Molecular diversity and genetic relationships in Secale.

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.

The role of two superoxide dismutase mRNAs in rye aluminium tolerance.

Aluminium (Al) is the main factor that limits crop production in acidic soils. There is evidence that antioxidant enzymes such as superoxide dismutase (SOD) play a key role against Al-induced oxidative stress in several plant species. Rye is one of the most Al-tolerant cereals and exudes both citrate and malate from the roots in response to Al. The role of SOD against Al-induced oxidative stress has not been studied in rye. Al accumulation, lipid peroxidation, H2O2 production and cell death were significantly higher in sensitive than in tolerant rye cultivars. Also, we characterised two genes for rye SOD: ScCu/ZnSOD and ScMnSOD. These genes were located on the chromosome arms of 2RS and 3RL, respectively, and their corresponding hypothetical proteins were putatively classified as cytosolic and mitochondrial, respectively. The phylogenetic relationships indicate that the two rye genes are orthologous to the corresponding genes of other Poaceae species. In addition, we studied Al-induced changes in the expression profiles of mRNAs from ScCu/ZnSOD and ScMnSOD in the roots and leaves of tolerant Petkus and sensitive Riodeva rye. These genes are mainly expressed in roots in both ryes, their repression being induced by Al. The tolerant cultivar has more of both mRNAs than the sensitive line, indicating that they are probably involved in Al tolerance.

On the consequences of aluminium stress in rye: repression of two mitochondrial malate dehydrogenase mRNAs.

Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al-tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al-induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al-tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.

Is the Mnr locus of Triticeae species the same as the Ndh and Dia loci?

The menadione reductase (MNR), the nicotinamide adenine dinucleotide dehydrogenase (NDH) and diaphorase (DIA) isozymes were studied in the allohexaploid Triticum aestivum cv "Chinese Spring" and in five diploid Triticeae species. The Mnr1, Ndh3and Dia1 loci were located on the chromosome arms 3AL, 3BL and 3DL of T. aestivum, respectively. These loci were also located on the 3H chromosome of Hordeum vulgare cv "Betzes", the 3L chromosome of Aegilops longissima and the 6RL chromosome arm of Secale cereale cv "Imperial". The chromosomal location results together with the segregation studies support a tetrameric behaviour of the MNR1, NDH3 and DIA1 isozymes. The Ndh1 and Dia3 loci were located on homoeologous group 4 showing a monomeric behaviour. The chromosomal locations and linkage data of the Mnr, Ndh and Dia loci suggest that Mnr1=Ndh3=Dia1; Ndh1=Dia3 and Ndh2=Dia2.

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye.

To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08 415 and OPR01 600 RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08 415 and SCR01 600 ) and linked to Alt1 gene (SCR01 600 -2.1 cM-Alt1-33.5 cM-SCA08 415 ). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat.

Sex influence on recombination frequency in Secale cereale L.

The variation in recombination frequency (rf) is important to plant breeders since their major objective is to obtain favorable recombinants of linked genes. One source of variation in rf is sex. Sex differences for recombination frequencies were studied in four of the seven chromosomes of Secale cereale L. cv 'Ailés' using isozyme and storage protein loci and were determined on the basis of reciprocal crosses between heterozygous plants of cv. 'Ailés' and homozygous plants of the inbred line 'Riodeva'. The differences were found to be strongly segmentspecific. In some cases the level of crossing-over in male and female meiosis was about the same (between Pgm1 and Ndh1 loci on chromosome arm 4RS). However, for most of the chromosome segments in 1R, 3RL and 6RL the male rf was significantly higher than the female rf. Different hypotheses about the mechanisms of plant sex differences for recombination are discussed.

A map of rye chromosome 4R with cytological and isozyme markers.

The progeny of two crosses between a structural heterozygote for a reciprocal translocation (4RL/5RL) and a homozygote for the standard chromosome arrangement and of four crosses between standard chromosome homozygotes were analysed in rye (Secale cereale L. cv 'Ailés') for the electrophoretic patterns of five different leaf and endosperm isozymes (LAP, PGM, NDH, ADH and EPER). The presence or absence of the quadrivalents at metaphase I (MI) was also tested. Loci Adh-1, Pgm-1 and Ndh-1 were located on chromosome arm 4RS, and locus Eper-1 on chromosome arm 4RL. Locus Lap-2 was located on the 4RS chromosome arm. The estimated distances among the different linked loci support the following gene order: Eper1¨ (breakpoint-centromere)¨Lap-2¨ ¨Adh-1 ¨Pgm-1¨Ndh-1. These results provide evidence for the chromosomal location of Lap-2 locus on chromosome arm 4RS in cv 'Ailés'. A high negative interference was detected between the zones delimited by centromere and Lap-2, and Lap-2 and Pgm-1 in plants with the 4RL/5RL translocation.

