Elimination and duplication of particular Hordeum vulgare chromosomes in aneuploid interspecific Hordeum hybrids

Summary Seeds formed in crosses Hordeum lechleri (6x) x H. vulgare (2x and 4x), H. arizonicum (6x) x H. v. (2x), H. parodii (6x) x H. v. (2x), and H. tetraploidum (4x) x H. v. (2x) produced plants at high or rather high frequencies through embryo rescue. Giemsa C-banding patterns were used to analyze chromosomal constitutions and chromosomal locations on the methaphase plate. Among 100 plants obtained from H. vulgare (2x) crosses, 32 plants were aneuploid with 2n=29 (1), 28 (3), 27 (13), 26 (5), 25 (4), 24 (4), or 22 (2); 50 were euploid (12 analyzed), and 18 were polyhaploid (5 analyzed). Four plants had two sectors differing in chromosome number. Two of four hybrids with H. vulgare (4x) were euploid and two were aneuploid. Parental genomes were concentrically arranged with that of H. vulgare always found closest to the metaphase centre. Many plants showed a certain level of intraplant variation in chromosome numbers. Except for one H. vulgare (4x) hybrids, this variation was restricted to peripherally located non-H. vulgare genomes. This may reflect a less firm attachment of the chromosomes from these genomes to the spindle. Interplant variation in chromosome numbers was due to the permanent elimination or, far less common, duplication of the centrally located H. vulgare chromosomes in all 34 aneuploids, and in a few also to loss/gain of non-H, vulgare chromosomes. This selective elimination of chromosomes of the centrally located genome contrasts conditions found in diploid interspecific hybrids, which eliminate the peripherally located genome. The difference is attributed to changed genomic ratios. Derivatives of various H. vulgare lines were differently distributed among euploid hybrids, aneuploids, and polyhaploids. Chromosomal constitutions of hypoploid hybrids revealed a preferential elimination of H. vulgare chromosomes 1, 5, 6, and 7, but did not support the idea that H. vulgare chromosomes should be lost in a specific order. H. vulgare SAT-chromosomes 6 and 7 showed nucleolar dominance. Aneuploidy is ascribed to the same chromosome elimination mechanism that produces haploids in cross-combinations with H. vulgare (2x). The findings have implications for the utilization of interspecific Hordeum hybrids.     

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)

Summary In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F₁ plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC₁ plants were obtained. Three of the BC₁ plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC₁ plants (BC₁-5) in BamHI-digésted genomic Southern blots. Plant BC₁-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC₁ plants (BC₁-1 and BC₁-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC₂ plants from plant BC₁-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 11, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC₁-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC₁ plants, randomly sampled from the 79 BC₁ plants, also segregated 2320 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 11 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.     

Genome relationships between Hordeum vulgare L. and H. bulbosum L.

Interrelationships between H. vulgare (2x=14) and H. bulbosum (2x=14; 4x=28) were estimated on the basis of the karyotypes and the pairing behaviour of the chromosomes in diploid, triploid and tetraploid hybrids obtained with the aid of embryo culture. — A comparison of the karyotypes of the two species revealed similarities as well as differences. It was concluded that at least 4 or more of the chromosomes were similar in morphology and probably closely related. — Diploid and tetraploid hybrids are rarely obtained and their chromosome numbers tend to be unstable whereas triploid hybrids (1 vulgare + 2 bulbosum genomes) were stable and relatively easy to produce. In the diploid hybrid only 40% of the meiotic cells contained 14 chromosomes while the numbers ranged from 7 to 16 in other cells. All hybrids exhibited pairing between the chromosomes of the two species. Diploid hybrids had a mean of 5.0 and a maximum of 7 bivalents per cell in those cells having 14 chromosomes. Triploid hybrids from crosses between 2x H. vulgare and 4x H. bulbosum exhibited a mean of 1.5 and a maximum of 5 trivalents per cell. In a hexaploid sector found following colchicine treatment of a triploid the mean frequencies of chromosome associations per cell were: 5.5_I+8.0_II+0.7_III+3.7_IV+0.3_V+0.4_VI. One unstable 27 chromosome hybrid obtained from crosses between the autotetraploid forms had a mean of 1.1 and a maximum of 4 quadrivalents per cell. The chromosome associations observed in these hybrids are consistent and are taken as evidence of homoeologous pairing between the chromosomes of the two species. Interspecific hybridization between these two species also reveals that chromosome stable hybrids are only obtained when the genomes are present in a ratio of 1 vulgare2 bulbosum. Based upon the results obtained, the possibility of transferring genetic characters from H. bulbosum into cultivated barley is discussed.     

