Daryl L. Klindworth

Targeted Introgression of a Wheat Stem Rust Resistance Gene by DNA Marker-Assisted Chromosome Engineering

Chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of chromosome engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between wheat chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87–9.15% of the translocated chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of chromosome engineering in genome manipulation for plant improvement.

Chromosomal location of genes for novel glutenin subunits and gliadins in wild emmer wheat (Triticum turgidum L. var. dicoccoides)

The glutenin and gliadin proteins of wild emmer wheat, Triticum turgidum L. var. dicoccoides, have potential for improvement of durum wheat (T. turgidum L. var. durum) quality. The objective of this study was to determine the chromosomes controlling the high molecular weight (HMW) glutenin subunits and gliadin proteins present in three T. turgidum var. dicoccoides accessions (Israel-A, PI-481521, and PI-478742), which were used as chromosome donors in Langdon durum-T. turgidum var. dicoccoides (LDN-DIC) chromosome substitution lines. The three T. turgidum var. dicoccoides accessions, their respective LDN-DIC substitution lines, and a number of controls with known HMW glutenin subunits were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), urea/SDS-PAGE, and acid polyacrylamide gel electrophoresis (A-PAGE). The results revealed that all three T. turgidum var. dicoccoides accessions possess Glu-A1 alleles that are the same as or similar to those reported previously. However, each T. turgidum var. dicoccoides accession had a unique Glu-B1 allele. PI-478742 had an unusual 1Bx subunit, which had mobility slightly slower than the 1Ax subunit in 12% SDS-PAGE gels. The subunits controlled by chromosome 1B of PI-481521 were slightly faster in mobility than the subunits of the Glu-B1n allele, and the 1By subunit was identified as band 8. The 1B subunits of Israel-A had similar mobility to subunits 14 and 16. The new Glu-B1 alleles were designated as Glu-B1be in Israel-A, Glu-B1bf in PI-481521, and Glu-B1bg in PI-478742. Results from A-PAGE revealed that PI-481521, PI-478742, and Israel-A had eight, 12, and nine unique gliadin bands, respectively, that were assigned to specific chromosomes. The identified glutenin subunits and gliadin proteins in the LDN-DIC substitution lines provide the basis for evaluating their effects on end-use quality, and they are also useful biochemical markers for identifying specific chromosomes or chromosome segments of T. turgidum var. dicoccoides.

Introgression and Characterization of a Goatgrass Gene for a High Level of Resistance to Ug99 Stem Rust in Tetraploid Wheat

The transfer of alien genes to crop plants using chromosome engineering has been attempted infrequently in tetraploid durum wheat (Triticum turgidum L. subsp. durum). Here, we report a highly efficient approach for the transfer of two genes conferring resistance to stem rust race Pgt-TTKSK (Ug99) from goatgrass (Aegilops speltoides) to tetraploid wheat. The durum line DAS15, carrying the stem rust resistance gene Sr47 derived from Ae. speltoides, was crossed, and backcrossed, to durum 5D(5B) aneuploids to induce homeologous pairing. After a final cross to ‘Rusty’ durum, allosyndetic recombinants were recovered. The Ae. speltoides chromosomal segment carrying Sr47 was found to have two stem rust resistance genes. One gene conditioning an infection type (IT) 2 was located in the same chromosomal region of 2BS as Sr39 and was assigned the temporary gene symbol SrAes7t. Based on ITs observed on a diverse set of rust races, SrAes7t may be the same as Sr39. The second gene conditioned an IT 0; and was located on chromosome arm 2BL. This gene retained the symbol Sr47 because it had a different IT and map location from other stem rust resistance genes derived from Ae. speltoides. Allosyndetic recombinant lines carrying each gene on minimal alien chromosomal segments were identified as were molecular markers distinguishing each alien segment. This study demonstrated that chromosome engineering of Ae. speltoides segments is feasible in tetraploid wheat. The Sr47 gene confers high-level and broad spectrum resistance to stem rust and should be very useful in efforts to control TTKSK.

