Dissection of barley chromosome 5H in common wheat

We dissected barley chromosome 5H added to common wheat by a genetic method or the gametocidal system. Firstly, we induced chromosomal breaks in the offspring of a 5H addition line of common wheat carrying a gametocidal chromosome and cytologically screened for plants with structural chromosomal changes involving 5H, such as deletions and translocations. Secondly, we screened the progeny of such plants to establish common wheat lines carrying structurally changed chromosomes containing single segments of the dissected 5H. Using 23 representative 5H dissection lines, we physically mapped 97 barley EST markers assigned to 5H. The ESTs fell into 20 regions of 5H between the breakpoints of the 23 dissected segments, distributing rather evenly along the chromosome, with significantly higher frequency in the distal region of the long arm. The ESTs, in turn, allowed us to distinguish the breakpoints of dissected 5H segments. We demonstrated by PCR (polymerase chain reaction), as well as by in situ hybridization, that these dissected 5H segments were stably transmitted in the dissection lines. We discuss the usefulness of the 5H dissection lines for physical mapping of DNA markers. These 5H dissection lines are available from National BioResource Projects-Wheat, Japan.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.     

Genetic induction of bivalent interlocking in common wheat

The frequency of interlocking bivalents at first meiotic metaphase of common wheat Triticum aestivum L., which is normally very low, is significantly increased by raising the dosage (from two to three, four and six) of the Ph1 gene, located on the long arm of chromosome 5B (5BL). In several cells more than three bivalents were interlocked in one chain configuration indicating involvement of non-homoeologous bivalents. Plants with reduced dose (one or zero) of Ph1 also exhibited an increased frequency of interlocking but to a lesser extent than those with high gene dosage. However, chains of more than three interlocked bivalents were never observed in these plants, suggesting that with one or zero doses of Ph1 interlocking is restricted to homoeologous bivalents only. Chromosomal arm 5BS affected interlocking in an opposite manner to 5BL; namely, two and four doses of 5BS markedly reduced interlocking frequency. The modification in the frequency of interlocking bivalents by these genetic manipulations represents the first successful attempt to affect interlocking by genetic means. The results are explained on the basis of the hypothesis that this gene system controls somatic and premeiotic association of both homologous and homoeologous chromosomes.     

Deletion-based physical mapping of barley chromosome 7H

Chromosomal mutations in barley (Hordeum vulgare, 2n=2x=14, HH) chromosome 7H added to the common wheat (Triticum aestivum, 2n=6x=42, AABBDD) cultivar Chinese Spring were induced genetically by the gametocidal activity of certain alien chromosomes derived from wild species of the genus Aegilops. The rearranged barley chromosomes were characterized by C-banding, FISH and GISH. Twenty two deletion or translocation chromosomes in a hemizygous condition were selected for deletion mapping of 17 AFLP and 28 STS markers that are specific to 7H. Of the 22 breakpoints in chromosome 7H, seven involved the short arm (7HS), 12 the long arm (7HL) and three were in the centromeric region. The seven 7HS breakpoints separated all four 7HS-specific AFLP markers and split the 21 STS markers into six groups. One breakpoint occurred between two STS markers formerly occupying the same position in the genetic map. All seven 7HS breakpoints were separated from each other by either the AFLP or STS markers. The 12 breakpoints in 7HL divided the 13 7HL-specific AFLP markers into seven groups, and the seven STS markers into three groups. On the other hand, the 12 breakpoints in 7HL were divided into six groups by the AFLP markers and into two groups by the STS markers. This deletion-based map was in accordance with previously published genetic and physical maps using the same STS markers. The breakpoints, AFLP markers and STS markers were arrayed in a consistent order. Received: 5 February 2001 / Accepted: 19 February 2001     

An efficient screening for terminal deletions and translocations of barley chromosomes added to common wheat

