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Key message GWAS on a bread wheat panel with high D genome diversity identified novel alleles and QTLs associated with resilience to combined heat and drought stress under natural field conditions. Abstract As heat (H) and drought stresses occur concurrently under field conditions, studying them separately offers limited opportunities for wheat improvement. Here, a wheat diversity panel containing Aegilops tauschii introgressions was evaluated under H and combined heat–drought (HD) stresses to identify quantitative trait loci (QTLs) associated with resilience to the stresses, and to assess the practicability of harnessing Ae. tauschii diversity for breeding for combined stress resilience. Using genome-wide analysis, we identified alleles and QTLs on chromosomes 3D, 5D, and 7A controlling grain yield (GY), kernel number per spike, and thousand-kernel weight, and on 3D (521–549 Mbp) controlling GY alone. A strong marker–trait association (MTA) for GY stability on chromosome 3D (508.3 Mbp) explained 20.3% of the variation. Leaf traits—canopy temperature, vegetation index, and carbon isotope composition—were controlled by five QTLs on 2D (23–96, 511–554, and 606–614 Mbp), 3D (155–171 Mbp), and 5D (407–413 Mbp); some of them were pleiotropic for GY and yield-related traits. Further analysis revealed candidate genes, including GA20ox, regulating GY stability, and CaaX prenyl protease 2, regulating canopy temperature at the flowering stage, under H and HD stresses. As genome-wide association studies under HD in field conditions are scarce, our results provide genomic landmarks for wheat breeding to improve adaptation to H and HD conditions under climate change.

In this study, we investigated the chromosome breakage caused by gametocidal (Gc) chromosome 3Ct and its interaction with the suppressor gene Igc1 (inhibitor of gametocidal gene 1) on wheat chromosome 3B. We demonstrated cytologically that patterns of 3Ct-induced chromosomal fragmentation in microspores differed from patterns observed for other Gc genes. Uninuclear microspores of the monosomic 3Ct addition line had high frequencies of micronuclei, possibly explaining its low fertility. Chromosome fragmentation was observed in prometaphase and metaphase of the first pollen mitosis in the monosomic 3Ct addition line. Patterns of chromosome fragmentation were different from those previously reported for Gc chromosomes 2S of Aegilops speltoides, 4Ssh of Ae. sharonensis and 2Ccy of Ae. cylindrica; many chromosome fragments were observed in prometaphase of the first pollen mitosis in the monosomic 3Ct addition plants. In anthers at the binuclear stage, many microspores at the uninuclear stage coexisted with normally developed microspores.

Developing wheat–alien chromosome introgression lines to improve bread wheat’s resistance to stresses, such as drought, salinity stress and diseases, requires reliable markers to identify and characterize the alien chromatins. Leymus mollis is a wild relative of bread wheat resistant to salinity and economically important diseases of wheat, but its genome sequence and cytological markers are not available. We devised a molecular marker-assisted strategy for L. mollis chromosome identification and applied it to produce 10 wheat–L. mollis chromosome addition lines. Using 47 L. racemosus genome polymorphic PCR markers and DArTseq genotyping, we distinguished the L. mollis chromosomes and differentiated disomic and monosomic lines by progeny test. DArTseq genotyping generated 14,530 L. mollis SNP markers and the chromosome-specific SNP markers were used to determine the homoeologous groups of L. mollis chromosomes in the addition lines. To validate the marker-based results, genomic in situ hybridization was applied to confirm the presence and cytological status of L. mollis chromosomes in the lines. This study demonstrates that adequate molecular markers allow the production and characterization of wheat–alien addition lines without in situ hybridization, which saves considerable time and effort.

The protein content and its type are principal factors affecting wheat (Triticum aestivum) end product quality. Among the wheat proteins, glutenin proteins, especially, high molecular weight glutenin subunits (HMW-GS) are major determinants of processing quality. Wheat and its primary gene pool have limited variation in terms of HMW-GS alleles. Wild relatives of wheat are an important source of genetic variation. For improvement of wheat processing quality its wild relative Thinopyrum elongatum with significant potential was utilized. An attempt was made to replace Th. elongatum chromosome long arm (1EL) carrying HMW-GS genes related to high dough strength with chromosome 1AL of wheat with least or negative effect on dough strength while retaining the chromosomes 1DL and 1BL with a positive effect on bread making quality. To create chromosome specific translocation line [1EL(1AS)], double monosomic of chromosomes 1E and 1A were created and further crossed with different cultivars and homoeologous pairing suppressor mutant line PhI. The primary selection was based upon glutenin and gliadin protein profiles, followed by sequential genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH). These steps significantly reduced time, efforts, and economic cost in the generation of translocation line. In order to assess the effect of translocation on wheat quality, background recovery was carried out by backcrossing with recurrent parent for several generations and then selfing while selecting in each generation. Good recovery of parent background indicated the development of almost near isogenic line (NIL). Morphologically also translocation line was similar to recipient cultivar N61 that was further confirmed by seed storage protein profiles, RP-HPLC and scanning electron microscopy. The processing quality characteristics of translocation line (BC4F6) indicated significant improvement in the gluten performance index (GPI), dough mixing properties, dough strength, and extensibility. Our work aims to address the challenge of limited genetic diversity especially at chromosome 1A HMW-GS locus. We report successful development of chromosome 1A specific translocation line of Th. elongatum in wheat with improved dough strength.