Molecular cytogenetic characterization and seed storage protein analysis of 1A/1D translocation lines of durum wheat

Two durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husn.] lines carrying the high-molecular-weight (HMW) glutenin subunits (GS) 1Dx5 + 1Dy10 encoded by Glu-D1d, L252 and S99B34, were characterized using fluorescent genomic in-situ hybridization (FGISH) and microsatellite markers. These two durum lines were derived from the crosses in which the common wheat (T. aestivum L.) ‘Len’ and durum wheat ‘Langdon’ (LDN) and ‘Renville’ were involved. FGISH patterns of the mitotic chromosomes indicated that these two durum lines have one pair of 1AS·1AL-1DL translocated chromosomes in which the terminal region of 1AL was replaced by a homoeologous segment of 1DL. The 1DL segment spans approximately 31% of the long arm of the translocated chromosome. Microsatellite marker analysis confirmed the 1AS·1AL-1DL translocation and determined the translocation breakpoint to be distal to Xgwm357 on 1AL. Seed storage proteins (GS and gliadins) were analysed in these two 1AS·1AL-1DL translocation lines and three sib lines (L092, S99B19 and S99B33) using SDS-PAGE and A-PAGE. The SDS-PAGE and A-PAGE profiles demonstrated that the two low yielding lines (L252 and S99B19) had the low-molecular-weight (LMW) −1 GS encoded by Glu-A3k and Glu-B3s and 1B-encoded gliadins from LDN, and the other three lines (L092, S99B33 and S99B34) with higher yield had LMW-2 GS and 1B-encoded gliadins from Renville, suggesting that undesirable genetic components from LDN might limit substantial improvement of yield. Thus, the translocation lines with 1Dx5 + 1Dy10 and LMW-2, which are associated with good bread-making and pasta qualities, respectively, in a good genetic background will be useful for developing durum cultivars with dual-purpose end-use. Results from this study demonstrate that the D-genome could play an important role in the genetic improvement of durum wheat and evolution of the A- and B-genomes in tetraploid wheat.

Homoeology of Thinopyrum junceum and Elymus rectisetus chromosomes to wheat and disease resistance conferred by the Thinopyrum and Elymus chromosomes in wheat

Thirteen common wheat “Chinese Spring” (CS)-Thinopyrum junceum addition lines and three common wheat “Fukuhokomuji”(Fuku)-Elymus rectisetus addition lines were characterized and verified as disomic additions of a Th. junceum or E. rectisetus chromosome in the wheat backgrounds by fluorescent genomic in situ hybridization. Another Fuku-E. rectisetus addition line, A1048, was found to contain multiple segregating E. rectisetus chromosomes. Seven partial CS-Th. junceum amphiploids were identified to combine 12–16 Th. junceum chromosomes with CS wheat chromosomes. The disomic addition lines AJDAj5, 7, 8, 9, and HD3508 were identified to contain a Th. junceum chromosome in homoeologous group 1. Two of them, AJDAj7 and AJDAj9, had the same Th. junceum chromosome. AJDAj2, 3, and 4 contained a Th. junceum chromosome in group 2, HD3505 in group 4, AJDAj6 and AJDAj11 in group 5, and AJDAj1 probably in group 6. The disomic addition lines A1026 and A1057 were identified to carry an E. rectisetus chromosome in group 1 and A1034 in group 5. E. rectisetus chromosomes in groups 1–6 were detected in A1048. The homoeologous group of the Th. junceum chromosome in HD3515 could not be determined in this study. Several Th. junceum and E. rectisetus chromosomes in the addition lines were found to contain genes for resistance to Fusarium head blight, tan spot, Stagonospora nodorum blotch, and stem rust (Ug99 races). Understanding of the homoeology of the Th. junceum and E. rectisetus chromosomes with wheat will facilitate utilization of the favorable genes on these alien chromosomes in wheat improvement.

