Cecile T. Olano

AFLP analysis of genetic diversity within a jackfruit germplasm collection

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.

A Composite Linkage Map from Three Crosses Between Commercial Clones of Cacao, Theobroma cacao L.

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.

Analysis of molecular diversity in Crinipellis perniciosa with AFLP markers

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.

Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis

A specific reverse transcription‐polymerase chain reaction (RT‐PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single‐strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE‐SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Sequence Diversity among Avocado Sunblotch Viroids Isolated from Single Avocado Trees

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.

Mitochondrial DNA Restriction Map for the Caribbean Fruit Fly, Anastrepha suspensa, and Occurrence of Mitochondrial DNA Diversity Within Highly Inbred Colonies

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.