Dry pea (Pisum sativum L.) production in many areas of the world may be severely diminished by soil inhabiting pathogens such as Fusarium oxysporum f. sp. pisi race 1, the causal organism of fusarium wilt race 1. Our objective was to identify closely linked marker(s) to the fusarium wilt race 1 resistance gene (Fw) that could be used for marker assisted selection in applied pea breeding programs. Eighty recombinant inbred lines (RILs) from the cross of Green Arrow (resistant) and PI 179449 (susceptible) were developed through single-seed descent, and screened for disease reaction in race 1 infested field soil and the greenhouse using single-isolate inoculum. The RILs segregated 38 resistant and 42 susceptible fitting the expected 1:1 segregation ratio for a single dominant gene (χ2 = 0.200). Bulk segregant analysis (BSA) was used to screen 64 amplified fragment length polymorphism (AFLP) primer pairs and previously mapped random amplified polymorphic DNA (RAPD) primers to identify candidate markers. Eight AFLP primer pairs and 15 RAPD primers were used to screen the RIL mapping population and generate a linkage map. One AFLP marker, ACG:CAT_222, was within 1.4 cM of the Fw gene. Two other markers, AFLP marker ACC:CTG_159 at 2.6 cM linked to the susceptible allele, and RAPD marker Y15_1050 at 4.6 cM linked to the resistant allele, were also identified. The probability of correctly identifying resistant lines to fusarium wilt race 1, with DNA marker ACG:CAT_222, is 96% percent. These markers will be useful for marker assisted breeding in applied pea breeding programs.
Melissa T. McClendon, Debra A. Inglis, Kevin E. McPhee, and Clarice J. Coyne
David N. Kuhn, Giri Narasimhan, Kyoko Nakamura, J. Steven Brown, Raymond J. Schnell, and Alan W. Meerow
Identifying genetic markers linked to disease resistance in plants is an important goal in marker-assisted selection. Using a candidate-gene approach, we have previously developed genetic markers in cacao (Theobroma cacao L.) for two families of genes involved in disease resistance: non-TIR-NBS-LRR (Toll/Interleukin-1 Receptor-nucleotide binding site-leucine rich repeat) resistance gene homologues and WRKY transcription factor genes; however, we failed to isolate TIR-NBS-LRR genes. Using a novel algorithm to design degenerate primers, we have now isolated TIR-NBS-LRR loci as determined by DNA sequence comparison. These loci have been developed as genetic markers using capillary array electrophoresis (CAE) and single-strand conformational polymorphism (SSCP) analysis. We have mapped three distinct TIR-NBS-LRR loci in an F2 population of cacao and demonstrated that one is located on linkage group 3 and the other two on linkage group 5.
Fruit and ornamental breeders were surveyed about their use of molecular markers in either their breeding programs or in their related research programs. Responses were obtained from over 100 fruit and ornamental breeding programs from throughout the world. Of these, less than 50% used molecular markers in their programs. The two most common uses of these markers were for studies in plant identification and diversity. These were followed by the use of markers in developing molecular maps, in discovering molecular tags and/or trying to identify the genes for specific plant traits, for marker assisted selection, and finally, for the elucidation of plant taxonomy. In conclusion, although there is much research in this area, few programs are actually using markers in the context of an applied breeding program. The major reason for this situation is the lack of available markers and the cost of using these markers to screen large numbers of progeny. Those that use markers in their breeding tend to use them to verify the genotype of the parents or confirm the genotype of selected seedlings rather than screen unselected seedlings.
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.
Honglin Chen, Shawn A. Mehlenbacher, and David C. Smith
Eastern filbert blight (EFB), caused by Anisogramma anomala (Peck) E. Müller, is a devastating disease to european hazelnut (Corylus avellana L.) orchards in the Willamette Valley of Oregon. Selection OSU 408.040 showed no symptoms or signs of the fungus following greenhouse inoculations, and enzyme-linked immunosorbant assays (ELISAs) were negative. Segregation ratios in three progenies indicate that a single dominant gene controls the resistance. A total of 64 amplified fragment length polymorphism (AFLP) primer combinations were screened using three resistant and three susceptible individuals as well as the parents of the cross OSU 245.098 × OSU 408.040. Primer combinations that showed no more than one recombinant in these six seedlings were investigated in 30 additional seedlings. Markers that showed <15% recombination with resistance were amplified in the remaining seedlings of the population. Five AFLP markers linked in coupling to resistance were identified. B2-125 was located on one side of the resistance locus at a distance of 4.1 centimorgans (cM), while A4-265 (9.2 cM), C2-175 (5.9 cM) and D8-350 (2.5 cM) were on the other side, and A8-150 cosegregated with resistance. Three of these markers (B2-125, C2-175, and D8-350) were also linked in coupling in a similar order in seedlings from a second progeny. These markers may be useful in marker-assisted selection for eastern filbert blight resistance from hazelnut selection OSU 408.040.