Characterization of a mitotic mutant of durum wheat.

An ethyl methanesulfonate-induced mitotic mutant of durum wheat (Triticum turgidum L. var. durum; 2n=4x=28) was found. We have characterized the mutant to determine the mechanism of abnormal cell division and to test for temperature effects on abnormal cell division. Stained root-tip meristems and pollen mother cells were studied with brightfield, phase contrast, and immunofluorescence microscopy. Abnormal cells included metaphase cells with a multiple of the normal complement (8x=56, or 16x=112), multinucleate cells, 4C, 8C, or 16C mononucleate cells, and cells exhibiting incomplete cytokinesis. The mutant had three classes of pollen mother cells: euploid with normal bivalent pairing, multiploid with bivalent pairing, and multiploid with multivalent pairing. Preprophase bands and spindles were normal in mononucleate cells. Some cells had asymmetrical phragmoplasts and phragmoplast dismantling that produced incomplete cytokinesis. Failure of cytokinesis followed by nuclear fusion were the mechanisms of abnormal cell division. To test for temperature sensitivity of the mutant, seedlings were germinated under six different temperature regimes. As germination temperature increased, the frequency of abnormal cells increased. When the mutant was crossed as the female with durum wheat, 3% of hybrids were hexaploid, indicating that functional–unreduced gametes had formed in megaspores.

Marker-assisted characterization of durum wheat Langdon–Golden Ball disomic substitution lines

The durum wheat cultivar ‘Golden Ball’ (GB) is a source of resistance to wheat sawfly due to its superior solid stem. In the late 1980s, Dr. Leonard Joppa developed a complete set of 14 ‘Langdon’ (LDN)–GB disomic substitution (DS) lines by using GB as the chromosome donor and LDN as the recipient. However, these substitution lines have not been previously characterized and reported in the literature. The objectives of this study were to confirm the authenticity of the substituted chromosomes and to analyze the genetic background of the 14 LDN–GB DS lines with the aid of molecular markers, and to further use the substitution lines for chromosomal localization of DNA markers and genes conferring the superior stem solidness in GB. Results from simple sequence repeat marker analysis validated the authenticity of the substituted chromosomes in 14 LDN–GB DS lines. Genome-wide scans using the target region amplification polymorphism (TRAP) marker system produced a total of 359 polymorphic fragments that were used to compare the genetic background of substitution lines with that of LDN. Among the polymorphic TRAP markers, 134 (37.3%) and 185 (51.5%) were present in LDN and GB, respectively, with only 10 (2.8%) derived from Chinese Spring. Therefore, marker analysis demonstrated that each LDN–GB DS line had a pair of chromosomes from GB with a genetic background similar to that of LDN. Of the TRAP markers generated in this study, 200 were successfully assigned to specific chromosomes based on their presence or absence in the corresponding LDN–GB DS lines. Also, evaluation of stem solidness in the substitution lines verified the presence of a major gene for stem solidness in chromosome 3B. Results from this research provides useful information for the utilization of GB and LDN–GB DS lines for genetic and genomic studies in tetraploid wheat and for the improvement of stem solidness in both durum and bread wheat.

Physical mapping of DNA markers linked to stem rust resistance gene Sr47 in durum wheat

