.
Journal of Clinical Endocrinology & Metabolism, 1993
Recent studies indicate that mutations in the androgen receptor gene are associated with androgen... more Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling.
The WRKY gene family of transcription factors is involved in several diverse pathways and include... more 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. Published by Elsevier Inc.
Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 1999
Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyke... more 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.
ADDITIONAL INDEX WORDS. QTL mapping, witchesʼ broom disease, resistance mapping, genomic maps, T.... more ADDITIONAL INDEX WORDS. QTL mapping, witchesʼ broom disease, resistance mapping, genomic maps, T. cacao ABSTRACT. A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F 2 population produced by selfi ng an F 1 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 signifi cant 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 witchesʼ 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 fl anked the minor QTL for witchesʼ broom resistance, implying possible association. QTLs mapped in F 2 populations produce estimates of additive and dominance effects, not obtainable in F 1 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 F 2 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 Theo... more 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.
We investigated the reliability of capillary array electrophoresis-single strand conformation pol... more 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°C, 25°C, 30°C, and 35°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 ≤250 bp, samples should be analyzed at two different temperatures between 18°C and 30°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.
Avocado (Persea americana Mill.) possesses a unique flowering mechanism, thought to promote outcr... more Avocado (Persea americana Mill.) possesses a unique flowering mechanism, thought to promote outcrossing, 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.
In this paper, we report the construction of the first composite map of cacao from linkage data o... more 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.
Coconut (Cocos nucifera L.) WRKY sequences containing single nucleotide polymorphisms (SNPs) and ... more Coconut (Cocos nucifera L.) WRKY sequences containing single nucleotide polymorphisms (SNPs) and one microsatellite repeat were used to develop 10 informative markers. These markers were evaluated in 15 genotypes representing six coconut cultivars. SNP-containing alleles were detected by single-strand conformation polymorphism (SSCP) analysis. The number of detected alleles ranged from two to four. Five pairs of loci were in linkage disequilibrium in the test population. These markers are currently being evaluated in more individuals/cultivars to determine their value in estimating the genetic diversity of this species.
For well-studied plant species with whole genome sequence or extensive EST data, SNP markers are ... more For well-studied plant species with whole genome sequence or extensive EST data, SNP markers are the logical choice for both genotyping and whole genome association studies. However, SNP markers may not address the needs of researchers working on specialty crops with limited available genomic information. Microsatellite markers have been frequently employed due to their robustness, but marker development can be difficult and may result in few polymorphic markers. SSCP markers, such as microsatellites, are PCR-based and scored by electrophoretic mobility but, because they are based on SNPs rather than length differences, occur more frequently and are easier to develop than microsatellites. We have examined how well correlated the estimation of genetic diversity and genetic distance are in a population or germplasm collection when measured by 13 highly polymorphic microsatellite markers or 20 SSCP markers. We observed a significant correlation in pairwise genetic distances of 82 individuals in an international cacao germplasm collection (Mantel test Rxy=0.59, p<0.0001 for 10 000 permutations). Both sets of markers could distinguish each individual in the population. These data provide strong support for the use of SSCP markers in the genotyping of plant species where development of microsatellites would be difficult or expensive.
Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for ... more 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.
Two large mapping populations of avocado consisting of 1582 trees were genotyped with 5050 SNP ma... more Two large mapping populations of avocado consisting of 1582 trees were genotyped with 5050 SNP markers from transcribed genes using an Illumina Infinium SNP chip. A Florida mapping population consisted of 527 progeny from 'Tonnage' x 'Simmonds' and 249 from 'Simmonds' x 'Tonnage'. A California mapping population consisted of 576 progeny from 'Hass' x 'Bacon' and 230 progeny from 'Bacon' x 'Hass'. Microsatellite marker data from the Florida mapping populations that had been used to produce a moderately resolved genetic recombination map (Borrone et al. 2009) were included with the SNP data to anchor the new maps to the old. Individual maps were created for each population and joined using JoinMap4.1. The same twelve linkage groups identified in the previous map (Borrone et al. 2009) were found. The genetic map for avocado has been significantly expanded. The expanded, saturated map will be useful for the assembly of the ...
A saturated genetic linkage map can aid greatly in efforts to improve avocado (Persea americana M... more A saturated genetic linkage map can aid greatly in efforts to improve avocado (Persea americana Mill.) via breeding using a Marker Assisted Selection (MAS) approach. A genetic linkage map for avocado can be generated using molecular genetic markers and a known family structure. Currently, approximately 300 microsatellite markers have been developed for avocado. This number of microsatellite markers should give suitable coverage for the avocado genome.
