Eight species and 57 selections/cultivars of Penstemon were compared for genetic variability using Random Amplified Polymorphic DNAs (RAPDs). The RAPD technique was used to help understand the genetic relationships in species and cultivars in the genus Penstemon. Ten RAPD primers (from Operon) were screened to identify polymorphisms among these eight species and 57 selections. More than 100 RAPD polymorphic bands were obtained. A principle component analysis was used to study genetic relationships. Variation among species was greater than variation among selections/cultivars within species. RAPD markers distinguished differences between most cultivars tested. DNA fingerprints generated by RAPDs should be useful to distinguish cultivars of Penstemon, as well as to assist in determining genetic relationships between species.
Guenhwa Jung, Dale T. Lindmen, and Dermot P. Coyne
L.H. Zhang, D.H. Byrne, R.E. Ballard, and S. Rajapakse
Microsatellite or simple sequence repeat (SSR) markers were developed from Rosa wichurana Crépin to combine two previously constructed tetraploid rose (Rosa hybrida L.) genetic maps. To isolate SSR-containing sequences from rose a small-insert genomic library was constructed from diploid Rosa wichurana and screened with several SSR probes. Specific primers were designed for 43 unique SSR regions, of which 30 primer pairs gave rise to clear PCR products. Seventeen SSR primer pairs (57%) produced polymorphism in the tetraploid rose 90-69 mapping family. These markers were incorporated into existing maps of the parents 86-7 and 82-1134, which were constructed primarily with AFLP markers. The current map of the male parent, amphidiploid 86-7, consists of 286 markers assigned to 14 linkage groups and covering 770 cm. The map of the female tetraploid parent, 82-1134, consists of 256 markers assigned to 20 linkage groups and covering 920 cm. Nineteen rose SSR loci were mapped on the 86-7 map and 11 on the 82-1134 map. Several homeologous linkage groups within maps were identified based on SSR markers. In addition, some of the SSR markers provided anchoring points between the two parental maps. SSR markers were also useful for joining small linkage groups. Based on shared SSR markers, consensus orders for four rose linkage groups between parental maps were generated. Microsatellite markers developed in this study will provide valuable tools for many aspects of rose research including future consolidation of diploid and tetraploid rose genetic linkage maps, genetic, phylogenetic and population analyses, cultivar identification, and marker-assisted selection.
J.G. Tivang, J. Nienhuis, O.S. Smith, and J.S.C. Smith
The statistical properties associated with molecular markers are important when used to characterize germplasm. Evaluation of these properties are necessary for informed selection of one marker system over another. Five different molecular marker systems, Amplified Fragment Length Polymorphism (AFLPs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Random Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphism (RFLPs), and Isozymes were used to evaluate 39 elite corn belt inbreds. Each system was characterized for fragment frequency distribution, and band correlation distribution as a measure of independence. A regression model estimating resolution and rate of information addition was constructed using the sampling variance. All marker systems were evaluated according to this model. The model facilitated genetic relationships among the inbreds to be compared at equivalent performance level among all marker systems. Four performance levels resulted in 10 comparisons. Pairwise test of significance were conducted using t tests where the null-distributions were obtained by the bootstrap procedure. The maker system were ranked, assisting breeders in selecting marker systems for germplasm organization.
Ruth S. Kobayashi, Stephen L. Sinden, and Lind L. Sanford
Leptine glycoalkaloids found in certain genotypes of Solanum chacoense, a wild potato relative, are resistance factors against the Colorado potato beetle (CPB). To efficiently introgress CPB resistance through leptine production into the cultivated potato, an understanding of leptine inheritance is important. Analysis of sibs within PI lines revealed a wide segregation for level of leptines. Leptine levels ranged from not detectable to 120 mg/100 g fresh weight among six sibs in a PI line, suggesting leptine production may be controlled by a few major genes. TLC analysis of F2 and backcross progeny, however, indicated that several genes probably control leptine level. This apparent multigene control of leptine level may make it difficult to incorporate leptine synthesis into the cultivated potato. Therefore, we are presently identifying microsatellite and RAPD markers associated with leptine synthesis to enable marker-assisted selection and facilitate the incorporation of leptine synthesis into the cultivated potato.