Key messageMarkers linked to stem rust resistance geneSr47were physically mapped in three smallAegilops speltoideschromosomal bins. Five markers, including two PCR-based SNP markers, were validated for marker-assisted selection.AbstractIn durum wheat (Triticum turgidum subsp. durum), the gene Sr47 derived from Aegilopsspeltoides conditions resistance to race TTKSK (Ug99) of the stem rust pathogen (Puccinia graminis f. sp. tritici). Sr47 is carried on small interstitial translocation chromosomes (Ti2BL-2SL-2BL·2BS) in which the Ae. speltoides chromosome 2S segments are divided into four bins in genetic stocks RWG35, RWG36, and RWG37. Our objective was to physically map molecular markers to bins and to determine if any of the molecular markers would be useful in marker-assisted selection (MAS). Durum cultivar Joppa was used as the recurrent parent to produce three BC2F2 populations. Each BC2F2 plant was genotyped with markers to detect the segment carrying Sr47, and stem rust testing of BC2F3 progeny with race TTKSK confirmed the genotyping. Forty-nine markers from published sources, four new SSR markers, and five new STARP (semi-thermal asymmetric reverse PCR) markers, were evaluated in BC2F2 populations for assignment of markers to bins. Sr47 was mapped to bin 3 along with 13 markers. No markers were assigned to bin 1; however, 7 and 13 markers were assigned to bins 2 and 4, respectively. Markers Xrwgs38a, Xmag1729, Xwmc41, Xtnac3119, Xrwgsnp1, and Xrwgsnp4 were found to be useful for MAS of Sr47. However, STARP markers Xrwgsnp1 and Xrwgsnp4 can be used in gel-free systems, and are the preferred markers for high-throughput MAS. The physical mapping data from this study will also be useful for pyramiding Sr47 with other Sr genes on chromosome 2B.

Interactions of Genotype and Glutenin Subunit Composition on Breadmaking Quality of Durum 1AS•1AL-1DL Translocation Lines

ABSTRACT Dual-purpose durum (Triticum turgidum L. subsp. durum) wheat, having both good pasta and breadmaking quality, would be an advantage in the market. In this study, we evaluated the effects of genotype and varying HMW and LMW glutenin subunit composition on durum breadmaking quality. Genotypes included five near-isogenic backgrounds that also differed by variability at the Glu-D1d (HMW subunits 1Dx5+1Dy10), Glu-B1 (presence or absence of subunit 1By8), and Glu-B3 (LMWI or LMWII pattern) loci. Quality tests were conducted on genotypes grown at five North Dakota locations. Genotype had a stronger influence on free asparagine content than glutenin subunit composition. Genotypes carrying Glu-D1d had higher glutenin content than lines that did not carry Glu-D1d. Among Rugby translocation genotypes, lines carrying LMWI had higher gliadin content and better loaf volume than genotypes carrying LMWII. Absence of 1By8 produced major reductions in loaf volume in nontranslocation lines regardless of whether LMW...

Resistance to recombinant stem rust race TPPKC in hard red spring wheat

The wheat (Triticum aestivum L.) stem rust (Puccinia graminis Pers.:Pers. f.sp. tritici Eriks. and Henn.) resistance gene SrWld1 conditions resistance to all North American stem rust races and is an important gene in hard red spring (HRS) wheat cultivars. A sexually recombined race having virulence to SrWld1 was isolated in the 1980s. Our objective was to determine the genetics of resistance to the race. The recombinant race was tested with the set of stem rust differentials and with a set of 36 HRS and 6 durum cultivars. Chromosomal location studies in cultivars Len, Coteau, and Stoa were completed using aneuploid analysis, molecular markers, and allelism tests. Stem rust differential tests coded the race as TPPKC, indicating it differed from TPMKC by having added virulence on Sr30 as well as SrWld1. Genes effective against TPPKC were Sr6, Sr9a, Sr9b, Sr13, Sr24, Sr31, and Sr38. Genetic studies of resistance to TPPKC indicated that Len, Coteau, and Stoa likely carried Sr9b, that Coteau and Stoa carried Sr6, and Stoa carried Sr24. Tests of HRS and durum cultivars indicated that five HRS and one durum cultivar were susceptible to TPPKC. Susceptible HRS cultivars were postulated to have SrWld1 as their major stem rust resistance gene. Divide, the susceptible durum cultivar, was postulated to lack Sr13. We concluded that although TPPKC does not constitute a threat similar to TTKSK and its variants, some cultivars would be lost from production if TPPKC became established in the field.