IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.     

Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents

Hexaploid tritordeum (H^chH^chAABB; 2n?=?42) results from the cross between Hordeum chilense (H^chH^ch; 2n?=?14) and cultivated durum wheat (Triticum turgidum ssp. durum (AABB; 2n?=?28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70 % of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop.     

Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621

Imidazolinone herbicides resistant varieties, induced by mutations at the AHAS gene (acetohydroxyacid synthase), have been developed in many crops. Hexaploid tritordeum (Tritordeum Asch. & Graebn.) is the amphiploid derived from the cross between Hordeum chilense (H^chH^ch) and durum wheat Triticum turgidum L. (Thell) (AABB). Tritordeums have the potential to become a new crop with high added-value for food or feed. Mutagenesis with EMS was conducted to obtain imidazolinone resistant lines derived of the tritordeum HT621. Eleven M₃ plants were selected after imidazolinone treatment and five descendants of two of these lines (HT621-M3R1-3 and HT621-M3R10-1) were analyzed at the molecular level. Partial sequences of the three homologous AHAS loci in genomes A, B, and H^ch were obtained as well as those of HT621. A partial sequence of the AHAS gene in Hordeum chilense is first described in this work, and the designation ahasL-H ^ch 1 is proposed. A single Ser-Asn₆₂₇ substitution at the AHAS locus in the B genome is responsible of resistance in both lines. We propose the name AhasL-B2 for this resistance allele. This is the first report of the selection of imidazolinone resistant lines of tritordeum and the molecular characterization of the mutation conferring this resistance.     

Effects of reciprocal crosses on agronomic performance of tritordeum

Tritordeums (Tritordeum Ascherson et Graebner) are the amphiploids derived from the crosses between Hordeum chilense and durum or bread wheats. Primary tritordeums are obtained using H. chilense as female parent and therefore they exhibit H. chilense cytoplasm. The effect of wheat cytoplasm on agronomic performance of tritordeums was investigated. We developed four pairs of reciprocal F₁ lines only differing in their cytoplasm, donated from wheat or H. chilense alternatively. The agronomic performance of reciprocal F₁ lines contrasting for their cytoplasm was evaluated. The following traits were assessed: leave and tillers number one month after sowing, plant height, anthesis date, total number of ears, number of spikelets per spike, fertility of the main spike, length and wide of the flag leaf in the main stem and thousand kernel weight. Reciprocal F₁ lines did not differ for any of the agronomic traits evaluated with the exception of anthesis date in the pair THC1726/HTC1727. Therefore, both wheat and H. chilense cytoplasms can be used in tritordeum breeding. The text was submitted by the authors in English.     

Variation in the high-molecular-weight glutenin subunits coded at the Glu-Hch1 locus in Hordeum chilense

Hordeum chilense Roem. et Schult. is a native South American diploid wild barley included in the section Anisolepis Nevski. H. chilense occurs exclusively in Chile and Argentina and has been used in the synthesis of a new amphiploid named tritordeum (×Tritordeum Ascherson et Graebner). The HMW glutenin subunits of H. chilense have a great influence on gluten strength of tritordeum. The variability of these proteins has been analysed electrophoretically, and up to ten allelic variants have been detected in a world collection of this species. This genetic variability has been included in 121 lines of tritordeum and could be used for widening the genetic basis of tritordeum and wheat. Received: 22 March 2000 / Accepted: 14 April 2000     

Organic nitrogen content and nitrate and nitrite reductase activities in tritordeum and wheat grown under nitrate or ammonium

Tritordeum is a fertile amphiploid derived from durum wheat (Triticum turgidum L. conv. durum) × a wild barley (Hordeum chilense Roem. et Schultz.). The organic nitrogen content of tritordeum grain (34 mg g^-1 DW) was significantly higher than that of its wheat parent (25 mg g^-1 DW). Leaf and root nitrogen content became higher in tritordeum than in wheat after four weeks of growth, independently of the nitrogen source (either NO₃ ^- or NH₄ ⁺). Under NO₃ ^- nutrition, tritordeum generally exhibited higher levels of nitrate reductase (NR) activity than wheat. Nitrite reductase (NiR) levels were however lower in tritordeum than in its wheat parent. In NH₄ ⁺-grown plants, both NR and NiR activities progressively decreased in the two species, becoming imperceptible after 3 to 5 weeks of growth. Results indicate that, in addition to a higher rate of NO₃ ^- reduction, other physiological factors must be responsible for the greater accumulation of organic nitrogen in tritordeum grain.     