To study the influence of genes from Thinopyrum intermedium on traits affecting the bread-making quality of wheat, two derivatives from a putative disomic addition line in cultivar ‘Vilmorin 27’ were used in cytological, biochemical and molecular characterization. Cytological analysis suggested that one of the derivatives (Line-1) had a terminal deletion involving the long arm of chromosome 1D (2n = 42, Del-1DL”), and the other (Line-2) was a conventional addition line, but also carried the same deletion on chromosome 1D (2n = 44, Thi”+Del-1DL”). Amplification and sequencing of high-molecular-weight glutenin subunit (HMW-GS) genes coded by the Th. intermedium chromosome in Line-2 indicated the presence of one x-type with an extra cysteine and four (rather than one) unique y-type genes. Rheological studies of Line-1 showed significantly lower dough strength compared to ‘Vilmorin 27’, confirming the recognized role of Glu-1D coded HMW-GSs. Line-2 showed significantly higher dough strength compared to the background cultivar, indicating a significant potential of Th. intermedium for improvement of bread-making quality in wheat.

In the genus Carthamus (2n=20, 22, 24, 44, 64; x=10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S-26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S-26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n=20, and the two botanical varieties of B genome C. tinctorius (2n=24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S-26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n=20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n=44), and C. lanatus is one of the progenitors for the hexaploid (2n=64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2-10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.

Aluminum (Al) toxicity is the key factor limiting wheat production in acid soils. Soil liming has been used widely to increase the soil pH, but due to its high cost, breeding tolerant cultivars is more cost-effective mean to mitigate the problem. Tolerant cultivars could be developed by traditional breeding, genetic transformation or introgression of genes from wild relatives. We used 30 wheat alien chromosome addition lines to identify new genetic resources to improve wheat tolerance to Al and to identify the chromosomes harboring the tolerance genes. We evaluated these lines and their wheat background Chinese Spring for Al tolerance in hydroponic culture at various Al concentrations. We also investigated Al uptake, oxidative stress and cell membrane integrity. The L. racemosus chromosomes A and E significantly enhanced the Al tolerance of the wheat in term of relative root growth. At the highest Al concentration tested (200 μM), line E had the greatest tolerance. The introgressed chromosomes did not affect Al uptake of the tolerant lines. We attribute the improved tolerance conferred by chromosome E to improved cell membrane integrity. Chromosome engineering with these two lines could produce Al-tolerant wheat cultivars.

Gametocidal (Gc) system is a novel method of inducing chromosome mutations in wheat by using Gc genes of wheat-related species. Many deletion lines were efficiently produced by this system and used for physical mapping of genes and DNA markers. A large stocks with chromosome deletions in homozygous state as well as the lines with Gc genes are available in the gene bank of Japan (KOMUGI). In this chapter, I describe the method of inducing breakage in a target chromosome using lines with the Gc gene on chromosome 2C of Aegilops cylindrica (a wheat-related wild species) and nullisomic-tetrasomics.

We studied the seed storage protein composition and dough strength of chromosome deletion (CD) lines involving group-1 chromosomes. The presence or absence of genes and protein bands corresponding to glutenin and gliadin was assessed by using locus-specific DNA markers, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and acid-polyacrylamide gel electrophoresis (A-PAGE). In this study, we were able to map the physical positions of several glutenin and gliadin genes in detail. Dough strength was evaluated by SDS sedimentation volume and protein content. We found that protein composition affected dough strength. The absence of chromosome arm 1AL, which carries the truncated glutenin gene Glu-A1c, significantly increased dough strength, although the protein composition did not change when the size of the deleted chromosome region was varied. In contrast, the presence of chromosome arm 1DL, which carries Glu-D1a (the gene for glutenin subunits 2 and 12), significantly increased dough strength. We did not find any known seed storage protein loci in any of the other chromosomal regions that significantly affected dough strength.

Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization. The six Elymus species are all naturally growing species in northwest China, namely, E. sibiricus, E. nutans, E. barystachyus, E. xiningensis, E. excelsus, and E. dahuricus. An StStHH genome constitution was revealed for E. sibiricus and StStHHYY for the remainder species. Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA, 5S rDNA, Afa-family, and AAG repeats, and be allocated to a certain genome by genomic in situ hybridization. Two 5S rDNA sites, each in the H and St genomes, and three 18S-26S rDNA sites, two in the St genome and one in the Y genome, were uncovered in most of the species. The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes. The H and Y genome carried more AAG repeats than St. A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E. barystachyus, E. xiningensis, E. excelsus and E. dahuricus. Comparison of molecular karyotypes strongly suggests that they can be classified into three groups, namely, E. sibiricus, E. nutans, and others.