Kirk W. Pomper, Anita N. Azarenko, Joel W. Davis, and Shawn A. Mehlenbacher
Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles and assist in cloning the locus responsible for the sporophytic SI displayed in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 27 progeny of the cross OSU 23.017 (S1 S12) × VR6-28 (S2 S26). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from six trees each were pooled into four “bulks,” one for each of the following: S1 S2, S1 S26, S2 S12, and S12 S26. “Super bulks” of twelve trees each for S1, S2, S12, and S26 then were created for each allele by combining the appropriate bulks. The DNA from these four super bulks and also the parents was used as a template in the PCR assays. Amplification products were electrophoresed on 2% agarose gels and photographed under UV light after ethidium bromide staining. 200 primers were screened and one RAPD marker each was identified for alleles S2 (OPI-07700) and S1 (OPJ-141700).
R.C. Grube, Y. Zhang, B. Huang, and M.M. Kyle
Resistance to cucumber mosaic virus (CMV) in Capsicum from two sources is being transferred into three commercial types (bell, jalapeno, and Anaheim) using a backcross breeding scheme. We have optimized our CMV seedling screening protocol, which involves multiple inoculations beginning at the cotyledon stage with a severe CMV serogroup I isolate. Both sources of resistance, C. annuum `French Perennial' and a C. frutescens accession (BG2814-6), exhibit oligogenic recessive inheritance and share some but not all resistance alleles. Selection for type in the BCF1 generation had no effect on the frequency of resistant individuals in the BCF2 generation. We have determined that it is necessary to self-pollinate every other backcross generation to screen for resistance. Occasionally disease symptoms appear in adult plants that were initially resistant to multiple inoculations at the seedling stage, and we are investigating the correlation between seedling resistance and adult plant resistance. We are also exploring the extent to which the different sources of resistance behave differently as a function of genetic background. Additionally, we are mapping quantitative trait loci (QTLs) for CMV resistance in pepper with the goal of converting RFLP and/or RAPD markers into PCR-based markers to facilitate molecular marker-assisted selection for CMV resistance.
Mikel R. Stevens, Shawn A. Chrisensen, Ammon B. Marshall, JoLynn J. Stevens, Peter Wenzl, Eric Hunter, Jason Carling, and Andrzej Killian
Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.
M.J. Striem, G. Ben-Hayyim, and P. Spiegel-Roy
Excluding seeded offspring at an early stage could be of great value to the breeder concerned with the development of seedless grapes (Vitis vinifera L.). We used the random amplified polymorphic DNA (RAPD) technique to identify molecular genetic markers, analyzing 82 individuals of a progeny resulting from a cross between `Early Muscat' (seeded) and `Flame Seedless'. Seven variables representing the traits of seedlessness were analyzed: mean fresh weight of one seed, total fresh weight of seeds per berry, perception of seed content, seed size categories evaluated visually, degree of hardness of the seed coat, degree of development of the endosperm, and degree of development of the embryo. Among 160 10mer primers, 110 gave distinct band patterns. Twelve markers yielded significant correlations with several subtraits of seedlessness, mainly with the mean fresh weight of one seed and the total fresh weight of seeds per berry. Multiple linear regression analysis resulted in high coefficients, such as R = 0.779 for fresh weight of seeds per berry, when the seven markers were included as independent variables in the model. Most of the seeded individuals, about 44% of the progeny, could be excluded using a two-step process of marker assisted selection.
Tong Geon Lee, Samuel F. Hutton, and Reza Shekasteband
unclear. To better use the br , the tomato breeding community needs genetic markers closely linked to the locus to improve selection efficiency. In addition, to understand the molecular mechanisms of the br gene, it needs to be cloned and this effort
Yiping Zhang and John R. Stommel
The carotenoids have an important influence on tomato fruit quality and enhance the fruit contribution to human nutrition. Expression of the high pigment (hp) locus in tomato results in increased total carotenoids and increased efficiency of utilization of the polyenes. A similar mutant, dark green (dg), contains higher level of chlorophyll in immature fruit and results in darker red pigmentation, both externally and internally in ripe fruit. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses were performed using two pairs of near isogenic lines (NILs) designed to be isogenic at the hp and dg loci. Sixty-four AFLP primer pairs and more than 1000 RAPD 10-mer primers were screened for polymorphism between each pair of the NILs. One RAPD marker was identified to be linked to the hp gene, and two AFLP primer pairs showed polymorphic fragments which distinguished the dg NILs. The markers identified in this study will be converted to allele specific SCAR (sequence characterized amplified region) markers, which are more useful in marker-assisted selection breeding programs.