As a prerequisite to determine physical gene distances in barley chromosomes by deletion mapping, a reliable, fast and inexpensive approach was developed to detect terminal deletions and translocations in individual barley chromosomes added to the chromosome complement of common wheat. A refined fluorescence in situ hybridization (FISH) technique subsequent to N-banding made it possible to detect subtelomeric repeat sequences (HvT01) on all 14 chromosome arms of barley. Some chromosome arms could be distinguished individually based on the number of FISH signals or the intensity of terminal FISH signals. This allowed the detection and selection of deletions and translocations of barley chromosomes (exemplified by 7H and 4HL), which occurred in the progeny of the wheat lines containing a pair of individual barley chromosomes (or telosomes) and a single so-called gametocidal chromosome (2C) of Aegilops cylindrica. This chromosome is known to cause chromosomal breakage in the gametes in which it is absent. Terminal deletions and translocations in barley chromosomes were easily recognized in metaphase and even in interphase nuclei by a decrease in the number of FISH signals specific to the subtelomeric repeat. These aberrations were verified by genomic in situ hybridization. The same approach can be applied to select deletions and translocations of other barley chromosomes in wheat lines that are monosomic for the Ae. cylindrica chromosome 2C.     

Replication delay along FRA7H, a common fragile site on human chromosome 7, leads to chromosomal instability

Common fragile sites are specific chromosomal loci that show gaps, breaks, or rearrangements in metaphase chromosomes under conditions that interfere with DNA replication. The mechanism underlying the chromosomal instability at fragile sites was hypothesized to associate with late replication time. Here, we aimed to investigate the replication pattern of the common fragile site FRA7H, encompassing 160 kb on the long arm of human chromosome 7. Using in situ hybridization on interphase nuclei, we revealed that the replication of this region is initiated relatively early, before 30% of S phase is completed. However, a high fraction ( approximately 35%) of S-phase nuclei showed allelic asynchrony, indicating that the replication of FRA7H is accomplished at different times in S phase. This allelic asynchrony is not the result of a specific replication time of each FRA7H allele. Analysis of the replication pattern of adjacent clones along FRA7H by using cell population and two-color fluorescent in situ hybridization analyses showed significant differences in the replication of adjacent clones, under normal growth condition and upon aphidicolin treatment. This pattern significantly differed from that of two nonfragile regions which showed a coordinated replication under both conditions. These results indicate that aphidicolin is enhancing an already existing difference in the replication time along the FRA7H region. Based on our replication analysis of FRA7H and on previous analysis of the common fragile site FRA3B, we suggest that delayed replication is underlying the fragility at aphidicolin-induced common fragile sites.     

Characterization of deletions in common wheat induced by an Aegilops cylindrica chromosome: detection of multiple chromosome rearrangements

An Aegilops cylindrica chromosome induces terminal deletions of chromosomes in wheat as identified by C-banding. We are constructing high-density physical maps of wheat chromosomes and have detected additional chromosome rearrangements. Among 63 lines with chromosomal subarm deletions in group 7 chromosomes, 7 lines (11.1%) were shown to harbor additional chromosome rearrangements. Two other lines were also omitted from the physical mapping because of the nature of the breakpoint calculations. The presence or absence of chromosome-specific restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) markers indicated that additional interstitial deletions are present in 3 lines (4.8%) with deletions in the short chromosome arms and in 4 lines (6.3%) with deletions in the long chromosome arms. We also used chromosome pairing analysis of F₁ plants of deletion lines with double ditelosomic lines of Chinese Spring wheat to detect small terminal deletions. The deletion of the most distal 1% of chromosome arm 7AL was associated with a pairing reduction of 60%.     

Genetic and physical mapping of a high recombination region on chromosome 7H(1) in barley

Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836. We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes (BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2–5 between molecular markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2–102 clones each with an average of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24 agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytogenetic identification of Triticum peregrinum chromosomes added to common wheat.

C-banded karyotypes of a complete set of 14 Triticum peregrinum whole chromosome addition lines and 25 telosomic addition lines are reported. The added T. peregrinum chromosomes were not structurally rearranged compared with the corresponding chromosomes of the donor accession. Comprehensive karyotypic analysis confirmed Triticum umbellulatum as the donor species of the Uv genome and identified Triticum longissimum as the donor species of the Sv genome of T. peregrinum. Neither the Uv nor Sv genome chromosomes of the T. peregrinum accession showed large modifications when compared with the ancestral U and S1 genomes. Key words : Triticum aestivum, Triticum peregrinum, Triticum umbellulatum, Triticum longissimum, chromosome addition lines, C-banding.     