Supernumerary (B) chromosomes of rye are not required for plant development and exhibit a reduced transcription activity. These special features inspired us to analyse whether there are differences between A and B chromatin organization in interphase nuclei. Applying fluorescence in situ hybridization, we found that both rye A and B chromosomes added to hexaploid wheat showed in meristematic nuclei a string-like shape and a clear Rabl orientation. In 4C differentiated leaf nuclei, a more relaxed chromatin structure, round-shaped chromosome territories and a less pronounced Rabl configuration were found. Also, the observed random association of homologues in 2C and 4C nuclei indicated in general a similar behaviour of A and B chromosomes. Whereas in differentiated 4C nuclei A sister centromeres are separated, B sister centromeres align in nearly all nuclei. In short, despite the different transcription activity of A and B chromosomes, both types of chromosomes exhibit a similar organization in meristematic and differentiated interphase nuclei. But the deletion of a B chromosome segment responsible for non-disjunction during gametogenesis induces released sister centromeres also in some interphase nuclei of somatic tissue. Hence, the control of rye B chromosome non-disjunction is also active in sporophytic cells.

Deficiencies of iron (Fe) and zinc (Zn) in afflict over three billion people worldwide, and deficiencies of these minerals in soil also limit crop production in one-third (Fe) and nearly half (Zn) of the world’s total cereal growing area. Screening genetic resources for improving wheat grain Fe and Zn contents and efficiency can contribute to both human health and crop production. We evaluated 47 wheat-Aegilops disomic addition lines derived from 6 Aegilops species to identify the chromosomes carrying genes for high grain Fe and Zn concentrations. Addition lines with chromosomes 1Sl and 2Sl of Ae. longissima, 1SS and 2SS of Ae. searsii, 2U and 6U of Ae. umbellulata, B of Ae. caudata, 4Sv of Ae. peregrina, and 5 Mg of Ae. geniculata showed increased grain Fe or Zn concentration of between 50% and 248% compared with the recipient cultivar, Chinese Spring. Most of alien chromosomes addition lines with significantly higher grain Fe and/or Zn concentrations belonged to the U and S genotypes and homoeologous groups 1 and 2 chromosomes. These lines could be used for the precise introgression of genes into elite wheat cultivars to improve wheat micronutrient concentrations.

Wide crossing is one of a number of practical methods that can be used to expand genetic variation in common wheat (Triticum aestivum). However, in crosses between wheat and distantly related species such as maize (Zea mays) and pearl millet (Pennisetum glaucum), non-wheat chromosomes are often eliminated from the hybrid during embryogenesis. In this study, we used pearl millet pollen to pollinate the pistils of a range of plants in the tribe Triticeae, as well as oat. Seven days after pollination, the dynamics of the pearl millet chromosomes in the embryos were observed using in situ hybridization, probing both the pearl millet genomic DNA and its centromere-specific repeats. In embryos from the crosses with oat, all seven of the pearl millet chromosomes were retained. However, in hybrids with the Triticeae species, chromosome elimination occurred during embryogenesis. Pearl millet chromosome showed chromosome rearrangements and non-disjunction together with micronuclei. These rearranged chromosomes and micronuclei derived from the breakage of bridges and retention of acentric fragments in anaphase, respectively. The cause of the chromosome elimination of wheat-pearl millet hybrid is not malfunction of the kinetochores binding to the spindles but the malfunction of the sister chromatids segregation at anaphase especially of chromosome arm.

After screening of 177 disomic addition lines (DALs) of wheat (Triticum aestivum) containing a pair of chromosomes from different alien species, we found that the chromosome 1E addition line of Agropyron elongatum, that is known to be a potential genetic resource for drought and salinity tolerance, showed potential for improvement of bread-making quality of wheat. This was indicated by increased SDS sedimentation, specific sedimentation, mixograph peak time and SE-HPLC analysis of polymeric proteins. This addition line spontaneously gave rise to a substitution line for chromosome 1D in subsequent generations that showed weak dough strength. Analysis of the x-type HMW-glutenin subunit sequence of Ag. elongatum from DAL1E indicated that it closely resembled the x-type sequence of the A and B genomes of wheat, and the y-type was intermediate between x- and y-type HMW-glutenin subunit genes. From these observations, it was inferred that 1E-encoded seed storage proteins have considerable potential for improvement of wheat end-product quality if transferred to specific chromosomes such as 1A of Chinese Spring (CS) wheat, which has a negative overall effect on bread-making quality.

To investigate the nature of plant chromosomes irradiated by heavy-ion beams, the effects of nitrogen (N) and neon (Ne) ion beams on hexaploid wheat chromosomes were compared with those of X-ray. Chromosome aberrations, such as short, ring and dicentric chromosomes appeared in high frequency. The average numbers of chromosome breaks at LD-50 by irradiation with X-ray, N and Ne ion beams were 32, 20 and 20, respectively. These values may be underestimated because chromosome rearrangement without change in chromosome morphology was not counted. Thus, we subsequently used a wheat line with a pair of extra chromosomes from an alien species (Leymus racemosus) and observed the fate of the irradiated marker chromosomes by genomic in situ hybridization. This analysis revealed that 50 Gy of neon beam induced about eight times more breaks than those induced by X-ray. This result suggests that heavy-ion beams induce chromosome rearrangement in high frequency rather than loss of gene function. This suggests further that most of the novel mutations produced by ion beam irradiation, which have been used in plant breeding, may not be caused by ordinary gene disruption but by chromosome rearrangements.