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction.

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalent to those obtained with standard DNA extraction protocols using either specific or random primers. Intra- and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.

High mutability in rye (Secale cereale L.).

Analysis of the rye cultivar Ailés of several descents derived from crosses between plants carrying specific genotypes and/or chromosome constitutions resulted in the detection of high chromosome (2.05 x 10(-2)) and gene (9.3 x 10(-3)) mutation frequencies. The existence of a transposon system responsible for this instability is suggested.

NADH dehydrogenase: a new molecular marker for homoeology group 4 in Triticeae. A map of the 4RS chromosome arm in rye.

Structural gene loci encoding the monomeric isozymes nicotin adenin dinucleotide dehydrogenase (NADH dehydrogenase or NDH) have been located on the 4AL, 4Bα, and 4DS chromosome arms of Triticum aestivum cv "Chinese Spring", on the 4RS chromosome arm of Secale cereale cultivars "Imperial", "King II", "Dakold," and "Ailes," on the 4S(1) S/7S(1) chromosome of Aegilops longissima, the 4E of Elytrigia elongata, and the CSU-A of Aegilops umbellulata. All the results support the homoeologous relationships among these chromosomes in the five species studied. In addition, a map of the 4RS chromosome arm in cv "Ailes" has been realized, linking loci Pgm-1 (located on the 4RS chromosome arm) and Ndh-1 (17.91 cM), with an estimated distance between both loci and the centromere of 20.00 cM and 32.12 cM, respectively.

Biochemical evidence of a translocation between 6 RL/7 RL chromosome arms in rye (Secale cereale L.). A genetic map of 6R chromosome.

The segregation of different isozymic loci was investigated in backcrosses and F2s in rye. The leucin aminopeptidase-1 (Lap-1), Aconitase-1 (Aco-1), Esterase-6 (Est-6), Esterase-8 (Est-8), and Endopeptidase-1 (Ep-1) loci were linked. The Aco-1, Est-6, and Est-8 loci have been previously located on the 6RL chromosome arm. The Lap-1 locus has been located on the 6RS chromosome arm. The results favor the gene order: Lap-1... (centromere)... Aco-1... Est-8... Est-6... Ep-1. The isoelectric focusing separations of aqueous extracts from mature embryo tissue of wheat-rye addition and substitution lines involving the chromosomes of cereal rye Secale cereale L. confirmed the gene location of locus Ep-1 on the 6RL chromosome arm. Screening of wheat-rye addition lines involving the chromosomes of Secale montanum revealed that Ep-1 locus is not located on chromosome 6R of S. montanum. These results are the first biochemical evidence of the translocation between chromosome arms 6RL/7RL in the evolution of S. cereale from S. montanum.

A map of rye chromosome 2R using isozyme and morphological markers.

The segregation of isozymic loci for leaf peroxidases (L2Per) has been investigated in backcrosses and F2 offspring of rye lines having purple seeds (Ps) and monstrosum ears (mo). The Ps, L2Per-3b, mo, and L2Per-2 loci were linked. The Ps and mo loci have been previously located on the 2R chromosome, and the L2Per-3b and L2Per-2 loci have been located on the 2RS chromosome arm. The results favor the gene order Ps ... L2Per-3b ... mo ... L2Per-2 or Ps ... mo... L2Per-3b ... L2Per-2. The position of the loci relative to the centromere is still not known, but the obtained results suggest that the mo locus could be located on the 2RS chromosome arm. On the basis of previously reported linkage groups, the most probable arrangement of the loci located on chromosome 2R is: dw2 ... Ps ... (L2Per-3a ... L2Per-3b ... mo) ... L2Per-2. It has not been possible to know the position of L2Per-4 loci (also located on 2RS chromosome arm) relative to L2Per-3a and L2Per-3b loci.

Chromosomal location of adenylate kinase isozymes in Triticeae species.