小麦染色体轴结构的观察

对普通小麦 ( Triticum aestivum L.)中期染色体进行常规制片银染的结果显示 ,染色体中存在着染色深的轴结构 ,每个染色单体一条 ,轴在有些部位似乎是螺旋的。研究结果对染色体轴结构的真实性提供了证据     

A new insight into application for barley chromosome addition lines of common wheat: achievement of stigmasterol accumulation

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.     

Dissection of rye chromosome 1R in common wheat

Rye chromosome 1R contains many agronomically useful genes. Physical dissection of chromosome 1R into segments would be useful in mapping 1R-specific DNA markers and in assembling DNA clones into contig maps. We applied the gametocidal system to produce rearranged 1R chromosomes of Imperial rye (1R(i)) added to common wheat. We identified rearranged 1R(i) chromosomes and established 55 1R(i) dissection lines of common wheat carrying a single rearranged 1R(i) chromosome. Fifty-two of the rearranged 1R(i) chromosomes had single breakpoints and three had double breakpoints. The 58 breakpoints were distributed in the short arm excluding the satellite (12 breakpoints), in the satellite (4), in the long arm (28), and in the centromere (14). Out of the 55 lines, nine were homozygous for the rearranged 1R(i) chromosomes, and the remaining lines were hemizygous. We developed 26 PCR-based EST markers that were specific to the 1R(i) chromosome, and nine of them amplified 1R(i) arm-specific PCR products without restriction-enzyme digestion. Using the nine EST markers and two previously reported 1R-specific markers, we characterized the 55 1R(i) dissection lines, and also proved that we can select critical progeny plants carrying specific rearranged 1R(i) chromosomes by PCR, without cytological screening, in 48 out of the 55 hemizygous dissection lines.     

Dissection of a malting quality QTL region on chromosome 1 (7H) of barley

Malting and brewing are major uses of barley (Hordeum vulgare L.) worldwide, utilizing 30–40% of the crop each year. A set of complex traits determines the quality of malted barley and its subsequent use for beer. Molecular genetics technology has increased our understanding of genetic control of the many malting and brewing quality traits, most of which are quantitatively inherited. The objective of this study was to further dissect and evaluate a known major malting quality quantitative trait locus (QTL) region of about 28 cM on chromosome 1 (7H). Molecular marker-assisted backcrossing was used to develop 39 isolines originating from a Steptoe / Morex cross. Morex, a 6–row malting type, was the donor parent and Steptoe, a 6–row feed type, was the recurrent parent. The isolines and parents were grown in four environments, and the grain was micro-malted and analyzed for malting quality traits. The effect of each Morex chromosome segment in the QTL target region was determined by composite interval mapping (CIM) and confirmed and refined by multiple interval mapping (MIM). One QTL was resolved for malt extract content, and two QTLs each were resolved for -amylase activity, diastatic power, and malt -glucan content. One additional putative malt extract QTL was detected at the plus border of the target region by CIM, but not confirmed by MIM. All QTLs were resolved to intervals of 2.0 to 6.4 cM by CIM, and to intervals of 2.0 cM or less by MIM. These results should facilitate marker-assisted selection in breeding improved malting barley cultivars.     

Genetic susceptibility to pre-eclampsia and chromosome 7q36

Pre-eclampsia is the most common serious medical disorder of human pregnancy. The human endothelial cell nitric oxide synthase (eNOS) gene is a candidate for pre-eclampsia/eclampsia (PE/E) susceptibility. A linkage study was performed on Australian PE/E families using 25 microsatellite markers from chromosome 7, one of which (eNOS-CA) resides within the eNOS gene. No significant linkage was found for the eNOS-CA marker using either parametric or non-parametric analysis. However, D7S 1805 from the eNOS gene region on 7q36, gave a suggestion of linkage using parametric analysis (maximum LOD score =2.143 at theta=0.14) and non-parametric APM analysis (T1/sqrt(p)=3.53; P=0.002). Further, an association study was performed on unrelated PE/E cases and controls from both Chinese and Australian populations to test for a relationship between the eNOS gene and PE/E. No association was found between the eNOS-CA marker and PE/E in either population. However, there was a significant difference in the allelic distribution of eNOS-CA between the two ethnic groups. The linkage results support the possibility that a susceptibility locus for pre-eclampsia resides in the 7q36 region, however, there is no definitive evidence to support the notion that the eNOS gene itself is responsible for susceptibility to pre-eclampsia.     