This study involved screening of wild species of wheat in search of functionally useful seed storage proteins for improvement of breadmaking quality of wheat (Triticum aestivum). After screening of 177 disomic addition lines (DALs) of wheat belonging to different wild species, Aegilops searsii DALs were selected and studied in detail. These DALs of Ae. searsii were from chromosome 1Ss to 7Ss in the background of cultivated wheat cv. Chinese Spring (CS). By analyzing these addition lines, genetic loci of actively expressed genes for the high molecular weight glutenin subunits (HMW-GS) and gliadin were found on the chromosome 1Ss for the first time and have been designated as Glu-Ss1 and Gli-Ss1, respectively. Disomic addition line of chromosome 1Ss (DAL1Ss) showed improved dough strength in different generations compared with CS. SDS sedimentation value and specific sedimentation of DAL1Ss were higher than CS. Mixograph peak height and band width were higher, with no difference in mixing peak time...

A monosomic addition line of Aegilops tauschii chromosome 1D in Triticum durum cv. PBW114 was produced in 1990. This line was self-pollinated and maintained for several generations while following the presence of chromosome 1D carrying the gene for red glume color. Cytological analysis indicated that two of the three derivative lines had substitution of chromosome 1D for 1A and another had substitution of chromosome 1D for 1B. One of these lines carried a pair of small chromosomes in addition to the 1D chromosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the derived lines showed the presence of high-molecular-weight (HMW) glutenin encoded by the Glu-D1 locus. The small chromosome found in one of the lines had nearly regular pairing and transmission to daughter nuclei. Fluorescent in situ hybridization (FISH) and analysis of molecular markers indicated that the small chromosome was derived from the short arm of chromosome 1A and carried the Glu-A3 locus. Microsatellite mapping based on the deletion bin map revealed that the small chromosome had terminal deletions on both the terminal and centromeric sides. The line with the small chromosome showed improvement of the sodium dodecyl sulfate (SDS)-sedimentation value as compared to parent durum. However, the increase in SDS-sedimentation value was more significant in the substitution line of chromosome 1D for 1A without the small chromosome. These facts suggest a negative effect of the Glu-A3 locus on dough strength. The sequence of the Glu-D1 locus from these lines showed that the HMW glutenin subunits were Ae. tauschii specific 2(t) + T2, which were previously found to be associated with poor rheological properties and bread loaf volume in synthetic hexaploid wheat by other workers. Thus, the significant improvement in the SDS-sedimentation value of the substitution line of 1D for 1A suggests that the absence of the negative effect of chromosome 1A on quality is more important than the presence of Glu-D1 of Ae. tauschii.

Under the changing climate, early flowering is advantageous to escape terminal heat and drought. Previously during evaluation of 14 chromosome introgression lines (ILs), we found three ILs that flowered a month earlier than their wheat background Chinese Spring (CS). This paper describes the cause of the early flowering in the ILs and provides insight into the evolution of spring wheat from the winter wheat. We used specific molecular markers for Vrn genes to determine its allelic composition. Phenotypic evaluations carried out under field conditions and in a growth chamber. Unlike the winter vrn-A1 allele of CS, the spring Vrn-A1 allele of the ILs had insertions of 222 and 131-bp miniature inverted-repeat transposable element (MITE) in the promoter region. Sequence analysis indicated that the 222-bp insertion is similar to an insertion in the spring genotype, Triple Dirk D. Our results ruled out any possibility of outcrossing or contamination. Without vernalization, Vrn-A1 is highly expressed in the ILs compared to CS. We attribute the early flowering of the ILs to the insertion of the MITE in the promoter of Vrn-A1. The alien chromosome might mediate this insertion.

Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity.

Torenia is a suitable model plant to study plant fertilization because of its protruding embryo sac. However, information on the genomes and chromosomes of this species is limited. We determined the genome sizes of T. fournieri Linden and T. baillonii Godefr as 1.71 pg x 10(8) bp and 1.67 x 10(8) bp, respectively. The small genome size of these species suggests their superiority as the targets for molecular cloning studies. Furthermore, karyotypes of T. fournieri and T. baillonii were determined using FISH probed with 5S rDNA, 45S rDNA and species-specific centromere repetitive sequences. Although the two species have similar genome size, number of chromosomes, centromere repeats and 5S rDNA loci were varied. Observation of meiosis in the F(1) hybrid revealed that all chromosomes except one of T. fournieri paired well with the chromosomes of T. baillonii throughout the entire length of the chromosomes including species-specific centromeric regions. One exceptional chromosome of T. fournieri behaved as a univalent and was not always required for gametogenesis. The present results provide the basis for the molecular genetics in Torenia.