One system of monomeric adenylate kinase isozymes, designated ADK, was observed in Triticum aestivum and in five diploid Triticeae species. The gene loci Adk-a, Adk-b and Adk-d were located on 7AL, 7BL and 7DL Triticum aestivum cv "Chinese Spring" chromosomal arms, respectively. Adk gene loci were also located on the 7RL chromosomal arm of Secale cereale cv "Ailés", the 7H chromosome of Hordeum vulgare cv "Betzes", 7X of Agropyron intermedium, 7E of Elytrigia elongata and CSU-E of Aegilops umbellulata. The results suport the notion of the conservation of gene synteny groups within Triticeae species.

Chromosomal identification and meiotic behavior of reciprocal translocations in a rye polymorphic population. Evolutionary implications.

The spontaneous interchange polymorphism of rye cultivar "Ailés" is composed, as can be deduced from the chromosomal identification of the interchanges analyzed, of several different reciprocal translocations in which the chromosomes of its haploid complement are involved with a similar frequency, except for chromosomes 4R and 6R. Several features of chromosome behavior at metaphase I, such as configuration and orientation of quadrivalents and frequency of chiasmata, were analyzed in structural heterozygotes for different interchanges. The two main factors affecting the orientation of quadrivalents at metaphase I proved to be the morphology of these chromosome associations at metaphase I and, in particular, the frequency of bound chromosome arms that they showed. A genotypic control of alternate orientation of quadrivalents independent of chiasmata frequency was not detected. In addition, the frequency of alternate orientation shows no relation to the fitness. Possible evolutionary implications of the results obtained are discussed.

Association of four isozyme loci with a reciprocal translocation between 1R/4R chromosomes in cultivated rye (Secale cereale L.).

The progeny of four crosses between a structural heterozygote for a reciprocal translocation and a homozygote for the standard chromosome arrangement were analyzed in rye (Secale cereale L. cv "Ailés") for the electrophoretic patterns of eight different leaf and endosperm isozymes and also for the meiotic configuration at metaphase I. The Pgi-1, 6-Pgd-2 and Mdh-1 loci are linked to each other and also to the reciprocal translocation. These loci have been located on chromosome 1R. The Mdh-1 locus is located in the interstitial segment of chromosome 1R, between the centromere and the breakpoint. The Pgm-1 locus has been located on chromosome arm 4RS and is linked to Pgi-1, 6-Pgd-2, Mdh-1 and the reciprocal translocation. The estimated distance between the Pgm-1 locus and the centromere is 14.98 ± 2.27 cM. Therefore, the reciprocal translocation involves the 1R and 4R chromosomes. Other linked loci detected have been Mdh-2b and Est-2 (7.40 ± 2.90 cM) and Got-3 and Est-2 (5.62 ± 3.07 cM). These three last loci are located on chromosome 3R and their order most probably is Mdh-2b - Est-2 - Got-3.

Location of genes coding isozyme markers on Aegilops umbellulata chromosomes adds data on homoeology among Triticeae chromosomes.

Zymogram analysis was used to identify the Aegilops umbellulata chromosomes that carry the structural genes for particular isozymes. Wheat, Aegilops and wheat-Aegilops hybrid derivative lines (which contained identified Aegilops chromosomes) were tested by gel electrophoresis for isozymes of particular enzymes. It was found that Aegilops chromosome A (nomenclature according to G. Kimber 1967) carries a structural gene for 6-phosphogluconate dehydrogenase, Aegilops chromosome B carries structural genes for glucose phosphate isomerase and phosphoglucose mutase, Aegilops chromosome D carries genes for leaf peroxidases, Aegilops chromosome E carries structural genes for endosperm peroxidases, acid phosphatases and leaf esterases, Aegilops chromosome F carries a gene for embryo plus scutellum peroxidases and Aegilops chromosome G carries structural genes for endosperm alkaline phosphatases, leaf alkaline phosphatases and leaf esterases. The results obtained indicate that chromosome B is partially homoeologous of the wheat chromosomes of group 1 and 4, and chromosome E is partially homoeologous of wheat chromosomes of groups 7 and 4. Circumstantial evidence is also provided about the possible association between chromosomes C, D and A of A. umbellulata respectively with chromosomes 5, 2 and 1 of wheat.

Genetics of rye phosphatases: evidence of a duplication.

