Detailed mapping of the species cytoplasm-specific (scs) gene in durum wheat

The compatibility-inducing action of the scs(ti) (species cytoplasm-specific gene derived from Triticum timopheevii) and Vi (vitality) genes can be observed when a durum (T. turgidum) nucleus is placed in T. longissimum cytoplasm. These two genes restore compatibility between an otherwise incompatible nucleus and cytoplasm. The objective of this study was to localize the scs(ti) gene on a linkage map of chromosome 1A, which could eventually be used to clone the gene. The mapping population consisted of 110 F2 individuals derived from crossing a Langdon-T. dicoccoides chromosome 1A substitution line with a euplasmic (normal cytoplasm) line homozygous for the scs(ti) gene. Through a series of testcrosses the genotypes of the 110 individuals were determined: 22 had two copies, 59 had one copy, and 29 had no copy of the scs(ti) gene. Data from RFLP, AFLP, and microsatellite analysis were used to create a linkage map. The flanking marker loci found for the scs(ti) gene were Xbcd12 and Xbcd1449-1A.2 with distances of 2.3 and 0.6 cM, respectively. Nearly 10% of individuals in this population were double recombinant for a genetic interval of <3 cM. A blistering phenotype reminiscent of the phenotype observed in maize brittle-1 mutable was also evident in these individuals. The higher frequency of double recombination within this region and seed-blistering phenotype could be an indication of a transposable element(s) in this locus.     

Interactions between the scs and Vi genes in alloplasmic durum wheat.

Two nuclear genes, vitality (Vi) on an A- or B-genome chromosome and species cytoplasm specific (scs) on a 1DL telosome from Triticum aestivum L. or a telosome from Aegilops uniaristata Vis. (un telosome), improved compatibility between the nucleus of Triticum turgidum L. var. durum and the cytoplasm of Ae. longissima S. &M. or Ae. uniaristata. To study interactions between Vi and scs and to determine the chromosomal location of Vi, 29-chromosome fertile plants were crossed with 13 D-genome disomic-substitution (d-sub) lines [except 5D(5A)] of 'Langdon' durum. F1 and backcross progenies were examined for meiotic chromosome number and pairing, fertility, and plant vigor. In 11 crosses, Vi restored seed viability but produced double-monosomics (d-monos) with greatly reduced growth and vigor. In contrast, crosses involving 1D(1A) and 1D(1B) d-sub lines produced d-monos with normal vigor and anthesis but nonfunctional pollen. A backcross of 1D + 1A d-mono F1 and 1D(1A) d-sub lines produced 11 male steriles; 3 had 13 II + 1 II 1D + 1 I 1A, 2 had 13 II + 2 I, 1 had 13 II + 1 II 1D(1A), and 5 were not examined. Crosses of 1D + 1A d-mono F1 with control durum, lo durum (with 1DL), and un durum (with un telosome) lines produced 16 male-sterile d-monos and 14 fertiles with 14 II + 1 I 1D, showing that 15-chromosome female gametes transmitted monosomes 1A and 1D. However, BC2F1's from 1D + 1B d-mono x fertile line with un telosome included 20 male-sterile d-monos, 6 fertile triple monosomics (13 II + 1 I 1D + 1 I 1B + t I un telosome), and 1 fertile plant with a 1B/1D translocation. Unlike d-mono 1A + 1D, d-mono 1B + 1D did not transmit 15-chromosome female gametes with monosomes 1D and 1B. Additional backcrosses also indicated that homozygous scs caused male sterility in 1D(1A) and 1D(1B) d-subs and that the procedure used was not suitable for the chromosomal location of Vi.     

Molecular cytogenetic characterization of an alloplasmic durum wheat line with a portion of chromosome 1D of Triticum aestivum carrying the scs(ae) gene.

