A PCR based SNPs marker for specific characterization of English walnut (Juglans regia L.) cultivars

Abstract

English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55-56.7 and 57.1-58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion-deletion (indel) at 5' end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.

Walnut: past and future of genetic improvement

“…The initial use of SNP markers to characterize J. regia cultivars (Ciarmiello et al 2011) employed the first and second internal transcribed spacer regions (ITS1 and ITS2) and 5.8S coding region of rDNA-repeat (ribosomal DNA) operon segment. Alignment of sequences revealed 244 SNPs and one short insertion-deletion (InDel).…”

Differences in Manioc Diversity Among Five Ethnic Groups of the Colombian Amazon

“…Information about these issues might be important for those interested in maintain and increase public manioc collections, and for those interested in the conservation of manioc diversity, to help develop strategies to conserve these materials in situ. There are already some experiences showing that it is possible to protect both crop diversity and the collective knowledge indigenous people have of crops by way of geographical indications or denominations of origin [52] in which the use of molecular tools such as SNPs to discriminate landraces from specific localities or ethnic groups is accurate [53][54][55]. Since it is difficult to maintain a real representation of diversity in worldwide crop collections, the possibility to acknowledge and incorporate local farmers in the conservation of diversity could be a real alternative for crop conservation and the preservation of independent cultures.…”

SSR and RAPD analysis of genetic diversity in walnut (Juglans regia L.) genotypes from Jammu and Kashmir, India

“…Previously, in addition to morphological identification (Zenelli et al 2005), various biochemical and molecular markers have been used for genetic characterization of walnut genotypes. These included isozymes (Fornari et al 2001;Busov et al 2002;Ninot and Aleta 2003;Vyas et al 2003,), restriction fragment length polymorphisms (RFLPs) (Fjellstrom et al1995), randomly amplified polymorphic DNAs (RAPDs) (Nicese et al1998;Yan-Min et al 2000;Zhang Li et al 2007), intersimple sequence repeats (ISSRs) (Potter et al 2002), simple sequence repeats (SSRs) Dangl et al 2005;Foroni et al 2005;Victory et al 2006;Robichaud et al 2006;Karimi et al 2010), amplified fragment length polymorphisms (Kafkas et al 2005;Bayazit et al 2007) and SNPs (Ciarmiello et al 2011). In our study, the average number of alleles per locus was 2 which is higher than 1.3 detected in J. regia with RAPDs (Nicese et al 1998) but lesser than 3.9 and 5.5 using ISSR (Potter et al 2002) and SSR (Foroni et al 2007) markers respectively.…”