Genetic analyses were conducted on alkaline phosphatases of the endosperm of dry kernels and leaf acid phosphatases in four open pollinated and one inbred line of cultivated rye (Secale cereale L.). A total of seven alkaline phosphatase isozymes were observed occurring at variable frequencies in the different cultivars analyzed. We propose that at least five loci control the alkaline phosphatases of rye endosperm - Alph-1, Alph-2, Alph-3, Alph-4 and Alph-5 - all of which have monomeric behaviour. The leaf acid phosphatases are controlled by one locus and have a dimeric quaternary structure. All loci coding for alkaline phosphatase isozymes showed one active, dominant allele and one null, recessive allele, except for the locus Alph-3 which showed two active, dominant alleles and one null, recessive one. The linkage analyses suggest the existence of two linkage groups for alkaline phosphatases: one of them would contain Alph-2, Alph-4, Alph-5 and the locus/loci coding isozymes 6 and 7. This linkage group is located in the 7RS chromosome arm. The other group would include Alph-1 and Alph-3 loci, being located in the 1RL chromosome arm. Leaf acid phosphatases have been previously located in the 7RL chromosome arm. Our data also support an independent relationship between loci controlling the endosperm alkaline phosphatases and leaf acid phosphatases.

Biochemical evidence of homoeology between Triticum aestivum and Agropyron intermedium chromosomes.

The alcohol dehydrogenase (ADH), phosphoglucose mutase (PGM), glucosephosphate isomerase (GPI), glutamic oxaloacetic transaminase (GOT), malate dehydrogenase (MDH), leaf esterases (ESTL), leaf acid (ACPH) and endosperm alkaline (PHE) phosphatases, leaf peroxidases (PERL) zymogram phenotypes of Triticum aestivum, Agropyron intermedium, Triticum aestivum - Agropyron intermedium octoploids and six Agropyron intermedium chromosome additions to Triticum aestivum and two ditelocentric addition lines were determined. It was found that the six disomic chromosome addition lines and one ditelocentric chromosome addition line could be distinguished from one another and from the other possible lines on the basis of the zymogram phenotypes of these isozymes. The structural gene Acph-X1 was located on Agropyron chromosome L1, the genes Got-X3 and Mdh-X2 on chromosome L2, the gene Gpi-X1 on chromosome L3, the genes Adh-X1, Pgm-X1 and Phe-3 on chromosome L4, gene Perl-1 on chromosome L5 and the gene Estl-2 on chromosome L7 and chromosome arm L7d2. These gene locations provide evidence of homoeology between Agropyron chromosomes L1, L2, L3, L4, L5 and L7 and the Triticum aestivum chromosomes of homoeologous groups 7, 3, 1, 4, 2 and 6, respectively.

Association of Isozymes with a Reciprocal Translocation in Cultivated Rye (SECALE CEREALE L.).

In rye (Secale cereale L. cv. "Ailés") the progeny of a cross between a structural heterozygote for a reciprocal translocation (involving the 1R chromosome) and a homozygote for the standard chromosome arrangement were analyzed for the electrophoretic patterns of eight different leaf isozymes and also for their meiotic configuration at metaphase I.--The Got-3 and Mdh-2b loci are linked to each other and also to the reciprocal translocation. The Mdh-2b locus is located in the interstitial segment of the 3Rq chromosome arm, with an estimated distance of 8 cM to the breakpoint. Therefore, the reciprocal translocation involves the 1R and 3R chromosomes.--Also, the Mdh-1 and 6-Pgd-2 loci are linked (16 +/- 3 cM) and have been located on the 2Rq arm. Finally, the Per-3 and Per-4 loci are located on the 2Rp chromosome arm at an estimated distance of 26 +/- 4 cM.

Pollen germination and tube growth in rye (Secade cereale L.) homozygous and heterozygous for reciprocal translocations.

To contribute to the knowledge of the role of reciprocal translocations in rye, a component of fertility was estimated by comparing germination and pollen tube growth in homozygous and heterozygous plants for reciprocal translocations. The results obtained indicate that there are no differences in germination and pollen tube growth rate when homozygous and heterozygous plants as a whole are compared. However, there are significant differences in pollen tube growth between plants carrying different translocations. This suggests that the chromosome constitution of a plant is relevant to these fitness-estimating parameters together with its particular genetic background.