Selective chromosomal elimination during haploid formation in barley following interspecific hybridization

Cytological observations were made on embryo and endosperm tissues with different genome combinations that were produced by crossing the diploid and tetraploid cytotypes of Hordeum vulgare and H. bulbosum. The high frequency of barley haploids results from hybridization followed by the selective elimination of bulbosum chromosomes during the early development of embryos which initially contained a ratio of 1 vulgare to 1 bulbosum genomes. Elimination is gradual as indicated by the increase in the percentage of cells with the gametic chromosome number. However, the balance between genetic factors of the two parents appears to regulate the stability or elimination of chromosomes. Triploid embryos containing 1 vulgare to 2 bulbosum genomes are relatively stable. The most stable endosperm tissues examined had a ratio of 1 vulgare to 4 bulbosum genomes. Evidence of genetic control in both the vulgare and bulbosum chromosomes and their interaction is discussed. As has been suggested by Lange (1971) and also found in mammalian somatic cell hybrids, the most probable basis for selective chromosome elimination relates to mitotic rhythm and the duration of cell cycle phases.     

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis

Summary Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1–7), but were mixoploid with respect to alien chromosomes (19–22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3–5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv Pallas resulted in a plant with 2n = 53–56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line (Haisa II), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line (9208/9) resulted in plant with 2n = 51–53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.     

Characterization of relic DNA from barley genome

Summary High-molecular-weight relic DNA fraction can be electrophoretically separated from the bulk of barley DNA digested with different restriction enzymes. We have cloned and analyzed a population of relic DNA fragments. The majority of AluI-relic DNA clones contained barley simple sequence satellite DNA and other families of repetitive DNA. One of these families, designated HvRT, has been analyzed in detail. This family is composed of tandemly arranged 118-bp monomers and is present in 7 × 10⁵ copies in the barley genome. Clones representing the HvRT family were sequenced. HvRT repeats were found to contain high levels of methylated cytosine. The HvRT family was found in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but not in H. marinum, H. geniculatum, and wheat. Different barley species and cultivars show restriction fragment length polymorphism with the HvRT probe. Chromosome-specific subfamilies of HvRT were found to be present on different barley chromosomes, providing the possibility of using the HvRT probe as a chromosome specific marker. HvRT fragments up to 810 kbp in length were resolved by pulsed field gel electrophoresis.     

The chromosomes and DNA of Allium cepa

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs₂SO₄-Ag⁺ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C₀t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a short period interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.     

Distribution,functional impact,and origin mechanisms of copy number variation in the barley genome

Background

There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys.

Results

A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley.

Conclusions

We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.     

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.     

Repeated DNA sequences isolated by microdissection. II. Comparative analysis in Hordeum vulgare and Triticum aestivum

The genomic organization of two satellite DNA sequences, pHvMWG2314 and pHvMWG2315, of barley (Hordeum vulgare, 2n=14, HH) was studied by comparative in situ hybridization (ISH) and PCR analysis. Both sequences are members of different RsaI families. The sequence pHvMWG2314 is a new satellite element with a monomer unit of 73 bp which is moderately amplified in different grasses and occurs in interstitial clusters on D-genome chromosomes of hexaploid wheat (Triticum aestivum, 2n=42, AABBDD). The 331-bp monomer pHvMWG2315 belongs to a tandemly amplified repetitive sequence family that is present in the Poaceae and preferentially amplified in Aegilops squarrosa (2n=14, DD), H. vulgare and Agropyron elongatum. (2n=14, EE). The first described representative of this family was pAs 1 from Ae. squarrosa. Different sequences of one satellite DNA family were amplified from Ae. squarrosa, A. elongatum and H. vulgare using PCR. Characteristic differences between members of the D and H genome occurred in a variable region which is flanked by two conserved segments. The heterogeneity within this element was exploited for the cytogenetic analysis of Triticeae genomes and chromosomes. Comparative ISH with pHvMWG2315 identified individual wheat and barley chromosomes under low (75%) and high (85%) hybridization stringency in homologous and heterologous systems. We propose the designation Tas330 for the Triticeae amplified sequence (Tas) satellite family with a 330 bp average monomer length.     

Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome

The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 × 10⁴. We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.     

Barley doubled-haploid production by uniparental chromosome elimination

Uniparental elimination of chromosomes, which occurs in interspecific crosses between Hordeum vulgare (cultivated barley) and H. bulbosum (bulbous barley grass), is a process which can be used to produce doubled-haploid barley plants in breeding programs. We review the procedure of haploid production and the mechanism underlying selective elimination of one of the genomes during the early development of species hybrid embryos.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.     

Features of Crossability, Haploidy and Polyembryony in Hybrid Combinations between Cultivated Barley Hordeum vulgare L. (2n = 14) and Wheat-Rye Substitution Lines Triticum aestivum L., Cultivar Saratovskaya 29/Secale cereale L., Cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between cultivated barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters—frequency of set grains and frequency of grains with embryos—was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare × T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between cultivated barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 784–792.Original Russian Text Copyright © 2005 by Pershina, Belova, Devyatkina, Rakovtseva, Kravtsova, Shchapova.     

