Download Marker Saturation and Construction of a High

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Marker saturation map of the potato cyst nematode resistance locus Grp1 on
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chromosome V of potato
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Beyene Amelework1*, Hussien Shimelis1
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1African
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Scottsville 3209, Pietermaritzburg, South Africa;
Centre for Crop Improvement, University of KwaZulu-Natal, Private Bag X01,
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*Corresponding author, Tel.:+27 837790105. E-mail address: [email protected] (B.
A. Amelework).
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Abstract
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Construction of a high-resolution map of the locus of interest is an important step in
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map-based cloning. The potato cyst nematode resistance gene (Grp1) confers
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resistance to the potato cyst nematode (PCN) species Globodera rostochiensis and G.
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pallida. This resistance is often quantitative and causes a reduced multiplication of the
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virulent nematode populations. Grp1 is previously located on the distal end of
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chromosome V, and flanked by the two RFLP markers GP21 and GP179. This interval
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is broad and a hotspot region for many resistance genes and the use of common
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markers to map different resistance genes superimpose the effect of the genes on each
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other. The objective of this study was to identify candidate markers in the GP21-GP179
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interval which are linked to the Grp1 gene. Cleaved Amplified Polymorphic Sequences
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(CAPS) analysis was performed and 25 primers designed based on the sequence
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information of Solanum demissum chromosome 5 BACs. A total of 1600 diploid
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mapping population derived from the cross of AM78-3778-16 x RH89-039-16 was used
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and progenies screened for recombination events. The designed markers were tested
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on 46 selected recombinant progenies. The analysis identified five CAPS markers within
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a distance of 3.4cM in the GP21-GP179 interval. The novel markers are useful for
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further high resolution mapping of the Grp1 locus.
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Key words: Cleaved Amplified Polymorphic Sequences, Grp1 locus, molecular
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markers, potato, potato cyst nematode, saturation map
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Introduction
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Potato belongs to the family Solanaceae, which consists of many important crop
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species. The cultivated potato (Solanum tuberosum L.) is tetraploid with four genome
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complements and 48 chromosomes (2n=4x=48). It exhibits a complex traits inheritance
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caused by the random pairing of the four chromosomes during meiosis. This tetrasomic
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inheritance may result in multi-allelism where combinations of four different alleles
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interact within a single locus (Luo et al., 2006; Ballvora et al., 2011). The international
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potato centre (CIP) maintains large genetic diversity of more than 7,000 wild and
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cultivated potatoes (www.cipotato.org). Over 200 wild and eight cultivated tuber bearing
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Solanum species are collected and characterized (www.cipotato.org).
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Plant nematodes are the fourth most important parasites and attack a wide range
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of crop plants worldwide. Out of the parasitic nematodes, the potato cyst nematodes
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(PCN) are the most widely known and economically important species (Lopez and
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Evans, 2004). In addition to PCN, other large numbers of parasitic nematode species
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have been identified (Decraemel and Hunt, 2006). The potato cyst nematode includes
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two species: Globodera pallida ("white" PCN) and G. rostochiensis ("yellow or golden"
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PCN), that attack potato universally.
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Molecular markers are powerful tools useful in various fields such as taxonomy,
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physiology, embryology, plant breeding and genetic engineering. The discovery of
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polymerase chain reaction (PCR) presented a unique and efficient process that brought
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a new class of DNA profiling markers. This facilitated the progress of marker-based
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gene tags, map-based cloning of agronomically important genes, genomic analysis
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(variability and phylogenetic studies and synteny mapping) (Chetelat et al., 2000) and
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marker-assisted selection of desirable genotypes (Tiwari et al., 2013).
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The genetic maps of potato have been constructed using RFLP (Ritter et al.,
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1991; Hosaka and Spooner, 1992; Leonards-Schippers et al., 1994; Jacobs et al., 1995)
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and AFLP markers (Isidore et al., 2003). So far 39,031 protein-coding genes have been
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mapped on potato genome, of which 2,642 genes are functional genes that encode
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traits essential for potato growth and tuber development (Potato Genome Consortium,
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2011).
