Sequence composition and gene content of the short arm of rye (Secale cereale) chromosome 1

Background

The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide.

Methodology/Principal Findings

Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice.

Conclusions

The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye.     

The Sec-1 locus on the short arm of chromosome 1R of rye (Secale cereale)

This paper describes a detailed sequence analysis of the ω-secalin gene array at theSec-1 locus on the short arm of chromosome 1 of rye. The analysis shows that the genes are separated by 8 kb of spacer sequence and that the gene/spacer units are arranged in a head to tail fashion. The boundaries of the array are identified, and a fragment containing the majority of the genes in the array is separated by PFG analysis. The sequence data of one 9.2 kb gene unit have been determined, and because of the similarity of the gene units within the array these data provide a detailed sequence analysis of 140 kb of theSec-1 locus. Fluorescence in situ hybridization, using lambda clones isolated for the structural analysis, identifies the position of the array on the rye chromosomes relative to the 5S rRNA genes. Edited by: W. Hennig     

Development of microsatellite markers specific for the short arm of rye (Secale cereale L.) chromosome 1

We developed 74 microsatellite marker primer pairs yielding 76 polymorphic loci, specific for the short arm of rye chromosome 1R (1RS) in wheat background. Four libraries enriched for microsatellite motifs AG, AAG, AC and AAC were constructed from DNA of flow-sorted 1RS chromosomes and 1,290 clones were sequenced. Additionally, 2,778 BAC-end-sequences from a 1RS specific BAC library were used for microsatellite screening and marker development. From 724 designed primer pairs, 119 produced 1RS specific bands and 74 of them showed polymorphism in a set of ten rye genotypes. We show that this high attrition rate was due to the highly repetitive nature of the rye genome consisting of a large number of transposable elements. We mapped the 76 polymorphic loci physically into three regions (bins) on 1RS; 29, 30 and 17 loci were assigned to the distal, intercalary and proximal regions of the 1RS arm, respectively. The average polymorphism information content increases with distance from the centromere, which could be due to an increased recombination rate along the chromosome arm toward’s the telomere. Additionally, we demonstrate, using the data of the whole rice genome, that the intra-genomic length variation of microsatellites correlates (r = 0.87) with microsatellite polymorphism. Based on these results we suggest that an analysis of the microsatellite length variation is conducted for each species prior to microsatellite development, provided that sufficient sequence information is available. This will allow to selectively design microsatellite markers for motifs likely to yield a high level of polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300–1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library. Received: 5 December 1998; in revised form: 15 April 1999 / Accepted: 29 April 1999     

RFLP-based genetic map of rye (Secale cereale L.) chromosome 1R

Summary A map of chromosome 1R of rye was constructed using 16 molecular and biochemical loci. From long arm to short arm, known-function loci were placed in the order: XAdh — XLee — Glu-R1[Sec-3] — XPpdk-1R — XEm-1R-1 — XEm-1R-2 — Centromere — XNor-R1 —Gpi-R1 — XGli-R1 [Sec-1a] along with six anonymous genomic and cDNA clones from wheat. The map, which spans 106 cM with 12 loci clustered in a 15-cM region around the centromere, shows reasonably good agreement with previously published maps for the centromeric region, whereas the XNor-R1 — Gpi-R1 region gives a much larger distance than previously reported.     

A consensus map of chromosome 6R in rye (Secale cereale L.)

Four F₂ mapping populations derived from crosses between rye inbred lines DS2×RXL10, 541×Ot1-3, S120×S76 and 544×Ot0-20 were used to develop a consensus map of chromosome 6R. Thirteen marker loci that were polymorphic in more than one mapping population constituted the basis for the alignment of the four maps using the JoinMap v. 3.0 software package. The consensus map consists of 104 molecular marker loci including RFLPs, RAPDs, AFLPs, SSRs, ISSRs, SCARs, STSs and isozymes. The average distance between the marker loci is 1.3 cM, and the total map length is 135.5 cM. This consensus map may be used as a source of molecular markers for the rapid development of new maps of chromosome 6R in any mapping population.     

New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat

Subcloning of a clone of the 120-bp family of rye, pSc119, has produced two extremely useful probes. pSc119.1 assays rye-specific dispersed repetitive sequence families. It is present on all seven rye chromosomes and hybridizes to the entire length of each chromosome, with the exception of some telomeres and the nucleolar organiser region. pSc119.2, in contrast, hybridizes predominantly to the telomeric regions of rye chromosomes, with some interstitial sites. Unlike pSc119.1, it assays similar repetitive sequence families in both wheat and rye chromosomes.     

