Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

CYTOGENETIC AND EVOLUTIONARY STUDIES IN SECALE. I. SOME NEW DATA ON THE ANCESTRY OF S. CEREALE

Khush , G. S., and G. L. Stebbins . (U. California, Davis.) Cytogenetic and evolutionary studies in Secale. I. Some new data on the ancestry of S. cereale. Amer. Jour. Bot. 48(8): 723–730. Illus. 1961.—Cultivated rye, Secale cereale, was crossed with all 4 wild species of the genus. It crosses readily with S. montanum, S. africanum, and S. vavilovii, but crossability of S. cereale and S. silvestre is extremely low. Meiosis in the hybrid S. cereale × silvestre is characterized by high frequency of univalents at metaphase I, reduced chiasma frequency at metaphase I, high frequency of PMC's with unequal separations and laggards at anaphase I and II, high frequency of microspores with micronuclei, and extremely low pollen fertility. These abnormalities occurred less frequently in other hybrids, and consequently the pollen fertility is fairly high: 19.1% in the hybrid S. cereale × vavilovii and 31.3% and 31.8%, respectively, in the hybrids S. cereale × montanum and S. cereale × africanum. An interesting cytogenetic feature of all these hybrids, however, was the formation of a translocation configuration of 6 chromosomes at metaphase I of meiosis. In the hybrid, S. cereale × silvestre, a translocation configuration of 8 chromosomes was observed in a few cells. It is evident, therefore, that the genome of S. cereale differs from the genomes of wild species by 2 translocations. Another small translocation may differentiate S. cereale and S. silvestre, in addition. Thus, on the basis of chromosome arrangements, no particular wild species is most likely to be ancestral to S. cereale. Similarly, Stutz's hypothesis of hybrid origin of S. cereale seems highly improbable. After considering ecological preferences, breeding habits, geographical distribution, morphological and cytological affinities of wild species and cultivated rye, it is concluded that S. cereale evolved from S. montanum as a result of progressive cytological and morphological differentiation and that this differentiation was facilitated, probably, by adaptive superiority of translocation heterozygotes and rearrangement homozygotes.     

Incomplete preferential pairing in a tetraploid Secale hybrid carrying translocations: Multivalent configuration frequencies and marker segregation

Meiotic configurations and segregation were studied in tetraploid hybrids (4n=28) between Secale cereale (2n=14) carrying a reciprocal translocation between chromosomes II and VI and a derivative of Secale montanum (2n=14), naturally differing from S.cereale in a translocation involving chromosomes I, III and V. Multivalent frequencies of the translocation complexes were reduced compared to expected but of still appreciable magnitude, which was ascribed to incomplete preferential pairing. Marker segregation (one of the S. cereale translocation chromosomes) also suggested partial preferential pairing.     

Identification by Giemsa technique of the translocations separating cultivated rye from three wild species of Secale

Complete identification of the translocations involved in evolution of S. vavilovii, S. africanum and S. cereale from S. montanum was attained by meiotic analysis after Giemsa banding technique. Based on the original mitotic karyotype of S. montanum, the different chromosome arms were determined by centromere position and banding pattern of chromosomes for the four species and all of the possible interspecific hybrids. This first consistent scheme of cytogenetic relationships reveals: one translocation each, separating S. montanum from S. vavilovii and S. africanum, two translocations each, separating S. cereale from S. vavilovii and S. africanum, and three translocations each, separating S. cereale from S. montanum and S. africanum, respectively.     

Identification of the three chromosomes involved in the translocations which structurally differentiate the genome of Secale cereale L. from those of Secale montanum Guss. and Secale vavilovii Grossh.

A standard tester set of reciprocal translocations in S. cereale and several primary trisomics have been used to identify the chromosomes involved in the natural translocation complexes between S. cereale and S. montanum, and S. cereale and S. vavilovii.Chromosomes I, III and V, in our classification system, were found to be involved in the translocations between cultivated rye, S. cereale, and both the wild species S. montanum and S. vavilovii.     

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

Summary 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.     

Homoeologous relationship between wheat and rye chromosomes. Present status

The work on methods for determining the homoeologous relationship between wheat and rye chromosomes has been reviewed. The results obtained for rye chromosomes belonging to different homoeologous groups have been discussed. It is proposed that chromosome 3R of Lee et al. (1969) should be designated as 1R/3R. It is pointed out that homoeology of all seven rye chromosomes may not be known in the future also, due to translocations. It is, therefore, suggested that Secale montanum should be used instead of S. cereale. Future lines of work have been suggested.     

<Emphasis Type="Italic">Aegilops-Secale</Emphasis> amphiploids: chromosome categorisation,pollen viability and identification of fungal disease resistance genes

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.     

