Physical analysis of the complex rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) <Emphasis Type="Italic">Alt4</Emphasis> aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library’s suitability for investigating Al tolerance genes. The library provides 6 × genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The ScAACT1 gene at the Q alt5 locus as a candidate for increased aluminum tolerance in rye (Secale cereale L.)

Soluble aluminum (Al³⁺) is a major constraint to plant growth in highly acidic soils, which comprise up to 50% of the world??s arable land. The primary mechanism of Al resistance described in plants is the chelation of Al³⁺ cations by release of organic acids into the rhizosphere. Candidate aluminum tolerance genes encoding organic acid transporter of the ALMT (aluminum-activated malate transporter) and MATE (multi-drug and toxic compound extrusion) families have been characterized in several plant species. In this study, we have isolated in five different cultivars the rye ScAACT1 gene, homolog to barley aluminum activated citrate transporter HvAACT1. This gene mapped to the 7RS chromosome arm, 25?cM away from the ScALMT1 aluminum tolerance gene. The gene consisted of 13 exons and 12 introns and encodes a predicted membrane protein that contains the MatE domain and at least seven putative transmembrane regions. Expression of the ScAACT1 gene is Al-induced, but there were differences in the levels of expression among the cultivars analyzed. A new quantitative trait locus for Al tolerance in rye that co-localizes with the ScAACT1 gene was detected in the 7RS chromosome arm. These results suggest that the ScAACT1 gene is a candidate gene for increased Al tolerance in rye. The phylogenetic relationships between different MATE proteins are discussed.     

Aluminium tolerance in triticale,wheat and rye as measured by root growth characteristics and aluminium concentration

Summary Screening large populations of plant species for Al tolerance requires simple and rapid tests. In this study, root characteristics of 12 cultivars of triticale (X Triticosecale, Witt Mack), wheat (Triticum aestivum L.), and rye (Secale cereale L.) were measured in nutrient solution with 0 or 6 ppm Al added. Aluminum injury to roots of triticale and wheat was characterized by decreases in root length, increases in the number of roots, and in Al-sensitive Redcoat and Arthur wheats by decrease in root weight. Root length and number of roots were correlated in triticale (r=−0.73^*) and in wheat (r=−0.85^*). Root length was also correlated with root weight in wheat (r=0.65^*); there was no relationship between the number of roots and weight. Differences in Al tolerance of cultivars of the three species were greater when the solution was adjusted to pH 4.8 only on the first day of the experiment than when pH was maintained at pH 4.8 throughout the growing period. Triticale and rye cultivars low in ability to increase solution pH gradually overcame Al toxicity by increasing the nutrient solution pH between 12 and 22 days. Aluminum sensitive triticale and wheat accumulated more Al in roots than tolerant cultivars when the solution pH was not adjusted daily; but no differences in Al accumulation were obtained between wheat cultivars at constant pH value. This study indicated that root length and number of roots can be reliably used for screening triticales for Al tolerance within 12 days of exposure to Al. Root length, Al concentration, and dry weight after 22 days of Al treatment were also reliable criteria for evaluating differential Al tolerances among triticale cultivars.     

Isolation and characterization of a new MATE gene located in the same chromosome arm of the aluminium tolerance (Alt1) rye locus

• Aluminium (Al) toxicity is the major constraint for crop productivity in acid soils. Wild rye species (Secale spp.) exhibit high Al tolerance, being a good source of genes related to this trait. The Alt1 locus located on the 6RS chromosome arm is one of the four main loci controlling Al tolerance in rye and is known to harbour major genes but, so far, none have been found.
• Through synteny among the short arm of the rye chromosome 6R and the main grass species, we found a candidate MATE gene for the Atl1 locus, later named ScMATE3, which was isolated and characterized in different Secale species.
• The sequence comparisons revealed both intraspecific and interspecific variability, with high sequence conservation in the Secale genus. SNP with replacement substitution that changed the structure of the protein and can be involved in the Al tolerance trait were found in ScMATE3 gene. The predicted subcellular localization of ScMATE3 is the vacuolar membrane which, together with the phylogenetic relationships performed with other MATE genes of the Poaceae related to Al detoxification, suggest involvement of ScMATE3 in an internal tolerance mechanism. Moreover, expression studies of this gene in rye corroborate its contribution in some Al resistance mechanisms.
• The ScMATE3 gene is located on the 6RS chromosome arm between the same markers in which the Alt1 locus is involved in Al resistance mechanisms in rye, thus being a good candidate gene for this function.