Triticum aestivum (2n = 6x = 42, AABBDD) with Triticum longissimum (2n = 2x = 14; S1S1) cytoplasm ((lo) cytoplasm) has normal fertility and plant vigor. However, the nucleus of durum wheat (Triticum turgidum (2n = 4x = 28, AABB)) is incompatible with the T. longissimum cytoplasm, producing non-viable progeny. This incompatibility is alleviated by scs(ae), a species cytoplasm-specific (scs) gene, on the long arm of chromosome 1D (1DL) of common wheat. The hemizygous (lo) durum scs(ae) line is male sterile and is maintained by crossing to normal durum wheat. After pollination, the seeds produced are either plump and viable (with scs(ae)) or shriveled and inviable (without scsae). Thus, the chromosome with scs(ae) is inherited as a whole without recombination. The objectives of this study were to characterize the chromosome carrying scs(ae) and to determine the process through which this gene was introgressed into the (lo) durum background. Molecular marker analysis with 27 probes and primers mapped to homoeologous group 1 and genomic in situ hybridization using differentially labeled total genomic DNA of durum wheat and Aegilops tauschii suggest the presence of a 1AL segment in place of the distal region of 1DL. Owing to the absence of any detectable duplications or deletions, homoeologous recombination is the most likely mechanism by which this introgression occurred.     

Genetic mapping of the gene affecting polyphenol oxidase activity in tetraploid durum wheat

The quality of durum wheat (Triticum turgidum ssp. durum) is influenced by polyphenol oxidase(PPO) activity and its corresponding substrates. A saturated molecular-marker linkage map was constructed previously by using a set of recombinant inbred (RI) lines, derived from a cross between durum wheat cultivars Jennah Khetifa and Cham 1. Quantitative trait loci (QTL) for PPO activity in seeds were mapped in this population. PPO activity in seeds of the parents and 110 RI lines was measured spectrophotometrically. The PPO activity of Cham 1 was significantly lower than that of Jennah Khetifa. QTL analysis of these data indicated that most of PPO activity was associated with major loci on the long arm of chromosome 2A. The trait was found to be strongly associated with the SSR marker Xgwm312@2A. With this knowledge, marker-assisted selection can be used to select genotypes with lower PPO activity in durum wheat populations.     

Genetic analysis of durable resistance against leaf rust in durum wheat

The Italian durum wheat cultivar Creso possesses a high level of durable resistance to leaf rust based on both hypersensitive and non-hypersensitive components. In order to investigate the genetic basis of this resistance, a segregating population composed of 123 recombinant inbred lines (RILs) derived from the cross Creso × Pedroso, was evaluated for disease severity in adult plants under field conditions. Furthermore, the resistance of parents and RILs was evaluated by assessing macroscopically the latency period and microscopically the number and type of pathogen colonies formed following artificial inoculation with a specific isolate. This experiment was performed at controlled conditions at two developmental stages. Besides some minor QTLs, one major QTL explaining both reduction of disease severity in the field and increased latency period was found on the long arm of chromosome 7B, and closely associated PCR-based and DArT markers were identified. Daniela Marone and Ana I. Del Olmo contributed equally to the work.     

RAPD markers linked to the nuclear gene from Triticum timopheevii that confers compatibility with Aegilops squarrosa cytoplasm on alloplasmic durum wheat.

Alien cytoplasms cause a wide range of phenotypic alterations in the nucleus-cytoplasm (NC) hybrids in the Triticeae. Nuclear genomes of timopheevii wheat (Triticum timopheevii and Triticum araraticum) are fully compatible with the cytoplasm of Aegilops squarrosa, while those of a majority of emmer or durum wheat cultivars and more than half the wild emmer wheats are incompatible, and a maternal 1D chromosome is required to restore seed viability and male fertility in the NC hybrids. A euploid NC hybrid of Triticum durum cv. Langdon with Ae. squarrosa cytoplasm produced by introgressing the NC compatibility (Ncc) gene from T. timopheevii was used to identify random amplified polymorphic DNA (RAPD) markers linked to it. After a survey of 200 random decamer primers, four markers were selected, all of which were completely linked in 64 individuals of a SB8 mapping population. One marker was derived from a single locus, while three others were from interspersed repetitive sequences. Also, the hybrid chromosomes and those of the parental T. durum had identical C-banding patterns. RAPD-PCR analysis of 65 accessions from wild and cultivated tetraploid wheat species showed the exclusive presence of the markers in timopheevii wheat. In conclusion, the chromosomal region flanking Ncc of T. timopheevii is highly conserved in the genome of this group of tetraploid wheats.     