Allelic variation of the D-prolamin subunits encoded at the Hch genome in a collection of primary hexaploid tritordeums

 Hexaploid tritordeum is the amphiploid derived from the cross between Hordeum chilense and durum wheat. The storage proteins synthesised by the H^ch genome have an influence on the gluten strength of this amphiploid. The D-prolamins of H. chilense are glutenin-like proteins. The variability has been analysed electrophoretically and up to 20 different patterns have been detected in a world collection of this species. This genetic variability of H. chilense could be a source of additional variation in tritordeum and in breeding wheat for quality. Received: 29 November 1998 / Accepted: 19 December 1998     

Ear and flag leaf photosynthesis of awned and awnless Triticum species

The net photosynthetic rates of ears and flag leaves of awned and awnless wheats were measured by infra-red gas analysis in a controlled environment experiment. At 107 W m^-2 the rates of net photosynthesis of the ears of two awned Triticum aestivum lines were two to three times greater than those of their isogenic awnless counterparts. The net rates of photosynthesis of the flag leaves of all four lines were, however, similar. Net photosynthetic rates of ears of T. durum and T. turgidum, which have much larger awns, were considerably higher than those of T. aestivum. Between 70 and 90% of carbon-14 assimilated by the flag leaves and by the ears of T. aestivum was translocated to the grains, with no effect of awns in this respect. In a glasshouse experiment with detached shoots the contributions of the component organs to gross photosynthesis were determined by carbon-14 labelling. Ears of awnless lines of T. aestivum contributed about 10% to the photosynthesis of the organs above the node of the penultimate leaf and awns increased this to about 18%. Fixation by awned ears of T. durum and T. turgidum was 21–29% and of this 78–86% was contributed by the awns. In both experiments the contribution of the flag leaf to shoot photosynthesis was greater in T. aestivum than in the other species.     

Photoperiod and Vernalization Responses in Triticum turgidumxT. tauschii Synthetic Hexaploid Wheats

Studies of synthetic hexaploid wheat developed from Triticumturgidum(AABB genomes) and T. tauschii(DD genome) can provideinformation on potentially useful characters in T. tauschiiand/or T. turgidum for genetic improvement of hexaploid wheat(T. aestivum). Synthetic hexaploid wheats and the T. turgidumand T. tauschii parents were assessed for their developmentalresponses to photoperiod and vernalization for days to ear emergence,final leaf number and the number of spikelets per spike. Theresponses to photoperiod and vernalization of the synthetichexaploids were generally intermediate between those of theparents but in some instances the levels of expression exhibitedby the T. tauschii or T. turgidum parents were epistatic inthe synthetic hexaploids. The relatively strong photoperiodresponse of the T. tauschii accessions was not expressed inthe synthetic hexaploids, but rather the synthetic hexaploidsreflected the photoperiod response of the respective T. turgidumparents. The synthetic hexaploids had vernalization responsesstronger than those of the T. turgidum and bread wheats usedin the study. The expression of ear emergence in response tovernalization of these synthetic hexaploids appeared to be modifiedby the T. turgidum parent. Copyright 2001 Annals of Botany Company Photoperiod, synthetic hexaploids, Triticum aestivum, Triticum tauschii, Triticum turgidum, vernalization     

Leaf development and phenology of Triticum aestivum and T. durum under different moisture regimes

