Evelopment 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: 2.6). 11/2010; 97(11):e124-9. DOI: 10.3732/ajb.1000280
Source: PubMed


• 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.

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    • "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. "
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    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.
    Full-text · Article · Oct 2013 · Tree Genetics & Genomes
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    • "CIBE****: These 34 markers were developed by Ollitrault and colleagues at CIRAD/IVIA (France/Spain) from a Clementine BAC end sequence database [27]. These markers are published in Ollitrault et al. [28]. Corresponding GenBank accession numbers can be found in Additional file 1. "
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    ABSTRACT: Background Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. Results Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Conclusions A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.
    Full-text · Article · Nov 2012 · BMC Genomics
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    • "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. "
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    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.
    Full-text · Article · Oct 2012 · Annals of Botany
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