Peach rootstock breeding may be accelerated by utilization of molecular markers linked to the root-knot nematode resistance locus (Mi) to screen segregating populations. A genetic linkage map was constructed using RFLP markers in an F2 population (PMP2) that is segregating for this locus. PMP2 is derived from a controlled cross of the relatively diverse peach rootstocks Harrow Blood (susceptible) and Okinawa (homozygous resistant). Bulked Segregant Analysis was applied using RAPD markers. A single small (227 base pairs) RAPD marker was found to be linked to the dominant resistant allele of Mi at a distance of 10 cM. This new marker joined the Mi locus to the RFLP linkage map and showed that two dominant RFLP markers are located between the RAPD marker and Mi. RFLPS are expensive, time-consuming and RAPD markers are unreliable, and therefore both are unsuitable for screening breeding populations. We attempted to convert the RAPD marker to a more breeder-friendly CAPS marker. The converted CAP marker was dominant. Attempts to convert the CAP marker to a co-dominant marker were not successful. The utility of the CAP marker was tested in an open pollinated F2 population derived from the F1 parent of PMP2 and in several rootstocks. The genetic linkage map was compared to other Prunus maps. The PMP2 linkage group containing the Mi locus can be related to the peach × almond linkage group which contains the phosphoglucomutase Pgm-1 locus.
Anne M. Gillen and Fred A. Bliss
An F2 population from a single F1 plant from the cross of peach [Prunus persica (L.) Batsch] rootstock cultivars Harrow Blood (HB) × Okinawa (Oki) was used to locate the Mi locus, which conditions resistance to Meloidogyne incognita (race 1) (Kofoid and White) Chitwood. These data and comparison of common markers among published genetic linkage maps placed the Mi locus on Prunus L. linkage group 2. Two restriction fragment length polymorphisms (RFLPs) [linked at 4.8 and 6.8 centimorgan (cM), repulsion phase] and one random amplified polymorphic DNA (RAPD) marker (linked at 9.5 cM, coupling phase) were linked to Mi. The RAPD marker was cloned, sequenced, and converted to a polymerase chain reaction (PCR)-based cleaved amplified polymorphic sequence (CAPs) marker. Clones of resistance gene analogs (RGA) developed from Oki were highly polymorphic when used as RFLP probes. The RGA's mapped to four linkage groups but clustered on two of the four linkage groups, providing limited coverage of the genome. Even so, they may be useful as markers for disease resistance genes that occur in other populations. The linkage maps of the HB × Oki F2 population and a peach × almond (Prunus amygdalus Batsch) F2 population were colinear in certain regions, however, a significant number of markers mapped to different linkage groups among the two populations. The locus for the blood-flesh trait (red-violet mesocarp) mapped to the top of linkage group 4.
M.A. Dalbó, G.N. Ye, N.F. Weeden, W.F. Wilcox, and B.I. Reisch
The efficiency of marker-assisted selection for powdery mildew (Uncinula necator (Schw.) Burr) resistance in grapes (Vitis L. sp.) was studied using molecular markers associated with a major QTL (quantitative trait loci) for this trait. Initially, genetic maps were constructed from a segregating population of the cross `Horizon' × Illinois 547-1 (a hybrid between V. rupestris Scheele and V. cinerea Engelm.). A major QTL from Ill. 547-1, the resistant parent, explained 41% of the variation. One RAPD (randomly amplified polymorphic DNA) marker and one AFLP (amplified fragment length polymorphism) marker, obtained by bulked segregant analysis, showed the highest association with powdery mildew resistance in the mapping population. Segregation of the QTL was followed in different crosses by CAPS (cleaved amplified polymorphic sequence) markers developed from these two markers. An allele-specific amplified polymorphism that segregates as present/absent was also developed from the CS25b locus. Powdery mildew resistance was evaluated visually on a 1 to 5 scale in four different seedling populations. Two populations originated from crosses using Ill. 547-1 as the resistant parent. Two other populations were from crosses with NY88.0514.03, a resistant seedling from the original `Horizon' × Ill. 547-1 mapping population. Segregation ratio distortions were observed in some crosses. In these cases, the allele associated with the QTL for powdery mildew resistance was less frequent than the alternate allele. In all crosses, the markers were closely associated with resistance. If selection were based on markers, the percentage of susceptible individuals (classes 4 and 5) would decrease from 24% to 52% to 2% to 18%. Selection efficiency was greatest in crosses where segregation distortion was most intense.