Wheat grain yield production in the rain-fed areas is limited by water deficits during crop growth. A greenhouse experiment was conducted during spring 1992 at ICARDA, Tel Hadya, Syria, with eight genotypes representing two Triticum species (Triticum turgidum var. durum and Triticum aestivum L.) under four soil-moisture regimes (95%, 75%, 55%, and 35% field capacity) to study the effect of water deficit on leaf development. The phyllochron was similar in the two species across the watering regimes. The range in variation in phyllochron among the genotypes was similar in the two species. Phyllochron response to water stress among genotypes was distinct in the driest regime in both species. Cham 6 (T. aestivum) and Gallareta (T. turgidum var. durum) had similar phyllochron across all moisture regimes whereas in other genotypes phyllochron was higher in the dries regime. Leaf area decreased with increasing moisture stress. Triticum turgidum var. durum genotypes were later in flowering as they had, on average, one leaf more than Triticum aestivum genotypes with similar leaf appearance rates.     

Subunits of tetrameric α-amylase inhibitors of Hordeum chilense are encoded by genes located in chromosomes 4Hch and 7Hch

Summary Three proteins (components 1, 2, and 4) of the non-prolamin, 70% ethanol soluble fraction from the endosperm of Hordeum chilense have been identified as putative subunits of the tetrameric inhibitors active against insect -amylases. In experiments carried out with the synthetic alloploid Tritordeum (H. chilense x Triticum turgidum conv. durum), previously described proteins from T. turgidum, designated CM2, CM3 and CM 16, have been also identified as subunits of -amylase inhibitors. Genes for components 1 and 4 of H. chilense have been located in chromosomes 4H^ch and 7H^ch, based on the analysis of H. chilense-T.turgidum addition lines. Subunits of the inhibitors from wheat and from cultivated barley had been previously assigned to chromosomes of the same homoeology groups.     

Chromosomal location of structural genes controlling isozymes in Hordeum chilense

Summary Polyacrylamide and starch gel electrophoresis of esterase (EST), glutamate oxaloacetate transaminase (GOT) and phosphoglucomutase (PGM) isozymes in Hordeum chilense, Triticum turgidum conv. durum, the amphiploid H. chilense X T. turgidum (Tritordeum), and the durum wheat/H. chilense monosomic addition lines revealed the chromosomal location of one EST locus, two GOT loci and one PGM locus. Loci Est-H ^ch1 and Got-H ^ch2 were found on chromosome 6H^ch,Got-H ^ch3 on chromosome 3H^ch, and Pgm-H ^ch1 on chromosome 4H^ch. These results lend evidence for the assumed homoeology relationships between chromosomes of Triticeae species.     

A core genetic map of Hordeum chilense and comparisons with maps of barley (Hordeum vulgare) and wheat (Triticum aestivum)

The first genetic map of the wild South Ameri- can barley species Hordeum chilense is presented. The map, based on an F₂ population of 114 plants, contains 123 markers, including 82 RAPDs, 13 SSRs, 16 RFLPs, four SCARs, two seed storage proteins and two STS markers. The map spans 694 cM with an average distance of 5.7 cM between markers. Six additional SSRs and seven additional SCARs which were not polymorphic were assigned to chromosomes using wheat/H. chilense addition lines. Polymorphisms were revealed by 50% of the RAPD amplifications, 13% of wheat and barley SSR primers, and 78% of the Gramineae RFLP anchor probes. The utility of SSR and RFLP probes from other Gramineae species shows the usefulness of a comparative approach as a source of markers and for aligning the genetic map of H. chilense with other species. This also indicates that the overall structure of the H. chilense linkage groups is probably similar to that of the B and D genomes of wheat and the H genome of barley. Applications of the map for tritordeum and wheat breeding are discussed. Received: 20 August 2000 / Accepted: 22 September 2000     

Linkage relationships between prolamin genes located on chromosome 1H<Superscript>ch</Superscript> in<Emphasis Type="Italic"> Hordeum chilense</Emphasis>