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Molecular markers are routinely used to identify and characterize novel
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resistance genes in crop plants (Michelmore, 1995; Phillips, 2006). Many single genes
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and quantitative trait loci (QTLs) that confer resistance to all the major classes of pests
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and pathogens have been mapped on the potato genome (van der Voort et al., 2000;
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van der Vossen et al., 2000; Paal et al., 2004). For instance, 14 potato cyst nematode
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resistance loci have been identified and mapped on potato genome (Grube et al., 2000;
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Caromel et al., 2003, 2005). Out of which ten loci confer partial resistance while the
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remaining four express absolute resistance for PCN. Several gene clusters have also
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been mapped on the potato genome (van der Voort et al., 2000; van der Vossen et al.,
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2000; Paal et al., 2004). The most conspicuous genetic clusters containing multiple
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genes for absolute and partial resistance to different pathogens are located on
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chromosome V, XI, and XII of potato genome (Gebhardt and Valkonen, 2001).
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The interval between the two RFLP markers GP21-GP179 is a novel region
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found on chromosome V of potato and known to harbor resistance genes to virus such
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as Nb and Rx2 (Ritter et al., 1991; De Jong et al., 1997), fungus (R1) (Leonards-
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Schippers et al., 1992) and Nematodes (Gpa, Gpa5 and Grp1) (Kreike et al., 1994; van
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der Voort et al., 2000; Caromel et al., 2003). In addition different CAPS markers such as
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SPUD839, SPUD237, and TG432 linked with these genes have been mapped within
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this interval (De Jong et al., 1997; Ballvora et al., 2002).
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The potato cyst nematode resistance gene (Grp1) identified from the tetraploid
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clone AM78-3778 reportedly confers absolute resistance to the potato cyst nematode
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species G. rostochiensis pathotype Ro5 and G. pallida pathotype Pa2. It also provides
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partial resistance to pathotype Pa3 (van der Voort et al., 1998). The resistance is often
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quantitative and causes a reduced multiplication of the virulent nematode populations.
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Grp1 is previously located on the distal end of chromosome V, and flanked by the two
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RFLP markers GP21 and GP179 (van der Voort et al., 1998).
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interval encompasses a wide region of the chromosome and considered as “a hotspot’
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region for many resistance genes. The use of very few and common markers to map
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different resistance genes may superimpose the effect of the genes on each other. The
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objective of this study was to identify candidate markers in the GP21-GP179 interval
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which are linked to the Grp1 gene. The results of the study may help for high-resolution
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mapping of the Grp1 gene to facilitate gene cloning and select desirable alleles for
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marker-assisted breeding.
The GP21-GP179
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Materials and methods
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Plant material and mapping population
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The diploid clone AM3778-16 used to map Grp1 is derived from AM78-3778, a
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tetraploid clone which combines the resistance introgressed from many wild Solanum
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species (Dellaert and Vinke, 1987). AM 78-3778 possesses resistance genes to both
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PCN species and is an interspecific hybrid between S. tuberosum and several wild
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species. Clones AM3778-16 and AM3778-14 are diploids and derived from crosses of
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AM78-3778 with a haploid inducer S. phureja (2n=24). In this study a mapping
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population of diploid potato derived from the crosses between AM78-3778-16 (AM) x
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RH89-039-16 (RH) (van der Voort et. al., 1998) was used. AM represents the resistance
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female parent and RH represents the fully susceptible male parent in the mapping
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population.
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A mapping population of 1600 AM x RH plants segregating for the Grp1 gene
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were identified and used for the study. Out of these 46 recombinant plants for GP21 and
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GP179 interval (spanning the Grp1 locus) have been identified. Consequently, the
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distance between these two markers is calculated as 3.4cM. The 46 recombinant plants
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in the GP21-GP179 interval were used for DNA extraction. DNA was extracted from
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frozen plant leaf tissue using the DNeasy Plant DNA Extraction kit from Qiagen
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(WWW.QIAGEN.COM).
