Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability in Citrus.

Centro de Protección Vegetal y Biotecnología, IVIA, Apartado Oficial 46113 Moncada (Valencia), Spain.
American Journal of Botany (Impact Factor: 2.46). 11/2010; 97(11):e124-9. DOI: 10.3732/ajb.1000280
Source: PubMed

ABSTRACT • Premise of the study: Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. • Methods and Results: Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata, C. maxima, C. medica, C. sinensis, C. aurantium, C. paradisi, C. lemon, C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra- and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. • Conclusions: These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps.

Download full-text


Available from: Javier Terol, Aug 14, 2015
1 Follower
  • Source
    • "The discrimination of somatic clones based on morphological traits can be very difficult for the citrus sharing high level of genetic similarity (Luro et al. 1995; Fang and Roose 1997). A number of studies have been performed using various molecular markers, such as RFLP, RAPD, SCAR, AFLP, SSR, ISSR, and SNPs, or cpDNA sequencing, in order to evaluate the level of genetic variability in Citrus (Luro et al. 1995; Herrero et al. 1996; Fang and Roose 1997; Federici et al. 1998; Nicolosi et al. 2000; Moore 2001; Barkley et al. 2006; Novelli et al. 2006; Luro et al. 2008; Ollitrault et al. 2010; Garcia–Lor et al. 2012). Among those, SNP markers gained popularity due to their widespread nature and potential direct link to genes responsible for the traits of agronomical importance. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in Citrus. SNP markers have been used for genetic diversity assessment using a variety of different methods. Recently, the availability of EST database and whole genome sequences has made it possible to develop more markers such as SNPs. In the present study, the high-resolution melting curve analysis (HRM) was used to detect SNPs or INDELs in Citrus genus for the first time. We aimed to develop a panel of SNPs to differentiate Citrus genotypes which can also be applied to Citrus biodiversity studies. The results showed that 21 SNP containing markers produced distinct polymorphic melting curves among the Citrus spp. investigated through HRM analysis. It was proved that HRM is an efficient, cost-effective, and accurate method for discriminating citrus SNPs as well as a method to analyze more polymorphisms in a single PCR amplicon, representing a useful tool for genetic, biodiversity, and breeding studies. SNPs developed based on Citrus sinensis EST database showed a good transferability within the Citrus genus. Moreover, HRM analysis allowed the discrimination of citrus genotypes at specific level and the resulting genetic distance analysis clustered these genotypes into three main branches. The results suggested that the panel of SNP markers could be used in a variety of applications in citrus biodiversity assessment and breeding programs using HRM analysis.
    Tree Genetics & Genomes 10/2013; 9(5). DOI:10.1007/s11295-013-0636-6 · 2.44 Impact Factor
  • Source
    • "In genetic studies of Citrus, SSR analysis (Gulsen and Roose, 2001; Luro et al., 2001, 2008; Barkley et al., 2006; Ollitrault et al., 2010) is seen as a powerful tool because SSRs are co-dominant, randomly dispersed throughout the plant genome, generally highly polymorphic and locusspecific . However, Barkley et al. (2009) showed that homoplasy might limit the usefulness of SSRs as tags to elucidate the phylogenetic origin of specific DNA fragments in citrus. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background and AimsDespite differences in morphology, the genera representing 'true citrus fruit trees' are sexually compatible, and their phylogenetic relationships remain unclear. Most of the important commercial 'species' of Citrus are believed to be of interspecific origin. By studying polymorphisms of 27 nuclear genes, the average molecular differentiation between species was estimated and some phylogenetic relationships between 'true citrus fruit trees' were clarified.Methods Sanger sequencing of PCR-amplified fragments from 18 genes involved in metabolite biosynthesis pathways and nine putative genes for salt tolerance was performed for 45 genotypes of Citrus and relatives of Citrus to mine single nucleotide polymorphisms (SNPs) and indel polymorphisms. Fifty nuclear simple sequence repeats (SSRs) were also analysed.Key ResultsA total of 16 238 kb of DNA was sequenced for each genotype, and 1097 single nucleotide polymorphisms (SNPs) and 50 indels were identified. These polymorphisms were more valuable than SSRs for inter-taxon differentiation. Nuclear phylogenetic analysis revealed that Citrus reticulata and Fortunella form a cluster that is differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). These results confirm the taxonomic subdivision between the subgenera Metacitrus and Archicitrus. A few genes displayed positive selection patterns within or between species, but most of them displayed neutral patterns. The phylogenetic inheritance patterns of the analysed genes were inferred for commercial Citrus spp.Conclusions Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.
    Annals of Botany 10/2012; 111(1). DOI:10.1093/aob/mcs227 · 3.30 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Citrus fruits (sweet orange, mandarin, pummelo, grapefruit, lemon, lime and assorted hybrids) are among the most widely grown and economically important fruit tree crops in the world. As fresh fruit, they are an important and nutrient dense food source for human diets; as processed juice products, primarily sweet orange juice, they represent a globally traded commodity. To support genetic improvement efforts for this important crop, the international citrus genetics community has collaborated with international sequencing centers in the development of freely available genomic resources, some of which are described herein. Most notable, two full-length annotated genome assemblies have been produced and made available to the global research community. The first genome, based exclusively on Sanger sequencing, is from a haploid plant derived from ‘Clementine’ mandarin, to serve as the reference genome for citrus. A second genome assembly was produced from the sweet orange clone ‘Ridge Pineapple’, this was done primarily with 454 technology. Extensive EST datasets and a number of microarray platforms for exploring the transcriptomic responses of citrus species and hybrids to a wide range of conditions have been shared, to support exploitation and utilization of genome sequence information. As many researchers in the citrus genomics community are also actively engaged in genetic improvement programs, there has been a natural integration of improvement efforts with the rapidly evolving genomic tools. Examples described below include work designed to better understand the control of gene expression in citrus polyploids, which are being used in development of seedless triploid selections or as rootstocks; unraveling the molecular mechanisms of host–pathogen interactions to devise novel genetic strategies to overcome a multitude of devastating diseases that currently threaten citrus production globally; and the mining of SSR and SNP markers for linkage studies to enable marker-assisted parental selection and breeding strategies, illustrated by work on two citrus traits, nucellar embryony and juvenility, which significantly impact breeding approaches. Citrus genome resources are available through publicly available web portals, through the USDOE-JGI ( and Tree Fruit Genome Database Resources (tfGDR, Work is continuing to expand and improve the citrus genome sequence resources and tools, to enable application of sequence-derived knowledge in improving citrus plants and to managing better their interactions with biotic and abiotic factors.
    Tree Genetics & Genomes 06/2012; 8(3). DOI:10.1007/s11295-012-0499-2 · 2.44 Impact Factor
Show more