The main objective of this study is to produce DNA markers of polymorphism between wheat and some important alien species. The applicability of 1,165 barley EST primer sets to amplify markers showing polymorphism between wheat and ten alien species, covering a wide range of variation in Triticeae, was investigated. These primers consisted of four series. Series 1 was randomly chosen from a pool of barley EST primer sets. The remaining three were pre-screened in a previous study and showed polymorphic and co-amplified patterns between barley and wheat. From 22% to 100% of the primer sets amplified single clear bands in the species; from 29% to 75% of which were polymorphic to wheat. The frequency of amplification corresponded with their phylogenetic distance from barley. Many DNA markers (78–859) showing polymorphism between each species and wheat were obtained. These markers are expected to be valuable in identifying the alien chromosomes added to a wheat genetic background. The usefulness of the markers in the basic and the applied studies of the wild species is discussed.

We produced ten wheat-Leymus racemosus chromosome addition lines. Eight chromosomes (A, C, F, H, I, J, k, and l) were recovered as disomic additions and two (E and n) as monosomic. Screening of the addition lines was done by fluorescence in situ hybridization using several repetitive sequences as probes, which allowed us to identify different L. racemosus chromosomes and find many aberrant L. racemosus chromosomes. RFLP analysis revealed partial conservation of homology between L. racemosus and wheat chromosomes, depending on the homologous groups. Chromosomes A and l belonged to group 2, chromosomes C and I to group 5, and chromosome k to group 6. Chromosomes H and J were a mixture of groups 1, 3, and 7, chromosome n of groups 3 and 7, and chromosomes E and F were of group 4 and others. Comparison of our addition lines with other addition lines showed large cytological differences.

Herein, we profile the first embryonic mitosis in a hybrid of wheat and maize by using a whole-mount genomic in situ hybridization method and immunofluorescence staining with a tubulin-specific antibody. We have successfully captured the dynamics of each set of parental chromosomes in the first zygotic division of the hybrid embryo 24-28 h after crossing. During the first zygotic metaphase, although both sets of parental chromosomes congressed into the equatorial plate of the zygote, the maize chromosomes tended to lag in comparison with the wheat chromosomes. During anaphase, each parental chromosome separated into its sister chromosomes; however, some of the maize chromosomes lagged around the metaphase plate as segregants. The maize sister chromosomes that did move toward the pole showed delayed and asymmetric movement as compared with the wheat ones. Immunological staining of tubulin revealed a bipolar spindle structure in the first zygotic metaphase. The kinetochores of the maize chromosomes that lagged around the metaphase plate did not attach to the spindle microtubules. These results suggest that factors on the kinetochores of maize chromosomes that are required to control chromosome movement are deficient in the zygotic cell cycle.Key words: whole-mount, GISH, chromosome elimination, hybrid embryogenesis.

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.

Tetrad analysis is a genetic method that can locate genes and centromeres on a linkage map with a high degree of precision. Despite its effectiveness and accuracy, application of this method is generally limited to fungi, algae and mosses. Here we demonstrate a new method of tetrad analysis that is applicable to other organisms. This combines tetrad analysis with fluorescence in situ hybridization (FISH), and is thus referred to as tetrad-FISH analysis. We demonstrate the effectiveness of this method using tetrads of rye, Secale cereale. The rye strain JNK contains interstitial heterochromatin in a region of Chromosome 2R. We have previously cloned the tandemly repeated sequence forming this heterochromatin in the plasmid pScJNK. We performed FISH using pScJNK as the probe on tetrads obtained from heterozygotes for the heterochromatin region. The frequency of tetrads demonstrating positive signals in two cells that are diagonally opposite one another must correspond to the frequency of recombination in the interval between the heterochromatin and the centromere. Comparison between the results of tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions. We discuss the effectiveness of tetrad-FISH analysis, particularly for the localization of functional centromeres in linkage maps.

Sixteen near-isogenic lines (NILs) carrying a marker gene were produced by the recurrent backcrossing method in the genetic background of common wheat (Triticum aestivum) cv. Chinese Spring (CS). Three genes from alien species showed segregation distortion. In NILs carrying a marker gene of rye (Secale cereale) or Aegilops caudata, the alien chromosome segments were detected by fluorescence in situ hybridization (FISH). The NILs were grown with replications and the effect of marker genes on plant morphology in the genetic background of CS was investigated. These NILs were further crossed with the corresponding monosomics of CS and 13 monosomic lines whose monosome carries a respective marker gene were established and named “marked monosomics.” Many of the marked monosomics were distinguishable from the disomic NILs because of the different dosage effect of the genes. The NILs are utilized for studies on gene isolation or gene regulation. Marked monosomics are useful not only for monosomic analysis but also for production of homologous chromosome substitution lines because chromosome observation is not required.

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

Tandem repetitive sequences consisting of 140-bp repetitive units were cloned from sugar cane genomic DNA and designated the SCEN family. In situ hybridization revealed that they were located on the centromeric region of almost all of the chromosomes of sugar cane. The 140-bp sequence included three CENP-B box-like sequences. Phylogenetic analysis of the members of the SCEN family revealed that the sequences had 75% homology with each other, on average, and that the sequences could not be further classified into smaller subfamilies. The copy number of the sequence was estimated to be 2.6 × 105 per haploid sugar cane genome and, therefore, 4.6 × 103 or 630 kb per chromosome on average.