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Primer design
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Based on the sequence information of Solanum demissum chromosome V BAC
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(http://www.ncbi.nlm.nih.gov/), 25 primers were designed. First each BACs sequences
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were cut into pieces of 5kb of DNA for ease of handling. The BAC piece was blasted to
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check if there is any R gene homologous in the region. Then one to three primers were
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designed from each BAC based on the size and the number of putative resistance gene
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present. A total of 25 primer pairs were designed. DNA sequence, the BACs origin,
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thermal condition used for each primer and size of the designed primers are shown in
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Table 1. The CAPS for potato RFLP loci GP21 and GP179 (van der Voort et. al., 1998)
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were used to identify recombinant plants and serve as a reference to identify additional
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marker that fits on this interval.
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Polymerase chain reaction (PCR) amplification
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Genomic DNA was routinely amplified using PCR in 50µl reaction mix containing 1µl
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genomic DNA template, in the presence of 39.3 MQ, 5µl 10*CAPS buffer, 2.5µl (5mM)
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dNTPs, 1µl
(5ng/µl) forward and reverse primer each and 0.2µl Supper Taq
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polymerase. The standard PCR condition was: initial denaturation step of 4 min at 94
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oC,
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appropriate temperature, 90 s amplification reactions at 72 oC and 5 min extension at 72
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oC.
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Dra I, Hinf I, Sau3A I, Bfa I, Hpa II) in a reaction mix of 15µl. The digested products
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were analyzed by electrophoresis on 4% agarose gels and visualized by DNA star.
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Table 1 List of RFLP markers, BACs origin, DNA sequences, thermal cycle and size of
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markers designed for the study.
followed by 35 cycles of 1 min denaturation at 94 oC, 1 min annealing at the
PCR products were then digested for 3 hours by restriction enzymes (Rsa I, Alu I,
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Result
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Markers linked to the Grp1 locus
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Forty-nine plants, previously selected for recombination events in the GP21-GP179
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interval, were used for CAPS analysis. All these plants were analyzed using the
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converted AFLP GP21 and GP179 markers to ensure the recombination events for the
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GP21-GP179 interval. Out of these, three plants denominated as AMRH1-F8, AMRH7-
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G3 and AMRH11-B8 were found to be none-recombinant for this interval. The CAPS
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analyses for the designed primers were made only on the forty-six recombinant plants
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and the parents. All the primers were first tested in a small set of plants including the
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parents to a range of temperature (55-650C) to get the appropriate condition for each
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primer. The PCR condition for each primer was then identified (Table 1).
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The diploid female parent AM carried the dominant allele of Grp1 in the
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heterozygous state (Rr). In this study, haplotype1 refers to the dominant Grp1 allele (R)
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and haplotype2 refers to the recessive allele (r). Similarly, the fully susceptible parent
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(RH) carries r allele in a homozygous state (rr). The profile generated specifically from
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the AM parent or the polymorphisms observed from plants inherited from resistance
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parent indicated haplotype1. The common fragments observed in both parents indicated
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haplotype2. Out of the 25 primers seven (AC6506a, AC6506b, AC9265a, AC9265b,
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AC9287a, AC9290a and AC2505) failed to amplify clear bands within a range of
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temperature (55-65 oC). Five primers (AC6506c, AC9301b, AC5288, AC9840, and
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AC9267c) showed polymorphisms to the AM parent of haplotype1 and were found
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within GP21-GP179 interval (Table 2). However, AC9266a, AC9266b and AC9288
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revealed polymorphisms to the susceptible (RH) parent (data not shown).
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Cleaved amplified polymorphic sequence (CAPS) analysis
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The segregation pattern of the CAPS markers among the recombinant plants are
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indicated in Fig. 1. The CAPS markers AC5288 and AC9267 were detected after
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digestion of the amplified PCR product using restriction endonuclease Alu I and Sau3A
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I, respectively. The other markers AC9840 and AC6506 were specifically amplified
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fragments and were only from plants which are inherited from the resistance haplotype1
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of the resistant parent.
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however, showed additional bands specific for the AM parent haplotype1. The CAPS
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marker AC5288, unlike the other four markers, showed the presence of the fragment
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correlated with the absence of haplotype1. On the other hand, the other four markers
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were placed in a way that the presence of the fragment indicates its association with
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haplotype1 of Grp1 gene.