Samia Lotfy, Francois Luro, Françoise Carreel, Yann Froelicher, Delphine Rist, and Patrick Ollitrault
Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.
Tong Geon Lee, Reza Shekasteband, Naama Menda, Lukas A. Mueller, and Samuel F. Hutton
20 s; 57 °C for 20 s; 72 °C for 15 s; and a 10-min extension at 72 °C. The products from a jointed and a jointless line were sequenced by the Sanger method to validate single-product amplification of the target region. Two software packages were used
Yuanfu Ji and John W. Scott
Resistance to begomoviruses tomato mottle virus (ToMoV) and tomato yellow leaf curl virus (TYLCV) has been introgressed to tomato (Lycopersicon esculentum) from L. chilense accessions LA 1932, LA 2779, and LA 1938. Resistance genes have been mapped to three regions on chromosome 6 using randomly amplified polymorphic DNA (RAPD) markers. We call these regions 1, 2, and 3. To facilitate breeding by marker assisted selection, advanced breeding lines with resistance from the above sources were assayed for the presence of RAPD markers to determine which were most tightly linked to begomovirus resistance. The best RAPD markers were then converted to sequence characterized amplified region (SCAR) markers or cleaved amplified polymorphic sequence (CAPS) markers. In addition, selected restriction fragment length polymorphism (RFLP) markers near the three regions were converted into CAPS markers, which were tested for association with the advanced breeding lines. Only LA 2779 derivatives have the L. chilense introgression in region 1, which is near the location of the Ty-1 gene and spans across CAPS markers 32.5Cla and TG118. Two region 1 RAPD markers UBC197 and UBC621 were converted co-dominant SCAR or CAPS markers, which were present in all 16 resistant breeding lines tested. Derivatives from all three accessions have introgressions in region 2. Further assays with more markers in this region are under way to determine the lengths and locations of the introgressions. No tightly linked RAPD markers have been found for the resistance gene from LA 1932 in region 3. RFLP and CAPS markers are being used to more precisely locate the region 3 gene.
K. Ikeda, A. Watari, K. Ushijima, H. Yamane, N.R. Hauck, A.F. Iezzoni, and R. Tao
S4′ is a pollen-part mutant in sweet cherry (Prunus avium L.) that is extensively used to develop self-compatible cultivars. The S4′-haplotype is known to have a functional stylar component and a nonfunctional pollen component. The pollen component in sweet cherry necessary for the specificity of the pollen reaction is believed to be an S-haplotype specific F-box protein gene, called SFB. This study describes two molecular markers that distinguish between SFB4 and SFB4′ by taking advantage of a four base pair deletion in the mutant allele. The resulting polymerase chain reaction (PCR) products can either be separated directly on a polyacrylamide gel or they can be subjected to restriction enzyme digestion and the different sized products can be visualized on an agarose gel. The latter technique utilizes restriction sites created in the PCR products from the SFB4′ allele, but not the SFB4 allele. Because the primer sets created differential restriction sites, these primer sets were termed dCAPS (derived cleaved amplified polymorphism sequence) markers. These molecular assays can be used to verify self-compatibility conferred by the S4′-haplotype.
John McCallum, Susan Thomson, Meeghan Pither-Joyce, Fernand Kenel, Andrew Clarke, and Michael J. Havey
BlastDigester ( Ilic et al., 2004 ) and if suitable polymorphisms could not be identified, potential for engineering these was identified using dCAPS finder 2.1 ( Neff et al., 2002 ). Cleaved amplified polymorphic sequence or derived cleaved amplified
Xinyi Zhang, Li Liao, Zhiyong Wang, Changjun Bai, and Jianxiu Liu
), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) ( Kalia et al., 2011 ). Intersimple sequence repeat markers can be amplified simply and directly without knowledge of the flanking sequences, thus enabling easier development
Karen R. Harris, W. Patrick Wechter, and Amnon Levi
sequence-tagged site (WRGA-STS), cleaved amplified sequence polymorphism (CAPS), and expressed sequence tag polymerase chain reaction (PCR) product (primers labeled watermelon unigene-WMU), and single nucleotide polymorphism (SNP) for ‘New Hampshire Midget