Near-isogenic lines of alloplasmic wheat for the Rf3 gene controlling fertility restoration against the cytoplasm of Triticum timopheevi were developed by successive backcrossing of Chinese Spring to the F1 plants between (timopheevi)-CS (male sterile) and T. spelta (carrying Rf3). The resultant three BC3F1 plants were self-pollinated so as to obtain 125 BC3F2 progenies. Using this population, we precisely mapped the Rf3 gene on the short arm of wheat chromosome 1B (1BS) of the RFLP linkage map. The Rf3 gene was localized at a position 1.2 cM and 2.6 cM distant from Xcdo388 and Xabc156, respectively. The genetic distances of Rf3 from Nor and Gli-B1 were calculated to be 22.3 cM and 18.6 cM, respectively, supporting previous data. Estimation of the physical distance of the region suggets that the Rf3 gene resides within 500 kbp from the adjacent RFLP markers. The marker order of the genetic map corresponded to that of the cytological map, except for the position of Xbcd98. Comparison between genetic and cytological maps clearly shows that RFLP markers are unevenly distributed throughout the chromosome arm, and recombinations took place unequally in the chromosome arm.

Wheat cultivars with wide introgression have strongly impacted global wheat production. Aegilops geniculata (MgUg) is an important wild relative with several useful traits that can be exploited for wheat improvement. Screening of Ae. geniculata addition lines indicated a negative effect of 1Ug and the positive effect of 1Mg chromosome on wheat dough strength. Negative effect of 1Ug is probably associated with variation in number and position of the tripeptide repeat motif in the high molecular weight glutenin (HMW-G) gene. To utilize the positive potential of 1Mg chromosome, three disomic substitution lines (DSLs) 1Mg(1A), 1Mg(1B) and 1Mg(1D) were created. These lines were characterized for morphological, cytogenetic properties and biochemical signatures using FISH, 1D-, 2D-PAGE and RP-HPLC. Contribution of wheat 1A, 1B and 1D chromosomes towards dough mixing and baking parameters, chapatti quality, Fe/Zn content and glume color were identified. Observed order of variation in the dough mixing and baking parameters {1Mg(1D) ≤wheat ≤1Mg(1B) ≤1Mg(1A)} indicated that chromosome specific introgression is desirable for best utilization of wild species’ potential.

Telomere formation of the normal and broken chromosomes of common wheat,Triticum aestivum, was investigated byin situ hybridization using the biotin-labeled probe of telomere repetitive sequences (pAtT4) ofArabidopsis thaliana with subsequent amplification by an antibody. After double and triple amplification, prominent signals appeared at all the telomeric regions of the normal chromosomes.

Psathyrostachys huashanica Keng (2n = 14; N(h)N(h)) is an endangered wheat-related species, with a distribution in the Huashan region of central China. It has many agronomically promising characters including resistance to disease and drought and winter hardiness. We produced hybrids between common wheat as the female parent and P. huashanica as the male parent. From the offspring, we selected chromosome addition lines of common wheat carrying each of all seven chromosomes of P. huashanica. Four chromosomes (B, D, E and F) were recovered in disomic lines and three (A, C and G) in monosomic addition lines. These alien chromosomes were distinguished from each other by cytological analyses. Chromosome A was characterized by a 45S rDNA site in the subtelomeric region of the short arm. Chromosome B carried one 5S and one 45S rDNA sites co-localized in an interstitial region of the short arm, and the expression of the alien high-molecular-weight glutenin was observed in the endosperm of line B. Chromosome D had a 45S rDNA signal in the interstitial region of the long arm. Chromosomes C, E, and F were distinguished by the EST-SSR markers Ltc0464, Ltc0096, and Xcfe175, respectively. The homoeologous group of each alien chromosome was implied from the results above, and the utilization of these addition lines for wheat breeding was discussed.

To clarify the DNA structure of the B chromosome of rye, Secale cereale L., we carried out in situ hybridization using probes of two highly repetitive sequences (pSc74 and pSc119) of rye A chromosome, of total genomic DNAs from plants with (4 B) or without (0 B) B chromosomes, and probes of fractionated genomic DNA (1.14-kb and 1.28-kb sequences) from a 4 B plant. Clones pSc74 and pSc119 did not hybridize to the B chromosomes except at small interstitial sites. A probe consisting of the total DNA of a 0 B plant hybridized over the entire length of all the A and B chromosomes except for the telomeric C-band region of the long arm of the B chromosome. A probe of total DNA from a 4B plant produced signals only in the terminal C-band region after prehybridization with the 0 B plant DNA. A probe consisting of DraI-digested 1.14-kb fragments from a 4B plant hybridized to the telomeric region of the B chromosome, whereas one of 1.28-kb fragments did so to an interstitial region of the A chromosome. On the basis of the in situ hybridization, and the cytological and molecular biological results, we assume that the B chromosome in rye originated once in the distant past and was then perpetuated in the populations.