Plant regeneration from protoplasts of durum wheat (Triticum durum Desf. cv. D6962)

Suspension cultures of durum wheat were established from embryogenic callus maintained in liquid medium for 30 months. Protoplasts were readily isolated from the suspension cultures with yields as high as 3 X 10⁷ protoplasts per g fresh weight suspension cells. When incubated in a modified MS medium containing half strength of the macroelements, 5 M 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.6 M glucose, protoplast-derived cells divided at frequencies ranging from 1.4 to 10.0 %. After transfer to a solid subculture medium, the protoplast-derived colonies formed embryogenic protuberances, from which green plants have been regenerated.     

High molecular weight glutenin subunit in durum wheat (T. durum)

Summary The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.     

The inheritance of fertility restoration in male-sterile wheat carrying cytoplasm derived from Triticum timopheevi

Summary The inheritance of the restoration of fertility in material carrying the cytoplasm of Triticum timopheevi was studied in F₂, F₃ and F₄ generations. In the material used the segregation of restoration was shown to fit the hypothesis of three major, dominant, partially dominant or additive genes each of which made a different contribution to restoration but which acted cumulatively to produce the phenotypic expression observed.From the material it was possible to extract homozygous lines carrying known combinations of these three genes which can be used as tester lines to investigate the inheritance of genes for restoration derived from other sources.     

Ethylene production and involvement during the first steps of durum wheat (Triticum durum) anther culture

The role of ethylene in anther culture of durum wheat ( Triticum durum Desf. cv. Ardente) was analyzed by testing the effects of 2-chloroethylphosphonic acid (ethrel) silver thiosulfate (Ag⁺), a -aminooxyacetic acid (AOA) and 1-aminocyclopropane-l-carboxylic acid (ACC) on microspore division observed after 21 days of culture and on development of calli estimated at day 45. The use of ethrel and Ag⁺ indicated a positive effect of ethylene on microspore division, whereas the use of AOA, and to a lesser extent ACC, snowed a negative effect. In contrast, the addition of ethrel or Ag⁺ indicated that ethylene inhibits the development of microspore-derived calli. AOA gave contradictory results. Ethylene production by anthers was about 7 pl anther⁻¹h⁻¹ and decreased during culture. ACC content in the anthers was maximal at day 9, whereas malonyl ACC (MACC) increased sharply from day 0 to day 3 and then decreased. The addition of AOA or ACC to the culture medium decreased or increased, respectively, ethylene production of anthers and the ACC and/or MACC content, but at concentrations higher than those that modified the formation of calli. This formation seems to occur in two successive phases: induction and initiation of microspore division, which was promoted by ethylene, followed by callus development, which was inhibited by ethylene.     

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP⁺-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.     

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm

Alterations of mitochondrial-encoded subunits of the F_oF₁-ATPsynthase are frequently associated with cytoplasmic male sterility(CMS) in plants; however, little is known about the relationshipof the nuclear encoded subunits of this enzyme with CMS. Inthe present study, the full cDNA of the gene TaF_Ad that encodesthe putative F_Ad subunit of the F_oF₁-ATP synthase was isolatedfrom the wheat (Triticum aestivum) fertility restorer ‘2114’for timopheevii cytoplasm-based CMS. The deduced 238 amino acidpolypeptide is highly similar to its counterparts in dicotsand other monocots but has low homology to its mammalian equivalents.TaF_Ad is a single copy gene in wheat and maps to the short armof the group 6 chromosomes. Transient expression of the TaF_Ad–GFPfusion in onion epidermal cells demonstrated TaF_Ad's mitochondriallocation. TaF_Ad was expressed abundantly in stem, leaf, anther,and ovary tissues of 2114. Nevertheless, its expression wasrepressed in anthers of CMS plants with timopheevii cytoplasm.Genic male sterility did not affect its expression in anthers.The expression of the nuclear gene encoding the 20 kDa subunitof F_o was down-regulated in a manner similar to TaF_Ad in theT-CMS anthers while that of genes encoding the 6 kDa subunitof F_o and the subunit of F₁ was unaffected. These observationsimplied that TaF_Ad is under mitochondrial retrograde regulationin the anthers of CMS plants with timopheevii cytoplasm. Key words: CMS, F_Ad subunit, F_oF₁-ATP synthase, retrograde regulation, wheat Received 8 October 2007; Revised 9 January 2008 Accepted 28 January 2008     

Genetic diversity of modern Russian durum wheat cultivars at the gliadin-coding loci

The allelic diversity at four gliadin-coding loci was studied in modern cultivars of the spring and winter durum wheat Triticum durum Desf. Comparative analysis of the allelic diversity showed that the gene pools of these two types of durum wheat, having different life styles, were considerably different. For the modern spring durum wheat cultivars, a certain reduction of the genetic diversity was observed compared to the cultivars bred in the 20th century.     

