Microsatellite-based molecular diversity of bread wheat germplasm and association mapping of wheat resistance to the Russian wheat aphid.

Genetic diversity of a set of 71 wheat accessions, including 53 biotype 2 Russian wheat aphid (RWA2)-resistant landraces and 18 RWA2 susceptible accessions, was assessed by examining molecular variation at multiple microsatellite (SSR) loci. Fifty-one wheat SSR primer pairs were used, 81 SSR loci were determined, and 545 SSR alleles were detected. These SSR loci covered all the three genomes, 21 chromosomes, and at least 41 of the 42 chromosome arms. Diversity values averaged over SSR loci were high with mean number of SSR alleles/locus = 6.7, mean Shannon's index (H) = 1.291, and mean Nei's gene diversity (He) = 0.609. The three wheat genomes ranked as A > D > B and the homoeologous groups ranked as 7 > 3 > 1 > 2 > 6 > 5 > 4 based on the number of alleles per locus. Xgwm136 on chromosome arm 1AS is the most polymorphic SSR locus with the largest number of observed and effective alleles and the highest H and He. Among all 2485 pairs of wheat accessions, genetic distance (GD) ranged from 0.054 to 1.933 and averaged 0.9832. A dendrogram based on GD matrix showed that all the wheat accessions could be grouped into distinct clusters. Most of the susceptible cultivars (13/18) were clustered into groups that contains all or mostly susceptible accessions. Most of the U.S. cultivars belong to a group that is distinguishable from all the different RWA2 resistant groups. Diversity analysis was also conducted separately for subgroups containing 53 RWA2-resistant accessions and 18 RWA2-susceptible accessions. Association mapping revealed 28 SSR loci significantly associated with leaf chlorosis, and 8 with leaf rolling. New chromosome regions associated with RWA2 resistance were detected, and indicated existence of new RWA resistance genes located on chromosomes of all other homoeologous groups in addition to the groups 1 and 7 in bread wheat. This information is helpful for development of mapping populations for RWA2 resistance genes from different phylogenetic groups, and for wise utilization of the RWA-resistant germplasm in wheat breeding programs.

Temperature Sensitivity and Efficacy of Wheat streak mosaic virus Resistance Derived from CO960293 Wheat.

Wheat yields often are limited by infection by Wheat streak mosaic virus (WSMV). Host plant resistance to WSMV can reduce losses. This study was conducted to characterize a new source of temperature-sensitive resistance found in CO960293 wheat. The source of the temperature-sensitive resistance in CO960293 is unknown. Parental and other wheat lines were tested for WSMV resistance using 51 WSMV isolates under different temperatures to determine the stability of the resistance, and yield trials were conducted in the field for 3 years. All parental wheat lines became infected by WSMV at all temperatures and were infective in back assay to 'Tomahawk' wheat. No WSMV isolate defeated the resistance of CO960293 at 18°C. Yield of CO960293 in field trials was reduced in only 1 of 3 years. Our data demonstrate that this wheat line can be a valuable source of resistance to WSMV in wheat programs, particularly in areas where temperatures are cool following planting in the fall.

Response of resistant and susceptible barley to infestations of five Diuraphis noxia (Homoptera: Aphididae) biotypes.

Since 2003, four new biotypes of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), RWA2-RWA5, have been discovered that have the ability to damage most of the wheat germplasm resistant to the original Russian wheat aphid population (RWA1). Barley germplasm lines with resistance to RWA1 have not yet been evaluated against the newest biotypes. Our study compared how biotypes RWA1-RWA5 affected the growth and leaf damage of RWA1-resistant germplasm (STARS 9301B, STARS 9577B), moderately resistant germplasm (MR-015), and susceptible varieties (Schuyler, Harrington, and Morex) under greenhouse conditions. Russian wheat aphid population levels also were determined 14 d after plant infestation. STARS 9301B exhibited strong resistance by showing only small differences in leaf damage and growth parameters from the feeding by the biotypes. STARS 9577B showed greater differences in damage by the Russian wheat aphid biotypes than STARS 9301B, yet, the ratings were still within the resistant category (e.g., chlorosis rating 2.3-4.9). Leaf chlorosis ratings for MR-015 ranged from 5.0 to 6.9 and fell within the moderately resistant to susceptible categories for all the biotypes. The greatest difference in leaf chlorosis occurred in Morex where RWA2 showed less virulence than the other biotypes. Feeding by the Russian wheat aphid biotypes produced only small differences in leaf rolling and plant growth within plant entries. Population levels of the Russian wheat aphid biotypes did not differ within barley entries (n = 610-971) at the termination of the study (14 d). From our research, we conclude that the new Russian wheat aphid biotypes pose no serious threat to the key sources of resistance in barley (STARS 9301B and 9577B).

Molecular cytogenetic and agronomic characterization of advanced generations of wheat x triticale hybrids resistant to Diuraphis noxia (Mordvilko): application of GISH and microsatellite markers.