The endosperm storage proteins of Hordeum chilense Roem. et Schult., a species used in the synthesis of the amphiploid tritordeum (×Tritordeum Ascherson et Graebner), have a great effect on the gluten strength of this amphiploid. We have analysed electrophoretically the heredity of these proteins, which are synthesised by genes located on chromosome 1H^ch, and detected up to five loci in a cross between two lines of H. chilense. These loci present a certain homology with loci synthesising the same proteins in wheat. The genetic distances between these loci were calculated.Communicated by H.F. Linskens     

Influence of Vernalization and Photoperiod on Supernumerary Spikelet Expression in Wheat

Two tetraploid (Triticum turgidum L.emend gr. turgidum and gr.durum) and five hexaploid wheats (Triticum x aestivum L. emendgr. aestivum) with reported tendencies for ‘branched heads’(supernurnerary spikelets) exhibited variation in its expressionunder different vernalization photoperiod and temperature regimes. Two main types of supernumerary spikelets were identified, multiplesessile spikelets (MSS) with two or more complete spikeletsat a rachis node and indeterminate rachilla spikelets (IRS)with two to 13 spikelets on an extended rachilla. The degree of supernumerary spikelet expression in wheats withvernalization response differed from those without. Short photoperiods(9–14 h) both outdoors and in a glasshouse environment,were more conducive to supernumerary spikelet expression than24 h photoperiod in both environments. The 24 h photoperiodglasshouse environment (higher mean temperatures) was leastconducive to its expression except in lines with a strong vernalizationresponse. The high stability of supernumerary spikelet expression in certaingenotypes in the different environments indicated the feasibilityof incorporating this character in breeding and selecting commercialwheats to increase grain number per head. Triticum, wheat, ear-branching, supernumerary spikelets, vernalization, photoperiod, temperature     

Grain protein and grain yield of tritordeum in comparison to wheat and triticale

The new species of cereal × Tritordeum Ascherson et Graebner (Hordeum chilense Roem. et Shultz × Triticum ssp.) has a grain protein concentration (GPC) of up to 25%. The relationship between GPC and yield, and the factors responsible for the high GPC of tritordeum were examined and compared in field experiments. Three experimental tritordeum lines, two early and a later released (recombined and secondary tritordeums) were compared to wheat (cv. Cajeme) and triticale cultivars (cv. Trujillo). GPC's were 19%–22% for recombined tritordeums, 16% for the secondary tritordeum, 12–15% for wheats and 11% for triticale. Grain yields of the recombined and secondary tridordeum were 17–33% and 45–57% that of the wheats and triticale, respectively. Reducing grain sink size by spikelet removal resulted in an increased GPC of remaining grains. Considering all species together there were a strong inverse relationship between GPC and grain yield (GY) per main ear (GPC=26–4.76 ln GY; r²=0.82). In another experiment, frost damage to an early sown treatment of wheat reduced sink size. Harvest index (HI) of early sown wheat was reduced from 0.45 to 0.19, values comparable to that of tritordeum. Having similar HI, the GPC of the early sown wheat was the same as an early sown tritordeum (around 18%). Data for total N uptake and the N concentration of plant tissue during the growing season indicated that enhanced N uptake and remobilisation were not responsible for tritordeum's high GPC. These results suggest that the high GPC of the early lines of tritordeum is a consequence of the small grain yield concentrating the grain protein.     

Structural chromosome differentiation between Triticum timopheevii and T. turgidum and T. aestivum

 Chromosome pairing at metaphase-I was analyzed in F₁ hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (A^tA^tGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AA^tBG, AA^tBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AA^tBGD hybrids. T. timopheevii chromosomes 1A^t, 2A^t, 5A^t, 7A^t, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6A^tS/1GS, 1GS/4GS, 4GS/4A^tL, and 4A^tL/3A^tL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats. Received: 15 June 1998 / Accepted: 19 August 1998     

Fertile transgenic plants obtained from tritordeum inflorescences by tissue electroporation