Wheat alien chromosome addition lines possess abundant genetic resources and they are usually used for transferring desired genes or traits into wheat. The screening and characterisation of addition lines for target traits is one of the prerequisites for efficient utilisation of the alien chromosomes. In order to understand the properties and potential utilisation of wheat addition lines, the effects of additional chromosomes on agronomic and photosynthetic traits of common wheat were evaluated using 34 addition lines with the same genetic background of Chinese Spring. The results showed that most of the alien chromosomes decreased plant height (61.8%) and grain number per spike (47.1%), whereas some increased spike length and tiller number. Alien chromosomes of Agropyron intermedium G, Elymus trachycaulus T5HL5HL, El. trachycaulus 5SS and Haynaldia villosa 1V performed well in improving yield components. None of the alien chromosomes studied had negative effects on photosynthetic traits. Higher net photosynthetic rates were observed in Aegilops umbellulata 5U, El. trachycaulus 5H and rye 1R addition lines. Regarding seedling traits, 21 lines (61.8%) showed improvement in different root traits, whereas 26.5% of the chromosomes decreased coleoptile length. Addition lines with better performance for some specific traits were identified and discussed.

Genome characterization of 14 hexaploid lines that spontaneously appeared in octoploid Triticales was carried out by sequential genomic in situ hybridization and fluorescence in situ hybridization, high molecular weight glutenin subunits and SSR marker analyses. All of the lines showed a chromosome constitution of complete A and B genomes, and a composite genome consisting of the chromosomes of D and R genomes. The composite genome of the 11 lines consisted of chromosomes 1R, 2D, 3R, 4R, 5R, 6R and 7R, that of the two lines were 1D, 2D, 3R, 4R, 5R, 6R and 7R, and that of one line was 1R, 2D, 3R, 4R, 5R, 6D and 7R. The incompatibility of the D and R genomes in common wheat genetic background, preferential retention of chromosome 2D and importance of these lines for the development of hexaploid Triticale are discussed in this report.

The cytoplasm of Triticum timopheevi causes cytoplasmic male sterility (CMS) in common wheat (T. aestivum) cv. ‘Chinese Spring’ (CS), and that of Aegilops kotschyi causes CMS in spelt wheat (T. spelta) var. duhamelianum (Sp). CS has fertility-restoring (Rf) genes against the latter cytoplasm and Sp has the ones against the former. To know the genetic system concerning to CMS, we crossed 66 F8 recombinant inbred lines (RILs) derived from a cross between CS and Sp as males to the alloplasmic lines of CS and Sp having the cytoplasms of T. timopheevi and Ae. kotschyi, respectively. The fertilities of respective F1 plants derived from the crosses were examined for QTL analysis. The major QTLs detected in both systems were located on the short arm of chromosome 1B. One minor QTL on chromosome 2B was also commonly detected in both of the systems, while other minor QTLs against T. timopheevi cytoplasm were distributed on the chromosomes 2A, 4B, and 6A.

When chromosomes are broken, the breakpoints become highly unstable and acquire the ability to fuse with other broken ends. The breakpoints are, however, eventually stabilized, and, therefore, the broken chromosomes are transmitted to the daughter cells without further morphological change. This phenomenon, known as “healing of breakpoints”, involves the addition of repetitive telomere sequences at the breakpoints by telomerase, the enzyme that normally synthesizes the telomere sequence at normal chromosome terminals. In many higher organisms, however, this property has not been well investigated. In this study, we examined the telomere sequences in wheat deletion lines with breakpoints on chromosome 1B. Lines that had breakpoints around the nucleolar organizer region were first selected on the basis of cytological observations, and the precise breakpoints were determined by mapping a fragment of rDNA and RFLP markers. In three lines – in addition to one previously reported – the DNA fragments encompassing the breakpoints were amplified by PCR using primers located in the rDNA and in telomere sequences. The DNA sequences provide insight into the properties of the telomerase activity at the breakpoints. The telomere sequences initiated from 2- to 4-nucleotide motifs in the original ribosomal DNA sequence which are also found in the repeat unit characteristic of telomere sequences. No specific sequences or structures were observed at or around the breakpoints. At all of the four breakpoints investigated, the newly synthesized telomere sequences contained considerable numbers of atypical telomere sequence units, particularly TTAGGG, which is the common unit of mammalian telomere sequences. Based on these results, we discuss the ability of plant telomerase to initiate the de novo synthesis of telomere sequences at internal breakpoints.

The Afa-family sequences in wheat-related species, Triticeae, are tandem repetitive sequences of 340 bp. All the analyzed Triticeae species carried the sequences in their genomes, though the copy numbers varied about 100-fold among the species. The nucleotide fragments amplified by PCR were cloned and sequenced, and their behavior in the evolution of Triticeae was analyzed by the neighbor-joining (NJ) method. The sequences in genomes with many copies of this family clustered at independent branches of the phylogenic tree, whereas the sequences in genomes with a few copies did not. This may suggest that Afa-family sequences had amplified several times in the evolution of Triticeae, each using a limited number of different master copies. In addition, the sequences of the A and B genomes of hexaploid common wheat indicated that the Afa-family sequences had not evolved in a concerted manner between the genomes. Furthermore, the sequences of each chromosome of the D genome of this species indicated that the sequences had amplified on all over the D-genome chromosomes in a short period.