     

A comparison of physical distribution of recombination in chromosome 1R in diploid rye and in hexaploid triticale

Summary Polymorphism for six C-bands on chromosome 1R was used to study the frequency and distribution of recombination along the chromosome in a diploid rye (Secale cereale L.) and in a hexaploid triticale (X Triticosecale Wittmack) derived from it. In rye, the total recombination frequency in five segments of chromosome 1R was 93.7%. Recombination was concentrated in the distal regions of both chromosome arms and was infrequent in the proximal regions. In hexaploid triticale the total recombination frequency in the same chromosome was reduced to 51.7%. In both backgrounds the distal half of the long arm showed similar recombination frequencies, 51.4% and 45.7% for rye and triticale, respectively. The remaining about two-thirds of the chromosome length showed 42.3% recombination in rye but only 6% recombination in triticale. The results demonstrate that the genetic background in which mapping is performed not only affects the total amount of recombination, but also its distribution along the chromosome length.     

Loss of chromosomes 2R and 5RS in octoploid triticale selected for agronomic traits

The advanced lines of octoploid triticale which have been bred for nearly a half century in China show significant improvements in agronomic traits such as plant height, fertility, threshability, maturity and seed plumpness, although no intentional cytological selection had been performed. In this study, eight primary and six advanced lines were analyzed by fluorescence and genomic in situ hybridization to elucidate their chromosome constitutions. In the advanced lines, about 70% of the plants examined had 2n = 56 chromosomes (range: 50 to 58). Almost all advanced lines, however, had lost rye chromosome 2R and the short arm of 5R (5RS). The exceptions were lines Y1005 and Y4683: The former had lost only the 2R chromosome and the latter only 5RS. The reduction of rye chromosomes was compensated by an extra pair of 2D or A-genome (possibly 2A) chromosomes in plants with 2n = 56. This suggests that the loss of 2R and 5RS chromosomes contributes to the improvement of octoploid triticale. Since the plants with chromosome 2R are non-free threshing and chromosome 2D of synthetic wheat is known to carry the Tg (tenacious glumes) gene, it is possible that chromosome 2R carries a gene affecting the threshability, and we carried out selection to remove it. We also discuss the possible relationshipbetween 5RS and the genetic stability of octoploid triticale.     

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.     

How a microbial drug transporter became essential for crop cultivation on acid soils: aluminium tolerance conferred by the multidrug and toxic compound extrusion (MATE) family

Background

Aluminium (Al) toxicity is a major agricultural constraint for crop cultivation on acid soils, which comprise a large portion of the world''s arable land. One of the most widely accepted mechanisms of Al tolerance in plants is based on Al-activated organic acid release into the rhizosphere, with organic acids forming stable, non-toxic complexes with Al. This mechanism has recently been validated by the isolation of bona-fide Al-tolerance genes in crop species, which encode membrane transporters that mediate Al-activated organic acid release leading to Al exclusion from root apices. In crop species such as sorghum and barley, members in the multidrug and toxic compound extrusion (MATE) family underlie Al tolerance by a mechanism based on Al-activated citrate release.