Analysis of QTLs for yield, yield components, and malting quality in a BC3-DH population of spring barley

Advanced backcross (AB)-quantitative trait locus (QTL) analysis has been successfully applied for detecting and transferring QTLs from unadapted germplasm into elite breeding lines in various plant species. Here, we describe the application of a modified AB breeding scheme to spring barley. A BC₃-doubled haploid (DH) population consisting of 181 lines derived from the German spring barley cultivar Brenda (Hordeum vulgare subsp. vulgare) as the recurrent parent and the wild species line HS213 (H. vulgare subsp. spontaneum) as the donor line was evaluated for yield and its components as well as malting quality traits. A set of 60 microsatellite markers was used to genotype the population, and phenotypic data were collected at two locations in Germany in continuous years. Altogether, 25 significant QTLs were detected by single-marker regression analysis and interval mapping. Most positive QTLs originated from the recurrent parent Brenda. A QTL, Qhd2.1, on chromosome 2HS from Brenda explained 18.3% and 20.7% of the phenotypic variation for yield and heading date, respectively. Due to the small percentage of donor-parent genome of 6.25%, the BC₃-DH lines could be directly used for the extraction of near-isogenic lines (NILs) for Qhd2.1. Consequently, it was possible to determine the precise location of the locus hd2.1 within a region of 6.5 cM, using an F₂ population consisting of 234 individuals developed from a cross between an NIL containing a defined donor segment at this locus and Brenda. The location of this QTL was consistent with the presence of a major photoperiod response gene, Ppd-H1, previously reported in this region, which is associated with pleiotropic effects on yield components. In summary, the analysis of a BC₃-DH population in barley provides a compromise between the analysis of QTLs by means of an AB scheme and the generation of defined substitution lines. Several lines carrying defined different donor segments for only one single chromosome or trait in the genetic background of Brenda could be selected for further genetic studies.     

Mitochondrial genome SNPs analysis of eight barley genotypes displaying hot spot regions,phylogenetic relationships and heteroplasmy

Abstract

Organellar genomes are small, circular entities that provide unique advantages as compared to the nuclear genome. The present study was aimed at evaluating the efficiency of utilizing mitochondrial single nucleotide polymorphisms (SNPs) approach in separating barley cultivars. Sequences generated via next-generation sequencing were further utilized to confirm the incidence of heteroplasmy in barley mitochondrial genome. The analysis involved seven cultivated barley (Hordeum vulgare subsp. vulgare) (VG) and one wild (H. vulgare subsp. spontaneum) (SP) genotypes. A total of 73 million paired-end reads per mitochondrial genomes across the eight barley genotypes were generated using Illumina HiSeq 2000 platform. Sequences of each genotype were separately aligned to the published barley mitochondrial reference genome, thus SNPs were detected. The overall results indicated the efficiency of using mitochondrial SNPs as a molecular marker in distinguishing among barley genotypes. Unique SNPs were determined in six out of the eight genotypes, where Giza131 and Giza129 had no specific mitochondrial SNPs, while Giza130 showed the largest number of unique mitochondrial SNPs. The phylogenetic tree indicated the close relationship between Giza129 and Giza130. Interestingly, SP was not clearly discriminated among genotypes.     

Analysis of chromosomal polymorphism in barley (Hordeum vulgare L. ssp. vulgare) and between H. vulgare and H. chilense using three-color fluorescence in situ hybridization (FISH)

The aim of the present work was to study chromosomal polymorphism within cultivated barley (Hordeum vulgare ssp. vulgare) using three-color fluorescence in situ hybridization (FISH). The physical distribution of the most frequently used, highly repetitive DNA sequences (GAA)₇ specific for pericentromeric heterochromatic regions, the ribosomal DNA clone pTa71, specific for the 45S rDNA, and the barley-specific telomere-associated sequence HvT01, was investigated to reveal genetic diversity in metaphase spreads of ten barley genotypes with diverse geographical origin, growth habit and row number. A wild relative of barley, Hordeum chilense was also studied in order to compare the polymorphism between and within Hordeum species. Significant differences in the hybridization patterns of all three DNA probes could be detected between the two related species, but only probes pTa71 and HvT01 showed variation in the intensity and/or position of hybridization sites among genotypes of H. vulgare ssp. vulgare. The extent of polymorphism was less than that earlier reported for molecular markers and was restricted to the long chromosome arms, with differences between the chromosomes. 1H and 3H proved to be the most variable chromosomes and 4H and 6H the most conserved.     

PCR detection of Hordeum bulbosum introgressions in an H. vulgare background using a retrotransposon-like sequence

Retrotransposon-like sequences are ideal tools for initial screening assays to distinguish between closely related species because of their ubiquitous presence, high copy number, chromosome coverage and rapid sequence evolution. A retrotransposon-like sequence, pSc119.1, cloned from Secale cereale (rye) has been used to obtain PCR primers that are capable of detecting small introgressions of Hordeum bulbosum (bulbous barley grass) chromatin in a Hordeum vulgare (cultivated barley) background. Combining this PCR-based assay with a crude but effective high-throughput DNA extraction has enabled the rapid identification of plants possessing H. bulbosum introgressions from large numbers of progeny from H. vulgare×H. bulbosum crosses. These plants are then further characterized by more-refined cytological, molecular and pathological techniques to locate and map the introgressed chromatin and to evaluate their disease resistance. Received: 18 April 2001 / Accepted: 23 August 2001