Identification and mapping of the Gli-R3 locus on chromosome 1R of rye (Secale cereale L.)

Summary The progenies of two different rye test-crosses were analyzed for secalin proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using unreduced and reduced aqueous ethanol extracts. Segregation for two high-molecular-weight secalin bands (Glu-R1 or Sec3), one -secalin band (Gli-R1 or Sec-1), two 40K -secalin bands (Gli-R1 or Sec1) and two -type secalin bands (new locus) were studied. One recombinant between - and -secalins was found in one test-cross. The new locus, designated Gli-R3 or Sec-4, was mapped between Glu-R1 and Gli-R1, more displaced towards Gli-R1. In test-cross 1 recombination between Glu-R1 and Gli-R3 was 33.80±3.22%, and between Gli-R3 and Gli-R1, 12.04±2.21%. In the other test-cross the map distances were relatively similar but smaller, likely due to less recombination within two different species of Secale. Genes coding for 40K -secalins at Gli-R1 were likely proximal to the centromere with respect to genes coding for -secalins at the same complex locus.     

Detection of quantitative trait loci on chromosome 5R of rye (Secale cereale L.)

 Progenies of an F₂ mapping population were analyzed for quantitative traits to detect QTLs by using marker information from F₂ plants for chromosome 5R. The mapping population was segregating for the major dwarfing gene Ddw1 and the gene Hp1 for hairy peduncle. The only QTL determining plant height was located between HP1 and Ddw1 on the distal part of chromosome 5RL. At the same position a QTL for peduncle length was found, and this trait was closely related to plant height (r=0.895). Since Hp1 and Ddw1 are dominant marker loci, no dominance effect could be estimated. The QTLs for spike length and the number of florets were located near the centromere on 5RL. These two traits were correlated with r=0.824 and showed partial dominance, but these traits were not correlated to plant height and peduncle length. Homoeologous relationships between the QTLs mapped for the first time in rye and those mapped in other Triticeae members are discussed. Received: 8 June 1998 / Accepted: 8 October 1998     

Organellar genome analysis of rye (Secale cereale) representing diverse geographic regions.

Rye (Secale cereale) is an important diploid (2n = 14, RR) crop species of the Triticeae and a better understanding of its organellar genome variation can aid in its improvement. Previous genetic analyses of rye focused on the nuclear genome. In the present study, the objective was to investigate the organellar genome diversity and relationships of 96 accessions representing diverse geographic regions using chloroplast (cp) and mitochondrial (mt) DNA PCR-RFLPs. Seven cpDNA and 4 mtDNA coding and noncoding regions were amplified using universal cpDNA and mtDNA primer pairs. Each amplified fragment was digested with 13 different restriction enzymes. mtDNA analysis indicated that the number of polymorphic loci (20) was low and genetic differentiation (GST) was 0.60, excluding the outgroups (hexaploid wheat, Triticum aestivum, 2n = 6x = 42, AABBDD; triticale, xTriticosecale Wittmack, 2n = 6x = 42, AABBRR). cpDNA analysis revealed a low level of polymorphism (40%) among the accessions, and GST was 0.39. Of the 96 genotypes studied, 70 could not be differentiated using cpDNA PCR-RFLPs even though they are from different geographic regions. This is most likely due to germplasm exchange, indicating that genotypes might have a common genetic background. Two cpDNA and 3 mtDNA fragments were significantly correlated to the site of germplasm collection. However, there was no clear trend. These results indicate that the level of organellar polymorphism is low among the cultivated rye genotypes. The cpDNA and mtDNA PCR-RFLP markers used in the present study could be used as molecular markers in rye genetics and breeding programs.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Hormone independent root organ cultures of rye (Secale cereale)

This work describes the growth of rye root organ cultures which were capable of being repeatedly subcultured in hormone-free medium. They showed morphological characteristics, growth rate, inability to produce shoots, and response to auxins and cytokinins similar to those of the Agrobacterium rhizogenes (Ri plasmid) transformed hairy root cultures of tobacco and red beet which were used for comparison. The root cultures of rye were initiated from callus produced on a medium containing the growth regulators (plant hormones) 2,4-d and kinetin, then transferred to hormone-free medium. However not all rye explants gave rise to callus that would differentiate into stable hairy root cultures and rye seedling root explants did not grow if placed directly on a hormone-free medium. Rice and wheat produced callus and roots on a medium containing hormones but root organ cultures could not be maintained on a hormone-free medium.     

Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.     

A genetic linkage map of rye (Secale cereale L.)

 A genetic linkage map of rye composed of 91 loci (88 RFLP, two morphological and one isozyme markers) has been developed using two reciprocal crosses. The RFLP loci covering all seven chromosomes were detected by a selection of rye, wheat, barley and oat cDNA and genomic DNA probes. The level of polymorphism was dependent on the source of the clones, with a ranking of rye>wheat>barley>oat. Distorted segregations were detected in linkage groups of chromosomes 1R, 4R, 5R and 7R. When the recombination of the two reciprocal crosses was compared, no systematic increase or decrease in one or the other direction was observed suggesting that a combination of populations of reciprocal crosses is possible. Received: 5 August 1997/Accepted: 2 September 1997     

Genetic composition of the short-stem rye populations (Secale cereale L.) for secaline genes

Genetic composition for secaline genes was studied in four short stem rye populations (Bolgarskaya korotkostebelnaya, Gnom 1, Gnom 2, and Gnom 3) using the methods of classical hybridological analysis and population genetics. Eight, three, and four alleles of the genes Sec-3, Sec-2, Sec-1 correspondingly have been revealed in the studied populations. The frequencies of the specific alleles in each population were determined. The allele transmission to the next generation is restricted through the linkage of the secaline genes with the genes of self-incompatibility, S1 and Z, located on the same chromosomes as the secaline genes.     

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.     

RFLP mapping of the dwarfing (Ddw1) and hairy peduncle (Hp) genes on chromosome 5 of rye (Secale cereale L.)

An F₂ population was established for mapping the two dominant genes for dwarfness (Ddw1) and hairy peduncle (Hp) on chromosome 5R. The location of both genes was shown to be on the segment of chromosome 5RL which was ancestrally translocated and is homoeologous to Triticeae 4L. Hp cosegregated with the wheat gDNA probe WG199, localised in wheat on chromosomes 5AL, 4BL and 4DL. No segregation was observed between the traits hairy peduncle and hairy leaf sheath. The locus for Ddw1 was found to map distally to Hp/Xwg199 but proximal to the isozyme marker -amy-R1. The genetical distances were 5.6 cM between Hp/Xwg199 and Ddw1 and ll.ScM between Ddw1 and -amy-R1, respectively. The map position of Ddw1 suggests that it is homoeologous to the wheat dominant dwarfing gene Rht12, present on chromosome 5AL and linked to -amy-A1.     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

Transfer to wheat (Triticum aestivum) of small chromosome segments from rye (Secale cereale) carrying disease resistance genes

One hundred wheat lines, derived from monosomic additions of chromosome 1R of rye inbred line R12 (Chinese rye), were detected by PCR amplification using rye-specific primer pairs. Only 5 wheat lines, 1R296, 1R330, 1R314, 1R725, and 1R734, were determined to contain rye chromatin. While 1R296 and 1R330 were highly susceptible to stripe rust and powdery mildew, 1R314, 1R725 and 1R734 were highly resistant to both diseases. Acid-polyacrylamide gel electrophoresis showed that the ω-secalin bands were absent in 1R314, but present in the other 4 wheat lines. Genomicin situ hybridization indicated that 1R296, 1R330, and 1R725 contained translocations involving the whole short arm of chromosome 1R. However, 1R314 and 1R734 contained a pair of wheat chromosomes with small, terminal, rye-derived chromosome segments. The results suggest that the translocation breakpoint of 1RS in 1R314 was located between theSec-1 locus and the disease-resistance loci, while in line 1R734, the breakpoint was located between theSec-1 locus and the centromere. Taking account of the improved disease resistance of 1R725, 1R314 and 1R734, the chromosome arm 1RS of R12 may represent new and valuable disease resistance resources for wheat improvement.     

Microdissection and Recovery of 1R Chromosome in Rye ( Secale cereale )

The technique of mieromanipulaion has been used to establish a system of single chromosome mierodissection. According to the standard karyotype of rye ( Secale cereale L. ), 1R chromosome carrying disease-resistant gene has been identified, microdissected and transferred into Ep-pendorf tube using mieromanipulator. The results showed that IR chromosome in cells pretreated with α-bromonaphthalene could be identified quickly and the efficiency of microdissection was greatly improved when the technique of wall degradation and hypotonic treatment was applied.