Avocado (Persea americana Mill.) farmers in South Florida have traditionally grown West Indian an... more Avocado (Persea americana Mill.) farmers in South Florida have traditionally grown West Indian and Guatemalan x West Indian hybrid cultivars because they are more suitable to prevalent growing conditions. Currently, there is a growing interest in expanding the avocado production to other areas of the state as an alternative crop to citrus. However, the West Indian and Guatemalan x West Indian hybrids are sensitive to cold damage. The objectives of this research were to a) evaluate cold tolerance in a F1 avocado mapping population of the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage' b) identify DNA markers associated with cold tolerance and flower type, and place them in their respective linkage groups. A total of 800 trees were used to collect phenotypic data for traits of horticultural interest during three years. A single QTL has been identified for cold tolerance using composite interval mapping. The cold tolerance QTL is located on LG1 and ac...
Supplementary material from: Borrone JW, Brown JS, Tondo CT, Mauro-Herrera M, Kuhn DN, Violi HA, ... more Supplementary material from: Borrone JW, Brown JS, Tondo CT, Mauro-Herrera M, Kuhn DN, Violi HA, Sautter RT, Schnell RJ (2009). An EST-SSR based linkage map of Persea americana Mill. (avocado). Tree Genetics & Genomes. 5: 553-560. doi: 10.1007/s11295-009-0208-y
The genetic improvement of tropical fruit trees is limited when compared to progress achieved in ... more 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 signi fi cant 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 dif fi cult 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.
Journal of Clinical Endocrinology & Metabolism, 1993
Recent studies indicate that mutations in the androgen receptor gene are associated with androgen... more Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling.
The WRKY gene family of transcription factors is involved in several diverse pathways and include... more 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. Published by Elsevier Inc.
Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 1999
Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyke... more 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.
ADDITIONAL INDEX WORDS. QTL mapping, witchesʼ broom disease, resistance mapping, genomic maps, T.... more ADDITIONAL INDEX WORDS. QTL mapping, witchesʼ broom disease, resistance mapping, genomic maps, T. cacao ABSTRACT. A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F 2 population produced by selfi ng an F 1 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 signifi cant 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 witchesʼ 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 fl anked the minor QTL for witchesʼ broom resistance, implying possible association. QTLs mapped in F 2 populations produce estimates of additive and dominance effects, not obtainable in F 1 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 F 2 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 Theo... more 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.
We investigated the reliability of capillary array electrophoresis-single strand conformation pol... more 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°C, 25°C, 30°C, and 35°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 ≤250 bp, samples should be analyzed at two different temperatures between 18°C and 30°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.
Avocado (Persea americana Mill.) possesses a unique flowering mechanism, thought to promote outcr... more Avocado (Persea americana Mill.) possesses a unique flowering mechanism, thought to promote outcrossing, 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.
In this paper, we report the construction of the first composite map of cacao from linkage data o... more 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.
Coconut (Cocos nucifera L.) WRKY sequences containing single nucleotide polymorphisms (SNPs) and ... more Coconut (Cocos nucifera L.) WRKY sequences containing single nucleotide polymorphisms (SNPs) and one microsatellite repeat were used to develop 10 informative markers. These markers were evaluated in 15 genotypes representing six coconut cultivars. SNP-containing alleles were detected by single-strand conformation polymorphism (SSCP) analysis. The number of detected alleles ranged from two to four. Five pairs of loci were in linkage disequilibrium in the test population. These markers are currently being evaluated in more individuals/cultivars to determine their value in estimating the genetic diversity of this species.
For well-studied plant species with whole genome sequence or extensive EST data, SNP markers are ... more For well-studied plant species with whole genome sequence or extensive EST data, SNP markers are the logical choice for both genotyping and whole genome association studies. However, SNP markers may not address the needs of researchers working on specialty crops with limited available genomic information. Microsatellite markers have been frequently employed due to their robustness, but marker development can be difficult and may result in few polymorphic markers. SSCP markers, such as microsatellites, are PCR-based and scored by electrophoretic mobility but, because they are based on SNPs rather than length differences, occur more frequently and are easier to develop than microsatellites. We have examined how well correlated the estimation of genetic diversity and genetic distance are in a population or germplasm collection when measured by 13 highly polymorphic microsatellite markers or 20 SSCP markers. We observed a significant correlation in pairwise genetic distances of 82 individuals in an international cacao germplasm collection (Mantel test Rxy=0.59, p<0.0001 for 10 000 permutations). Both sets of markers could distinguish each individual in the population. These data provide strong support for the use of SSCP markers in the genotyping of plant species where development of microsatellites would be difficult or expensive.
Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for ... more 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.
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Papers by James Borrone