The Afa-family repetitive sequences were isolated from barley (Hordeum vulgare, 2n = 14) and cloned as pHvA14. This sequence distinguished each barley chromosome by in situ hybridization. Double color fluorescence in situ hybridization using pHvA14 and 5S rDNA or HvRT-family sequence (subtelomeric sequence of barley) allocated individual barley chromosomes showing a specific pattern of pHvA14 to chromosome 1H to 7H. As the case of the D genome chromosomes of Aegilops squarrosa and common wheat (Triticum aestivum) hybridized by its Afa-family sequences, the signals of pHvA14 in barley chromosomes tended to appear in the distal regions that do not carry many chromosome band markers. In the telomeric regions these signals always placed in more proximal portions than those of HvRT-family. Based on the distribution patterns of Afa-family sequences in the chromosomes of barley and D genome chromosomes of wheat, we discuss a possible mechanism of amplification of the repetitive sequences during the evolution of Triticeae. In addition, we show here that HvRT-family also could be used to distinguish individual barley chromosomes from the patterns of in situ hybridization.

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.

Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1 (*)1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1 (*)1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1 (*)1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.

When gametocidal agents (gametocidal genes or chromosomes) derived from four Aegilops species, Ae. speltoides, Ae. sharonensis, Ae. longissima and Ae. triuncialis, are present in a hetero- or hemizygous state in wheat, they cause a syndrome characterized by male and female sterility, seed shriveling, chromosome breakage and mutation. However, the characteristics of the syndrome differ among the agents. Gametocidal gene, Gc1, originally from Ae, speltoides, induces sterility, seed shriveling and mutation in the F1 generation. In this case, seed shriveling is accentuated by low temperature, about 5 to 11 hrs after the pollination. Gametocidal chromosomes, 4Ssh of Ae. sharonesis and 4S1 of Ae. longissima, cause high rates of mutation and chromosome aberration in addition to sterility, although they do not induce seed shriveling. Chromosome 3C of Ae. triuncialis induces only sterility in the F1, though chromosome breakage and mutation are induced in the B11generation in the presence of its suppressor, Igc1 (from a common wheat cultivar Norin 26). The syndromes caused in wheat by these gametocidal agents are compared to hybrid dysgenesis observed in Drosophila melanogaster.

Chromosome 3C of Aegilops triuncialis, in the monosomic state, causes semisterility in common wheat cultivars (Triticum aestivum). The Japanese cultivar 'Norin 26' carries a gene that acts as dominant suppressor of the gametocidal gene on chromosome 3C. However, this suppressor gene does not alter the functions of the gametocidal gene on chromosome 4S1 of Ae. longissima or Ae. sharonensis. By means of monosomic analyses, the suppressor has been located on chromosome 3B and is designated Igc1. This gene is distributed widely among wheat cultivars bred in central and southwest Japan. In the presence of Igc1 chromosome 3C shows no preferential transmission, but is transmitted to offspring at a frequency similar to those of other alien chromosomes. The progeny of plants that carry a single chromosome 3C exhibit chromosome aberrations, and possibly mutations, at high frequencies. Thus, the gametocidal gene on chromosome 3C causes a syndrome similar to hybrid dysgenesis in common wheat.Key words: common wheat, Aegilops triuncialis, gametocidal gene, suppressor, hybrid dysgenesis.

All the alloplasmic common wheat lines having Agropyron trichophorum cytoplasm were found to carry one extra submetacentric or telocentric chromosome although they had been backcrossed seven times with common wheat as the pollen parent. Behavior of the extra chromosome and its interaction with the A, trichophorum cytoplasm were studied. A, trichophorum cytoplasm was found to induce seed shriveling and severe vigor reduction in common wheat. The extra chromosome originating from A, trichophorum carries a gene(s) for complete restoration of seed plumpness and normal plant vigor. Conscious selection of plump seeds and vigorous plants for backcrossing has resulted in the preferential transmission of the A. trichophorum chromosome in the present alloplasmic lines.

Four monosomic substitution lines of the Elytrigia elongata (=Agropyron elongatum) chromosomes, e2, e3, e6 and e7 for chromosome 4A of durum wheat were produced by the cross between monosomic 4A and the corresponding disomic addition lines of Stewart durum. Among them the monosomic substitution line e7(4A) was the most vigorous and fertile. Although this line showed a chromosome configuration of 13″+2′ at the first meiotic metaphase, it had 14 bivalents at diakinesis in most of its pollen mother cells. The fertilization rate of the four types of the egg cells produced by this monosomic substitution suggested a 50% elimination of both e7 and 4A univalents during the meiosis of durum wheat. Male transmission behavior indicated that chromosome e7 compensates for the function of chromosome 4A in the certation. A disomic substitution line was selected from the progeny of the monosomic substitution line e7 (4A). Plants of this line were vigorous and completely fertile. Chromosome e7 was found to have a structural gene for alcohol dehydrogenase; in wheat this gene is known to be located on the three chromosomes of homoeologous group 4. It was thus concluded that chromosome e7 of E. elongata belongs to homoeologous group 4. A comparison of the morphology of the substitution, addition and monosomic lines with those of the normal Stewart durum, showed that chromosome e7 carried genes for awn suppression, culm thickness and ear laxness.