Scope and Conclusions

The study of Al tolerance in plants as conferred by MATE family members is in its infancy. Therefore, much is yet to be discovered about the functional diversity and evolutionary dynamics that led MATE proteins to acquire transport properties conducive to Al tolerance in plants. In this paper we review the major characteristics of transporters in the MATE family and will relate this knowledge to Al tolerance in plants. The MATE family is clearly extremely flexible with respect to substrate specificity, which raises the possibility that Al tolerance as encoded by MATE proteins may not be restricted to Al-activated citrate release in plant species. There are also indications that regulatory loci may be of pivotal importance to fully explore the potential for Al-tolerance improvement based on MATE genes.     

Mapping the nucleolus organizer region,seed protein loci and isozyme loci on chromosome 1R in rye

Summary The nucleolus organizer region located on the short arm of chromosome 1R of rye consists of a large cluster of genes that code for ribosomal RNA (designated the Nor-R1 locus). The genes in the cluster are separated by spacer regions which can vary in length in different rye lines. Differences in the spacer regions were scored in two families of F₂ progeny. Segregation also occurred, in one or both of the families, at two seed protein loci and at two isozyme loci also located on chromosome 1R. The seed protein loci were identified as the Sec 1 locus controlling -secalins located on the short arm of chromosome 1R and the Sec 3 locus controlling high-molecular-weight secalins located on the long arm of 1R. The two isozyme loci were the Gpi-R1 locus controlling glucose-phosphate isomerase isozymes and the Pgd 2 locus controlling phosphogluconate dehydrogenase isozymes. The data indicated linkage between all five loci and map distances were calculated. The results indicate a gene order: Pgd 2 ... Sec 3 ... [centromere] ... Nor-R1 ... Gpi-R1 ... Sec 1. Evidence was obtained that rye possesses a minor 5S RNA locus (chromosome location unknown) in addition to the major 5S RNA locus previously shown to be located on the short arm of chromosome 1R.     

Cytogenetic analysis of forms produced by crossing hexaploid triticale with common wheat

Summary Hexaploid triticales were crossed with common wheats, and the resultant froms were selected for either triticale (AD 213/5-80) or common wheat (lines 381/80, 391/80, 393/80). The cytogenetic analysis showed that all forms differ in their chromosome composition. Triticale AD 213/5-80 and wheat line 381/80 were stable forms with 2n = 6x = 42. Lines 391/80 and 393/80 were cytologically unstable. In triticale AD 213/5-80, a 2R (2D) chromosome substitution was found. Each of the three wheat lines had a chromosome formed by the translocation of the short arm of IR into the long arm of the IB chromosome. In line 381/80, this chromosome seems to be inherited from the Kavkaz wheat variety. In lines 391/80 and 393/80, this chromosome apparently formed de novo since the parent forms did not have it. The karyotype of line 381/80 was found to contain rye chromosomes 4R/7R, 5R and 7R/4R. About 15% of the cells in line 391/80 contained an isochromosome for the 5R short arm and also a chromosome which arose from the translocation of the long arms of the 5D and 5R chromosomes. About one-third of the cells in the common wheat line 393/80 contained the 5R chromosome. This chromosome was normal or rearranged. Practical applications of the C-banding technique in the breeding of triticale is discussed.     