Marker AC9301 amplified fragments from both parents,
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The PCR fragment pattern of all the markers allowed detecting recombinant and
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non recombinant genotypes among the tested plants. The highest recombination was
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observed between GP21 and AC6506; and AC9267 and GP179 in the magnitude of 19
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and 13, respectively. AC9301 and AC9840; and AC9840 and AC5288 were separated
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by one recombination event in each case (Fig. 2). The marker orders on chromosome
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V, within the interval of markers GP21 and GP179 are AC6506, AC9301, AC9840,
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AC5288, and AC9267 in order of their position. The marker order is established based
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on the observed recombination events between them (Table 3).
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The distance between the markers was calculated based on previous
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experiments. The calculated distance between the markers is given in Table 3. The
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distance is calculated by multiplying the distance between GP21-GP179 intervals
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(3.4cM) by the recombination frequencies of each marker. The calculated distance
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between: GP21 and AC6506, AC6506 and AC9301, AC9301 and AC9840, AC9840, and
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AC5288, AC5288, and AC9267 and AC9267 and GP179 is found to be 1.40cM, 0.37cM,
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0.07cM, 0.07cM, 0.52cM and 0.96cM, respectively.
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Seven plants for marker AC9267 showed disagreement with the results of the
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other markers. Out of these plants five of them (AMRH5-C1, AMRH5-H5, AMRH12-C2,
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AMRH12-F5 and AMRH15-F2) were identified to be linked to haplotype2. However, the
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other plants tested for all markers were predicted to be linked to halpotype1 of
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resistance parent. Additionally two plants for the same marker (AMRH1-G4 and
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AMRH4-A8) were found to be linked to haplotype1, but they were predicted to link to
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haplotype2.
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Discussion
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In the field of traditional breeding, there are difficulties in fixing desirable agronomic
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traits because of the epigenetic and epistatic gene effects at complex loci (Cao et al.,
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2001; Lawson et al., 2013). Due to the critical need for genetic maps in modern
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breeding programs, constructions of genetic maps are vital. Analysis of the potato
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genome at the molecular level was started with the construction of genetic linkage maps
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using RFLP markers and development of diploid mapping populations (Gebhardt et. al.,
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1989 and 1991). The latter development has been important in that it avoids the
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complications associated with linkage analysis due to tetrasomic inheritance (Luo et al.,
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2001; Cao et al., 2005). Markers and maps were used to locate genetic factors
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controlling agronomic traits, resistance to pathogens, and genes affecting tuber yield or
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tuber quality in the potato genome (Potato Genome Consortium, 2011; Li et al., 2013).
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In this study forty-six recombinant plants in the GP21-GP179 interval were used
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to map novel markers in the specific locus. Although a number of markers (TG432,
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SPUD237, SPUD839, GM339 and GM637) were localized within this locus (Marano et
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al., 2002; van der Voort et. al., 2000; Ballvora et. al., 1995; Meksem et. al., 1995) none
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of them showed polymorphism among the AM x RH mapping population.
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Marker enrichment of chromosome segment around Grp1 locus using CAPS
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analysis resulted in the identification of five new CAPS markers within a distance of
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3.4cM in the GP21-GP179 interval. The CAPS markers include AC6506, AC9301,
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AC5288, AC9267 and AC9840 flank Grp1. Although 25 primers were tested, only 5 were
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successful to show polymorphism among the AM X RH mapping population. This is
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most likely because the introgression segment from the wild species to S. tuberosum
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may be larger in the Grp1 locus in one of the haplotypes. The recurring backcrossing
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might remove most of the wild species introgressions and creates homozygous
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haplotype for the S. tuberosum outside the Grp1 locus. According to Konieczny and
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Ausubel (1993), if the marker chosen are well dispersed in the genome, then linkage to
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marker can be established using a small number of markers and a small number of
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individuals. Therefore, the distance between the markers was calculated. The present
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study found that the distance between these markers are less than one cM from each
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other except GP21 and AC6506, which is 1.4cM.