Eric Stafne, John Clark, and Kim Lewers
Molecular markers have been used previously to identify linkages to important traits of interest. In this study two marker types, randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR), were used to find molecular markers linked to two morphological traits in blackberry (Rubus L. subgenus Rubus). Thorniness and floricane fruiting are both qualitative, recessive traits that are inherited tetrasomically. A cross of `Prime-Jim'® × `Arapaho' was made to create a population that segregated for the two traits. A random sample of 98 plants from a population of 200 were assayed to find molecular markers that co-segregate with the two traits. Three putative markers were identified for the floricane fruiting trait (two SSRs and one RAPD; χ2 = 4.09 to 9.99, P < 0.001 to 0.043). Five potential RAPD markers were found for the thorny trait (χ2 = 3.88 to 10.23, P < 0.001 to 0.048). Identification of markers linked to these traits could potentially be useful in marker-assisted selection.
Leigh K. Hawkins, Fenny Dane, Thomas L. Kubisiak, Billy B. Rhodes, and Robert L. Jarret
Isozyme, randomly amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers were used to generate a linkage map in an F2 and F3 watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai] population derived from a cross between the fusarium wilt (Fusarium oxysporum f. sp. niveum) susceptible `New Hampshire Midget' and resistant PI 296341-FR. A 112.9 cM RAPD-based map consisting of 26 markers spanning two linkage groups was generated with F2 data. With F3 data, a 139 cM RAPD-based map consisting of 13 markers covering five linkage groups was constructed. Isozyme and SSR markers were unlinked. About 40% to 48% of the RAPD markers were significantly skewed from expected Mendelian segregation ratios in both generations. Bulked segregant analysis and single-factor analysis of variance were employed to identify RAPD markers linked to fusarium wilt caused by races 1 and 2 of F. oxysporum f. sp. niveum. Current linkage estimates between the resistance trait and the marker loci were too large for effective use in a marker-assisted selection program.
X.Y. Zheng and David W. Wolff
Three randomly amplified polymorphic DNA (RAPD) markers (E07, G17, and 596) linked to the Fom-2 gene, which confers resistance to race 0 and 1 of Fusarium oxysporum f. sp. melonis, were evaluated by RAPD-polymerase chain reaction for their linkage to Fusarium wilt resistance/susceptibility in diverse melon cultigens (48 resistant, 41 susceptible). Primer 596 was identified in the multiple disease-resistant breeding line MR-1, whereas E07 and G17 were identified in the susceptible `Vedrantais'. The RAPD markers E07 (1.25 kb) and G17 (1.05 kb) correctly matched phenotypes in 88% and 81% of the cultigens. The validity of the RAPD scores was verified by Southern hybridization analysis for sequence homology and bulked segregant analysis of a selected cross population for the linkage. These results will facilitate the introgression of resistance genes into susceptible lines from multiple sources in marker-assisted selection.
Majid R. Foolad, Arun Sharma, Hamid Ashrafi, and Guoyang Lin
Early blight (EB), caused by the fungus Alternaria solani, is a destructive disease of tomato (Lycopersicon esculentum) worldwide. Sources of genetic resistance have been identified within related wild species, including green-fruited L. hirsutum and red-fruited L. pimpinellifolium. We have employed traditional protocols of plant breeding and contemporary molecular markers technology to discern the genetic basis of EB resistance and develop tomatoes with improved resistance. Backcross breeding has resulted in the development of germplasm with improved resistance; however, linkage drag has been a major obstacle when using L. hirsutum as a donor parent. To identify and map QTLs for EB resistance, we used several filial and backcross populations derived from interspecific crosses between L. esculentum and either L. hirsutum or L. pimpinellifolium. In each population, an average of seven resistance QTLs were detected. While similar QTLs were detected in different generations of the same cross, generally different QTLs were identified in populations derived from different crosses. The results suggested stability of QTLs across environments and generations but variation in QTLs in different interspecific populations. It is expected that marker-assisted pyramiding of QTLs from different sources results in development of germplasm with strong and durable resistance. Further inspection of the results led to the identification and selection of six QTLs with stable and independent effects for use in marker–assisted selection (MAS). However, to facilitate “clean” transfer and pyramiding of these QTLs, near-isogenic lines (NILs) containing individual QTLs in a L. esculentum background should be developed.
David B. Rubino
Segregating progenies from controlled pollinations of Eustoma grandiflorum Griseb. were investigated to determine the inheritance of diaphorase (DIA) and glucoses-phosphate isomerase (GPI) isozymes. Phenotypic data supported the hypotheses that DIA1 is tetrameric and is controlled by a single locus with two alleles (Dia1-1 and Dia1-2) and that GPI1 is dimeric and also is controlled by a single locus with two alleles (Gpi1-1 and Gpi1-2). Examination of isozyme phenotypes for over 70 cultivars of E. grandiflorum revealed polymorphism for DIA1 and GPI1. These isozymes may be useful for marker-assisted selection and cultivar identification.