A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/ time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with a reversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values were lower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin in fruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg -1 .
A single nucleotide polymorphism (SNP) associated with pungency was detected within an expressed sequence tag (EST) of 307 bp. This fragment was identified after expression analysis of the EST clone SB2-66 in placenta tissue of Capsicum fruits. Sequence alignments corresponding to this new fragment allowed us to identify an SNP between pungent and non-pungent accessions. Two methods were chosen for the development of the SNP marker linked to pungency: tetra-primer amplification refractory mutation system-PCR (tetra-primer ARMS-PCR) and cleaved amplified polymorphic sequence. Results showed that both methods were successful in distinguishing genotypes. Nevertheless, tetra-primer ARMS-PCR was chosen for SNP genotyping because it was more rapid, reliable and less cost-effective. The utility of this SNP marker for pungency was demonstrated by the ability to distinguish between 29 pungent and non-pungent cultivars of Capsicum annuum. In addition, the SNP was also associated with phenotypic pungent character in the tested genotypes of C. chinense, C. baccatum, C. frutescens, C. galapagoense, C. eximium, C. tovarii and C. cardenasi. This SNP marker is a faster, cheaper and more reproducible method for identifying pungent peppers than other techniques such as panel tasting, and allows rapid screening of the trait in early growth stages.
Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (F.o.m), is a worldwide soilborne disease of melon (Cucumis melo L.). The most eVective control measure available is the use of resistant varieties. Resistance to races 0 and 2 of this fungal pathogen is conditioned by the dominant gene Fom-1. An F 2 population derived from the 'Charentais-Fom1' £ 'TRG-1551' cross was used in combination with bulked segregant analysis utilizing the random ampliWed polymorphic DNA (RAPD) markers, in order to develop molecular markers linked to the locus Fom-1. Four hundred decamer primers were screened to identify three RAPD markers (B17 649 , V01 578 , and V06 1092 ) linked to Fom-1 locus. Fragments ampliWed by primers B17 649 and V01 578 were linked in coupling phase to Fom1, at 3.5 and 4 cM respectively, whereas V06 1092 marker was linked in repulsion to the same dominant resistant allele at 15.1 cM from the Fom-1 locus. These RAPDs were cloned and sequenced in order to design primers that would amplify only the target fragment. The derived sequence characterized ampliWed region (SCAR) markers SB17 645 and SV01 574 (645 and 574 bp, respectively) were present only in the resistant parent. The SV06 1092 marker ampliWed a band of 1092 bp only in the susceptible parent. These markers are more universal than the CAPS markers developed by Brotman et al. (Theor Appl Genet 10:337-345, 2005). The analysis of 24 melon accessions, representing several melon types, with these markers revealed that diVerent melon types behaved diVerently with the developed markers supporting the theory of multiple, independent origins of resistance to races 0 and 2 of F.o.m.
The quantitative inheritance of capsaicin and dihydrocapsaicin contents in fruits has been studied in
an intraspecific cross of Capsicum annuum L. across two different environments, namely, fruits
developed in spring and summer. A liquid chromatography-electrospray ionization/time-of-flight mass
spectrometry [HPLC-ESI/MS(TOF)] method was used to identify and quantify capsaicin and
dihydrocapsaicin in extracts of pepper fruits. The analytical method used was able to determine the
pungency of genotypes that, using other methods, would have been classified as non-pungent.
Capsaicin and dihydrocapsaicin contents varied largely among families, and families did not respond
similarly in producing these capsaicinoids when their fruits were grown in spring and summer, with
some families showing no increase, whereas in others, the increase was more than 2-fold. Heterosis
for the pungency trait, assessed by the capsaicin and dihydrocapsaicin contents in fruits, was found,
indicating the existence of epistasis, over-dominance, or dominance complementation. Non-pungent
parent alleles contributed to the capsaicin and dihydrocapsaicin contents since transgressive
segregation did occur. Furthermore, the type of gene action varied between capsaicin and
dihydrocapsaicin, and a seasonal effect during fruit development could affect gene action.
Keywords: Capsaicin; dihydrocapsaicin; family−environment interaction; HPLC; intraspecific cross;
mass spectrometry; quantitative inheritance
Potato Virus Y (PVY) is the only potyvirus infecting pepper ( Capsicum annuum L.) in Europe. Currently, the development of pepper varieties resistant to PVY seems to be the most-efficient method to control PVY damage. Among the sources of resistance, a monogenic dominant gene Pvr4 confers resistance against all known PVY pathotypes. In this work, bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pvr4 gene, using segregating progenies obtained by crossing a homozygous resistant ('Serrano Criollo de Morelos-334') with a homozygous susceptible ('Yolo Wonder') cultivar. Eight hundred decamer primers were screened to identify one RAPD marker (UBC19(1432)) linked in repulsion phase to Pvr4. This marker was converted into a dominant sequence characterised amplified region (SCAR) marker (SCUBC19(1423)). This marker was mapped into a dense Capsicum genetic map in a region where several genes for resistance to different diseases are located. This marker can be useful to identify PVY-resistant genotypes in segregating progenies of pepper in marker-assisted selection (MAS) breeding programs.
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