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The chromosome V region, between RFLP markers GP21 and GP179, is known
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to harbor a number of resistance genes for different pathogens. These two markers are
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used to delimit broader part and used to localize all the six resistance genes in that
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locus (Leonards-Schippers et. al., 1992, van der Voort et. al., 1998). Based on the
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mapping experiment of Leonards-Schippers et al. (1992) the genetic distance between
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the markers GP21 and GP179, flanking the R1 locus, was 6.6 cM. Contrary to this,
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other
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(https://gabi.rzpd.de/database).
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Nematology of Wageningen University on a mapping population of AM and RH parents,
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the calculated genetic distance was 3.4cM. This large difference is observed probably
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because of the differences in the mapping population.
researchers
found
that
the
genetic
Experiments
distance
conducted
in
is
the
at
about
Laboratory
3cM
of
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The profile generated from marker AC5288 after digestion by restriction
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endonuclease enzymes (Rsa I, Alu I, Sau3A I and Hinf I) were the same. However in
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this case the profile generated by Alu I and Rsa I showed partial digestion. This is either
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because of bad quality and insufficient amount of restriction enzyme or wrong buffer
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and temperature reaction condition. To ensure the complete digestion, new enzyme
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batch was used and the enzyme and the DNA molecule were incubated for longer time
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but the result remains the same. This is probably because in most cases the restriction
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endonuclease enzymes require specific buffer for their optimal activities. However in the
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CAPS analysis the 10*CAPS buffer was used and it might have reduced the optimal
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enzymatic activities.
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Markers, AC6506 and AC9840, seem allele specific primers. The allele specific
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primer matches perfectly with one haplotype and mismatch with the other haplotype.
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Because of the specificity of these primers, they only amplify the matched haplotype
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(Drenkard et. al., 2000). These types of markers are advantageous in that restriction
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digestion is not necessary after PCR amplification. On the other hand, in cases when
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PCR failed to amplify any fragment, there is a danger of counting genotypes as an allele
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specific marker. Furthermore the co-dominance nature of the CAPS marker helps to
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discriminate the homozygous and heterozygous individual (Kosman and Leonard,
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2005). However in this study no co-dominant primer was found.
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The association between a phenotype and the underling genotype can be
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measured by the relationship between flanking markers and a phenotypic trait in the
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progeny (Finkers-Tomczak et al., 2009; D’hoop et al., 2008; Bakker et al., 2004). To
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determine which marker is closely linked to the Grp1 gene and to make high-resolution
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map of the Grp1 gene a further nematode resistance assays will be conducted.
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Acknowledgement
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This work was supported by the Swedish International Development Agency (SIDA) and
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the Amhara Rural Development Program (ARDP) and financed by the Swedish
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government. Dr. Erin Bakker of the Laboratory of Nematology at Wageningen University
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is sincerely thanked for technical and material supports.
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List of Tables:
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Table 1
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List of RFLP markers, BACs origin, DNA sequences, thermal cycle and size of markers
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designed for the study.
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Table 2
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Details of markers identified and linked to potato cyst nematode resistance gene, Grp1.
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Table 3
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Summery of the distance between markers in cM within the GP21-GP179 interval.
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List of Figures
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Fig. 1.
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Performance of the identified CAPS markers. The linkage of both markers is shown by
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the profiles generated from the resistance parent AM, the susceptible parent RH and a
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subset of their progenies. B & E show the band patterns detected after digestion of the
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amplified product using restriction endonuclease Alu I and Sau3A I for markers AC5288
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and AC9267, respectively. A, C and D show the band patterns generated by the PCR
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products of markers AC9840, AC9301, and AC6506, respectively.
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Fig. 2.
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Mapping of the GP21–GP179 interval on chromosome V of the diploid potato clone AM.
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Markers are presented in the correct genetic order: GP21, AC6506, AC9301, AC9840,
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AC5288, AC9267, and GP179. Bold horizontal lines represent chromosomal regions
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derived from the haplotype2 harbouring Grp1 and light black horizontal lines represent
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chromosomal regions derived from the other haplotype.
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