Genetic adaptation for vernalization requirement in Nepalese wheat and barley

A large number of accessions of covered and naked barley from eastern Nepal were grown without vernalization, and it was found that naked barley accessions were predominantly spring varieties while covered barley accessions were predominantly winter varieties. Seven accessions were subjected to a range of vernalization periods. Four naked varieties were spring varieties, although one showed some response to vernalization, but the three covered barleys were winter varieties. Although the majority of naked barleys are spring forms, they are winter sown at high altitudes and this does not conform to the distribution of naked barley described by Takahashi (1955). Wheat accessions which came from villages situated at high altitudes tended to have higher vernalization requirements than those which came from lower altitudes. This was taken to indicate local adaptation and a low movement of seeds (gene-flow) between villages. The relationship between vernalization requirement and altitude was not found in barley. Marked but contrasting regional patterns for vernalization requirement occurred in the wheat and covered barley. It was concluded that gene-flow was greater within regions than between them. This regional isolation together with environmental heterogeneity are major diversity promoting mechanisms.     

Integration of the barley genetic and seed proteome maps for chromosome 1H, 2H, 3H, 5H and 7H

Two-dimensional gel electrophoresis was used to screen spring barley cultivars for differences in seed protein profiles. In parallel, 72 microsatellite (simple sequence repeat (SSR)) markers and 11 malting quality parameters were analysed for each cultivar. Over 60 protein spots displayed cultivar variation, including peroxidases, serpins and proteins with unknown functions. Cultivars were clustered based on the spot variation matrix. Cultivars with superior malting quality grouped together, indicating malting quality to be more closely correlated with seed proteomes than with SSR profiles. Mass spectrometry showed that some spot variations were caused by amino acid differences encoded by single nucleotide polymorphisms (SNPs). Coding SNPs were validated by mass spectrometry, expressed sequence tag and 2D gel data. Coding SNPs can alter function of affected proteins and may thus represent a link between cultivar traits, proteome and genome. Proteome analysis of doubled haploid lines derived from a cross between a malting (Scarlett) and a feed cultivar (Meltan) enabled genetic localisation of protein phenotypes represented by 48 spot variations, involving e.g. peroxidases, serpins, α-amylase/trypsin inhibitors, peroxiredoxin and a small heat shock protein, in relation to markers on the chromosome map. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome.

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.     

The chromosomal locations of leaf peroxidase genes in hexaploid wheat,rye and barley

Summary Eight leaf peroxidase isozymes were distinguished by IEF in Chinese Spring. Two genes which control the production of three of these isozymes were located on chromosome arms 1BS and 1DS by nullisomic analysis. These loci probably form part of a homoeoallelic series and have been designated Per-B1 and Per-D1 respectively. Analysis of chromosome 1B short arm terminal deletion stocks indicated that the Per-B1 locus is located between the nucleolar organiser region and another isozyme marker, Hk-B1. Two variant leaf peroxidase phenotypes were distinguished in a small sample of hexaploid wheat varieties. Analysis of wheat-alien addition and substitution lines identified homoeologous loci in rye (Per-R1) and barley (Per-H1).     

Waxy protein deficiency and chromosomal location of coding genes in common wheat

Deficiency of the wheat waxy (Wx) proteins (Wx-A1, Wx-B1 and Wx-D1) was studied in 1,960 cultivars derived from several countries. Gel electrophoretic analyses revealed that the null allele for the Wx-A1 protein occurred frequently in Korean, Japanese and Turkish wheats but was relatively rare in cultivars from other countries and regions. About 48% of the wheats deficient for the Wx-B1 protein were from Australia and India. One Chinese cultivar lacked the WxD1 protein. While 9 Japanese cultivars were deficient in both the Wx-A1 and Wx-B1 proteins, no cultivars lacked both the Wx-A1 and Wx-D1 proteins, both the Wx-B1 and Wx-D1 proteins or all three Wx proteins. Two-dimensional gel electrophoresis revealed polymorphisms of the three Wx proteins that varied according to isoelectric points or molecular weight. The Wx-A1 gene coding the Wx-A1 protein and the Wx-B1 gene coding the Wx-B1 protein were localized in the distal regions of chromosome arms 7AS and 4AL, respectively, by deletion mapping using the deletion lines developed in the common wheat cultivar Chinese Spring.