Genetic dissection of seminal root architecture in elite durum wheat germplasm

Although root architecture has been shown to play an important role in crop performance, particularly under drought conditions, no information is available on the genetic control of root traits in durum wheat, a crop largely grown in rainfed areas with low rainfall. In our study, a panel of 57 elite durum wheat accessions were evaluated under controlled conditions for root and shoot traits at the seedling stage. Significant genetic variability was detected for all the root and shoot traits that were investigated. Correlation analysis suggested that root and shoot features were only partially controlled by common sets of genes. The high linkage disequilibrium (up to 5 cM) present in the germplasm collection herein considered allowed us to use simple sequence repeat‐based association mapping to identify chromosome regions with significant effects on the investigated traits. In total, 15 chromosome regions showed significant effects on one or more root architectural features. A number of these regions also influenced shoot traits and, in some cases, plant height measured in field conditions. Major effects were detected on chromosome arms 2AL (at Xgwm294), 7AL (at Xcfa2257 and Xgwm332) and 7BL (at Xgwm577 and Xcfa2040). The accessions with the most remarkable differences in root features will provide a valuable opportunity to assemble durum wheat mapping populations well suited for ascertaining the effects of root architecture on water use efficiency and grain yield.     

The effect of specific chromosome and cytoplasm substitutions on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of four lines of hexaploid wheat (Triticum aestivum L. em. Thell), the euplasmic nuclear donor Chinese Spring, Chinese Spring in which both 4B chromosomes were substituted by those of the variety Cappelle-Desprez and two alloplasmic lines in which these nuclei were substituted into the cytoplasm of Aegilops ovata. The calli were found to differ in their initia growth rates and their ability to organise shoot primordia and regenerate shoots. The Cappelle 4B chromosomes had a very significant effect on all these characters. The potential for modelling genotypes for improved tissue culture characteristics is discussed.     

Radiation hybrid mapping of the species cytoplasm-specific (scsae) gene in wheat

Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage and analysis of chromosome segment retention or loss using molecular markers. In durum wheat (Triticum turgidum L., AABB), an alloplasmic durum line [(lo) durum] has been identified with chromosome 1D of T. aestivum L. (AABBDD) carrying the species cytoplasm-specific (scsae) gene. The chromosome 1D of this line segregates as a whole without recombination, precluding the use of conventional genome mapping. A radiation hybrid mapping population was developed from a hemizygous (lo) scsae--line using 35 krad gamma rays. The analysis of 87 individuals of this population with 39 molecular markers mapped on chromosome 1D revealed 88 radiation-induced breaks in this chromosome. This number of chromosome 1D breaks is eight times higher than the number of previously identified breaks and should result in a 10-fold increase in mapping resolution compared to what was previously possible. The analysis of molecular marker retention in our radiation hybrid mapping panel allowed the localization of scsae and 8 linked markers on the long arm of chromosome 1D. This constitutes the first report of using RH mapping to localize a gene in wheat and illustrates that this approach is feasible in a species with a large complex genome.     

Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2‐D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2‐ and 2.2‐fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI‐TOF and MALDI‐TOF‐TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress‐related proteins. Many of the heat‐induced polypeptides are considered to be allergenic for sensitive individuals.     

A PCR-marker for the CMS(1) inducing cytoplasm in chives derived from recombination events affecting the mitochondrial gene atp9

The complete coding and 3′-flanking region of the mitochondrial gene atp9 of chives (Allium schoenoprasum L.) was determined in order to develop primers that allow the identification of atp9-related sequences in subsequent PCR-amplifications. One of these sequences is of a chimerical nature, consisting of atp9-homologous regions on its end, interrupted by an insertion that is composed of one atp6-homologous part and one part of unknown origin. This PCR-fragment is 762 bp in size and exclusively amplified in the sterility inducing cytoplasm of CMS₁. Thus it can be used as a PCR-marker in order to distinguish this cytoplasm type from the remaining cytoplasm types of chives. The chimerical marker sequence forms a putative open reading frame (orfA501), from which CMS₁ might originate. Received: 6 June 2001 / Accepted: 3 August 2001