A new aluminum tolerance gene located on rye chromosome arm 7RS

Rye has one of the most efficient groups of genes for aluminum tolerance (Alt) among cultivated species of Triticeae. This tolerance is controlled by, at least, three independent and dominant loci (Alt1, Alt2, and Alt3) located on chromosome arms 6RS, 3RS, and 4RL, respectively. The segregation of Alt genes and several random amplified polymorphic DNA (RAPD), Secale cereale inter-microsatellite (SCIM), and Secale cereale microsatellite (SCM) markers in three F(2) between a tolerant cultivar (Ailés) and a non-tolerant inbred line (Riodeva) were studied. The segregation ratio obtained for aluminum tolerance in the three F(2) populations analyzed was 3:1 (tolerant:non-tolerant), indicating that tolerance is controlled by one dominant locus. SCIM811(1376) was linked to an Alt gene in the three F(2) populations studied, and three different SCIMs and one RAPD (SCIM811(1376), SCIM812(626), SCIM812(1138), and OPQ4(725)) were linked to the Alt gene in two F(2) populations. This result indicated that the same Alt gene was segregating in the three crosses. SCIM819(1434) and OPQ4(578) linked to the tolerance gene in one F(2) population were located using wheat-rye ditelosomic addition lines on the 7RS chromosome arm. The Alt locus is mapped between SCIM819(1434) and the OPQ4(578) markers. Two microsatellite loci (SCM-40 and SCM-86), previously located on chromosome 7R, were also linked to the Alt gene. Therefore, the Alt gene segregating in these F(2) populations is new and probably could be orthologous to the Alt genes located on wheat chromosome arm 4DL, on barley chromosome arm 4HL, on rye chromosome arm 4RL, and rice chromosome 3. This new Alt gene located on rye chromosome arm 7RS was named Alt4. A map of rye chromosome 7R with the Alt4 gene, 16 SCIM and RAPD, markers and two SCM markers was obtained.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

Development of commercially valuable traits in hexaploid triticale lines with Aegilops introgressions as dependent on the genome composition

Introgressive hybridization is an efficient means to improve the genetic diversity of cultivated cereals, including triticale. To identify the triticale lines with Aegilops introgressions, genotyping was carried out with ten lines obtained by crossing hexaploid triticale with genome-substitution forms of the common wheat cultivar Avrora: Avrolata (AABBUU), Avrodes (AABBSS), and Avrotika (AABBTT). The genome composition of the triticale lines was studied by in situ hybridization, and recombination events involving Aegilops and/or common wheat chromosomes were assumed for nine out of the ten lines. Translocations involving rye chromosomes were not observed. Substitutions for rye chromosomes were detected in two lines resulting from crosses with Avrolata. Genomic in situ hybridization (GISH) with Ae. umbellulata DNA and molecular genetic analysis showed that chromosome 1R was substituted with Ae. umbellulata chromosome 1U in one of the lines and that 2R(2U) substitution took place in the other line. Fluorescence in situ hybridization (FISH) with the Spelt 1 and pSc119.2 probes revealed a translocation from Ae. speltoides to the long arm of chromosome 1B in one of the two lines resulting from crosses with Avrodes and a translocation in the long arm of chromosome 7B in the other line. In addition, the pSc119.2 probe revealed chromosome 1B rearrangements in four lines resulting from crosses with Avrolata and in a line resulting from crosses with Avrotika. The lines were tested for main productivity parameters. A negative effect on all productivity parameters was demonstrated for Ae. umbellulata chromosome 2U. The overwinter survival in all of the lines was similar to or even higher than in the original triticale cultivars. A substantial increase in winter resistance as compared with the parental cultivar was observed for the line carrying the T7BS-7SL translocation. The line with the 1R(1U) chromosome substitution seemed promising for the baking properties of triticale.     

The influence of the rye genome on expression of heat shock proteins in triticale

Summary The heat shock protein profiles from Secale cereale L. cv Imperial, Triticum aestivum L. cv Chinese Spring, S. cereale x T. aestivum amphiploid, and the seven disomic S. cereale addition lines to T. aestivum were used to compare the wheat, rye, and triticale Heat Shock Protein profiles and to study the influence of the rye genome on heat shock protein expression in triticale. Three-day-old seedlings were heat shocked for 2 h at 40 °C in the presence of ³⁵S-methionine, and polypeptides from root tissues were subjected to one- or two-dimensional gel electrophoresis. The wheat and rye heat shock protein profiles each consisted of > 150 heat shock proteins, of which 94 were sufficiently reproducible to construct a standard map. There were 11 unique rye heat shock proteins compared to 22 unique wheat heat shock proteins. The triticale heat shock protein profile resembled the rye parent more than the wheat parent. There were 22 heat shock proteins expressed uniquely by wheat that were not expressed in triticale. Rye chromosomes 1 and 3 exhibited a substantial repressive influence on the expression of 95% of the unique wheat heat shock proteins in triticale, while rye chromosome 4 appeared to have the least repressive influence on expression of the unique wheat heat shock proteins in triticale.Mention of a trade name or proprietary product does not constitute a guarantee, warranty, or recommendation of the product by the United States Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitable     