 Tissue electroporation was applied to a member of the Triticeae family, namely tritordeum (Hordeum chilense Roem.×Triticum turgidum L. Conv. durum), for the generation of fertile transgenic plants. Two transgenic plants were recovered following the treatment of 361 explants of immature inflorescences (although they were subsequently found to result from the same transformation event). The expression of both inserted marker genes (uidA and bar) was confirmed using standard assays, while transgene integration was confirmed using PCR and Southern hybridization analyses. Integration pattern, segregation ratio and the inheritance of transgene expression in T₁ progeny were consistent for the presence of a single transgene locus containing five to ten plasmid insertions. Although this procedure has been applied to other cereal species, stable transformation of the Triticeae using tissue electroporation has not previously been reported. Received: 28 October 1999 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Solute adjustments in leaves of two species of wheat at two different stages of growth in response to salinity

Changes in leaf solute concentrations in response to salinity were measured at two growth stages in two species of wheat, Triticum turgidum L. cv. Aldura (Durum group) and Triticum aestivum L., cv. Probred that differed in their salt tolerances. Both species at 55 days of age were Na⁺-excluders, but the concentration of Na⁺ was 10 times higher in T. turgidum than T. aestivum at low to moderate levels of stress. The ratio then decreased until it was 2:1 at – 1.2 MPa. In T. turgidum, K⁺ concentrations decreased with increasing Na⁺ concentrations so that the sum of the two cations remained constant at all stress levels, but in T. aestivum K⁺ decreased more rapidly than Na⁺ increased. In both species growing in media at 0 to –0.6 MPa, the amounts of Mg²⁺ and Ca²⁺ in 55-day-old plants that could be extracted with hot water were below 0.1 mmol (g dry weight)^?1. Then, as osmotic potentials of media decreased further, hot water-extractable Ca²⁺ increased greatly until, at – 1.2 MPa, Ca²⁺ concentrations were almost equal to the sum of Na⁺ and K⁺. In the range of 0 to –1.0 MPa, the ratio of Cl^? to total cationic charge remained constant at 1:6 in T. aestivum and 1:2 in T. turgidum. However, at – 1.2 MPa, the ratio in both species had changed to 2:3. Sucrose and betaine concentrations were 4 and 48 μmol (g dry weight)^?1, respectively, in non-stressed plants of both species. At – 1.2 MPa, sucrose had increased 30-fold but betaine had increased only 2.5-fold. Proline increased exponentially relative to foliar Na⁺ in T. turgidum. In T. aestivum only plants grown at –1.2 MPa contained sufficient Na⁺ to stimulate the accumulation of proline. Although the quantities of the solutes in leaves of non-stressed 96-day-old plants differed from those in non-stressed younger plants, the patterns of change of organic solutes as the older plants were subjected to increasing saline stresses were the same as in younger plants with the exception of sucrose. Sucrose concentrations were much higher in leaves of non-stressed older plants and this sugar first increased and then decreased with decreasing osmotic potentials of media.     

Inhibitory activities against heterologous α-amylases and in vitro allergenic reactivity of Einkorn wheats

Salt extracts from seeds of 36 lines of Einkorn wheats were analyzed for their inhibitory activity towards two insect (Tenebrio molitor, Coleoptera, and Ephestia kuehniella, Lepidoptera) and one mammalian (human salivary) -amylases. Whereas all ten T. monococcum accessions tested were active towards the lepidopteran enzyme, they had no effect on the coleopteran or the mammalian ones. More variability was found among the 21 lines of T. boeticum analyzed, although none of them inhibited human -amylase. The five accessions of T. urartu showed even greater diversity. Among all Einkorn accessions tested, only two urartu lines affected the three -amylases. These lines displayed inhibition patterns similar to those of T. aestivum and T. turgidum cultivars. Since several breadwheat -amylase inhibitors are major allergens associated with baker's asthma, we also studied the in vitro allergenic activity of salt extracts from the Einkorn wheats under study. No significant differences in IgE-binding were found between these accessions and theT. aestivum or T. turgidum cultivars. Furthermore, putative allergens with molecular sizes in the range of 20–60 kDa were detected in these Einkorn wheats.