The PI 386148 triticale from Russia is among the highest resistant line to the Russian wheat aphid (RWA) (Diuraphis noxia (Mordvilko)). This triticale line was used as the male parent in crosses with Lamar wheat (Triticum aestivum L.). The F1 plants were backcrossed to Lamar wheat. The progenies were tested for RWA biotype 1 reaction for at least eight backcross and selfing generations. Five lines from these selections were identified for their resistance to the RWA and their seeds were increased for agronomic and other characterizations. Molecular and cytological analyses of these lines were performed using genomic in situ hybridization and rye chromosome-specific microsatellites markers. Three lines were cytologically stable and carried a pair of rye (Secale strictum (C. Presl) C. Presl) chromosomes as disomic addition lines of 1R. One line was unstable and showed a moderate level of mixoploidy with monosomic additions of 1R. Duplication of rye chromosome 1R was also identified. No wheat-rye chromosome interchange was detected, suggesting little homology between S. strictum and T. aestivum chromosomes. Specific microsatellite primers were used to identify the rye chromosomes present in each line. One rye chromosome, 1R, from the donor species contains genes for RWA resistance. Grain yield and test weight of three of the lines were similar to some adapted released wheat varieties under stress conditions.

Large-scale production of murine bone marrow cells in an airlift packed bed bioreactor.

Large-scale cultivation of murine bone marrow cells was accomplished in an airlift packed bed bioreactor system designed to mimic the in vivo bone marrow environment. The attachment-dependent stromal cell population, which provides the necessary microenvironment, including growth factors for subsequent hematopoietic activity, was first established within the bioreactor. This attachment-dependent cell growth occurred on the fiber-glass matrix packed in the annular region of the bioreactor. Once the stromal cell layer was established, fresh bone marrow cells were inoculated to initiate hematopoiesis. However, traditional culture medium was found to be inadequate for the initiation of hematopoiesis, but the use of stromal cell "conditioned" medium (with no exogenously added growth factors) yielded sustained cell production. The extent of stromal cell subculturing prior to inoculation into the bioreactor and the inoculation density were also important factors for the successful initiation of hematopoietic activity. A 500-mL perfusion culture experiment resulted in the production and harvest of 3.6 x 10(8) suspended bone marrow cells over the course of 11 weeks.

Identification of RAPD markers linked to a major rust resistance gene block in common bean.

Rust in bean (Phaseolus vulgaris L.), caused byUromyces appendiculatus (Pers.) Unger var.appendiculatus [ =U. phaseoli (Reben) Wint.], is a major disease problem and production constraint in many parts of the world. The predominant form of genetic control of the pathogen is a series of major genes which necessitate the development of efficient selection strategies. Our objective was focused on the identification of RAPD (random amplified polymorphic DNA) markers linked to a major bean rust resistance gene block enabling marker-based selection and facilitating resistance gene pyramiding into susceptible bean germplasm. Using pooled DNA samples of genotyped individuals from two segregating populations, we identified two RAPD markers linked to the gene block of interest. One such RAPD, OF10970 (generated by a 5'-GGAAGCTTGG-3' decamer), was found to be closely linked (2.15±1.50 centi Morgans) in coupling with the resistance gene block. The other identified RAPD, OI19460 (generated by a 5'-AATGCGGGAG-3' decamer), was shown to be more tightly linked (also in coupling) than OF10970 as no recombinants were detected among 97 BC6F2 segregating individuals in the mapping population. Analysis of a collection of resistant and susceptible cultivars and experimental lines, of both Mesoamerican and Andean origin, revealed that: (1) recombination between OF10970 and the gene block has occurred as evidenced by the presence of the DNA fragment in several susceptible genotypes, (2) recombination between OI19460 and the gene block has also occurred indicating that the marker is not located within the gene block itself, and (3) marker-facilitated selection using these RAPD markers, and another previously identified, will enable gene pyramiding in Andean germplasm and certain Mesoamerican bean races in which the resistance gene block does not traditionally exist. Observations of variable recombination among Mesoamerican bean races suggested suppression of recombination between introgressed segments and divergent recurrent backgrounds.

Heterogeneous inbred populations are useful as sources of near-isogenic lines for RAPD marker localization.

The development and use of RAPD markers for applications in crop improvement has recently generated considerable interest within the plant breeding community. One potential application of RAPDs is their use for "tagging" simply-inherited (monogenic) pest-resistance genes and enabling more efficient identification and selection of genotypes carrying specific combinations of resistance genes. In this report, we propose and describe the use of heterogeneous inbred populations as sources of near-isogenic lines (NILs) for targeting RAPD markers linked to major pest resistance genes. The development of these NILs for RAPD marker analyses involved a sequence of line and mass selection during successive generations of inbreeding. DNA bulks derived from the NILs were used to identify a RAPD marker (designated OK14620, generated by 5'-CCCGCTACAC-3' decamer) that was tightly linked (2.23±1.33 centiMorgans) to an important rust [Uromyces appendiculatus (Pers.) Unger var. appendiculatus] resistance gene (Ur-3) in common bean (Phaseolus vulgaris L.). The efficiency of this approach was demonstrated by a low rate of false-positives identified, the tightness of the linkage identified, and the ability to detect polymorphism between genomic regions that are representative of the same gene pool of common bean. This method of deriving NILs should find application by researchers interested in utilizing marker-assisted selection for one or more major pest resistance genes. The identification of OK14620 should help to facilitate continued use of the Ur-3 resistance source and will now enable marker-assisted pyramiding of three different bean rust resistance sources (two previously tagged) to provide effective and stable resistance to this important pathogen.