The mapping of highly-repeated DNA families and their relationship to C-bands in chromosomes of Secale cereale

The relationship between the chromosomal location of heterochromatin C-bands and of four non-homologous repeated sequence families constituting 8 to 12% of total rye DNA has been investigated in chromosomes of rye (Secale cereale) by in situ hybridisation. Three rye varieties, a set of rye disomic additions to wheat and a triticale were studied. Only centromeric and nucleolar organizer region (NOR) associated C-bands failed to display hybridisation to at least one of the sequences and many telomeric blocks of heterochromatin contained all four repeated sequence families. Both between-variety differences in the chromosomal distribution of repeated sequences, and intravarietal heterozygosities were frequently noted and are probably widespread. — Previously reported deletions of heterochromatin from King II rye chromosomes added to the Holdfast wheat complement were correlated with deletions of some, but not all, of the highly repeated sequence families. A previously unreported loss of some families from King II rye chromosome 4R/7R in a Holdfast wheat genetic background was detected. This loss was not associated with complete deletion of a C-band. A deletion has also probably occurred from the short arm telomere of 4R/7R in the triticale variety Rosner. It is suggested that the families of repeats in rye telomeric heterochromatin which are absent from wheat are selected against in the wheat genetic background.     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Genetic mapping of a 7R Al tolerance QTL in triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) is a relatively new cereal crop. In Poland, triticale is grown on 12 % of arable land (http://www.stat.gov.pl). There is an increasing interest in its cultivation due to lowered production costs and increased adaptation to adverse environmental conditions. However, it has an insufficient tolerance to the presence of aluminum ions (Al³⁺) in the soil. The number of genes controlling aluminum tolerance in triticale and their chromosomal location is not known. Two F2 mapping biparental populations (MP1 and MP15) segregating for aluminum (Al) tolerance were tested with AFLP, SSR, DArT, and specific PCR markers. Genetic mapping enabled the construction of linkage groups representing chromosomes 7R, 5R and 2B. Obtained linkage groups were common for both mapping populations and mostly included the same markers. Composite interval mapping (CIM) allowed identification of a single QTL that mapped to the 7R chromosome and explained 25 % (MP1) and 36 % (MP15) of phenotypic variation. The B1, B26 and Xscm150 markers were 0.04 cM and 0.02 cM from the maximum of the LOD function in the MP1 and MP15, respectively and were highly associated with aluminum tolerance as indicated by Kruskal–Wallis nonparametric test. Moreover, the molecular markers B1, B26, Xrems1162 and Xscm92, previously associated with the Alt4 locus that encoded an aluminum-activated malate transporter (ScALMT1) that was involved in Al tolerance in rye (Secale cereale) also mapped within QTL. Biochemical analysis of plants represented MP1 and MP15 mapping populations confirmed that the QTL located on 7R chromosome in both mapping populations is responsible for Al tolerance.     

C-banding pattern and meiotic pairing in five rye chromosomes of hexaploid triticale

Summary The meiotic behaviour of rye chromosomes 1R, 2R, 3R, 6R and 7R/4R of hexaploid triticale Cachirulo is analyzed using the C-banding technique. These chromosomes show different C-banding patterns and present different pairing levels at metaphase I. A decreasing effect of large telomeric heterochromatin bands on pairing is deduced from the following two main facts: i) The chromosome 7R/4R shows the highest pairing associated with the smallest amount of heterochromatin, ii) pairing levels of 2 R short arm and 3 R long arm which carry large telomeric bands are less than their respective long and short arms lacking telomeric heterochromatin. Possible desynaptic effects of heterochromatin are discussed although an asynaptic effect cannot be rejected.