Population genetics of colonizing success of weedy rye in Northern California

Summary Genetic parameters of 11 weedy rye populations located in California's northern mountain area and the adjoining Oregon border were compared with those of the putative parents, wild species Secale montamim and cultivated rye S. cereale. All weedy populations exhibited high levels of genetic variation as determined by isozyme analysis. On average, 44% of the isozyme loci were polymorphic, total genetic diversity was 0.30; and number of alleles per locus was 1.65. High genetic identities, averaging 0.994 ± 0.005 between populations, indicated that little genetic differentiation has occurred among these weedy populations since the initial colonization. Lack of population differentiation could be attributed to a wind-pollinated, self-incompatible breeding system resulting in extensive gene flow among weedy populations, and between weedy populations and local cultivars of rye. Multilocus outcrossing rates of weedy populations ranged from 0.86 to 0.97. The estimated levels of gene flow using the private-alleles method were high among weedy populations, and between cv Merced and weedy populations, with estimated Nm values of 14.50 and 8.21, respectively. The colonizing success of weedy rye is discussed and a strategy for its conservation recommended.     

The effect of reciprocal translocations on segregation and multivalent formation in autotetraploids of rye,Secale cereale

Chromosomal segregation, and the frequency of large multivalents in Secale cereale were studied in autotetraploid duplex translocation heterozygotes. Models for estimating expected segregations and frequencies of multivalents were developed incorporating the probabilities of different chromosomal segments being bound by chiasmata. It appeared that the segregation of the two translocations tested fitted quite well the expected corrected segregation ratio of approximately 1: 11.5: 1, suggesting that induced preferential pairing was not strong enough to enhance preferential segregation resulting from random translocation segregation. Interspecific hybrids with S. montanum carrying the same translocations showed strong preferential pairing, i.e. significant deviation from the expected ratios.Three translocations tested (two not tested for segregation) showed a decrease in multivalent frequency mainly attributable to preferential pairing, especially in cases where the breakpoint was near one chromosome end. Possible reasons why preferential pairing is expressed here and not in the segregations are discussed.     

The structure,amount and chromosomal localisation of defined repeated DNA sequences in species of the genus Secale

The structure, copy number and chromosomal location of arrays of four families of highly repeated sequences have been investigated in representative species of the genus Secale. The four unrelated families, previously characterised in Secale cereale, have repeating units of 480, 610, 630 and 120 base pairs respectively. The following general conclusions can be drawn in addition to detailed knowledge of the sequence content of heterochromatin in each accession studied: (1) Every species is unique in its complement or chromosomal distribution or both of the four highly repeated sequence families. S. montanum and S. cereale accessions studied here show the same complement of repeated sequences, but they differ substantially in the amounts they contain of the 610 and 630 base pair (bp) families, and in the distribution over the chromosomes of the 480 bp family. The structure of the repeating unit is also different in many members of the 480 bp family in S. montanum. — (2) The substantial differences between species in the amounts of the most highly repeated DNA sequences exist in the absence of any such conspicuous differences in most other repeated sequences which were detected as fluorescent bands after restriction enzyme digestion and gel electrophoresis. — (3) Each of the different highly repeated families can exist independently of the other families, though all the families have telomeric sites. Also, in the outbreeding species, heteromorphisms are frequent, and are particularly conspicuous in hybridisation detecting the 480 bp sequence family. — (4) The association of the highly repeated sequences with heterochromatin, discussed in the accompanying paper is generally true for other species in the genus, and the lower amounts of heterochromatin in other Secale species compared to S. cereale are associated with lower amounts of specific families of highly repeated DNA sequences. — (5) Analysis of highly repeated sequence families is likely to provide an easy method of identification of new accessions of Secale.     

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid <Emphasis Type="Italic">Secale cereale</Emphasis> × <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.     

Identification of sterility-inducing cytoplasms in rye using the plasmotype–genotype interaction test and newly developed SCAR markers

A series of 25 rye (Secale cereale L.) inbred lines was tested with respect to three mitochondrial sequence-characterized amplified region (SCAR) polymorphisms. The analysis revealed a close association between the marker-determined mitotypes and plasmotypes (cytoplasm types known from breeding data) represented by the inbreds. The mitochondrial markers also confirmed normal (N-) cytoplasmic character of three wild rye species: Secale montanum, S. vavilovii and S. kuprijanovii. For 186 plants from open-pollinated cultivars of Turkish and South American origin, cytoplasm identification was performed with the use of crossing with double non-restoring tester. In 77 plants the normal (N) cytoplasm was detected, and for 63 of these the PCR analysis was performed producing results which were consistent with the genetic data based on testcrossing and phenotype assessment. The mitochondrial markers also confirmed a presence of sterility-inducing cytoplasm in the remaining 109 plants. Moreover, the markers allowed for differentiation between Pampa (P-) and Vavilovii (V-) cytoplasmic individuals. For 60 plants the latter results were verified using crosses with a line maintaining P-cytoplasmic sterility and acting as a restorer of the V-cytoplasm. For two of these plants contradicting results were produced with the applied methods of cytoplasm identification and the basis of this discrepancy is discussed. Regardless of the identification method, widespread occurrence of a sterility-inducing cytoplasm was revealed, especially in South American populations.     

Two-dimensional leaf isozyme patterns of <Emphasis Type="Italic">Aegilops kotschyi ’ Secale cereale</Emphasis> amphiploids

Esterase, peroxidase, shikimic dehydrogenase, malic dehydrogenase and diaphorase isozymes in leaves of the amphiploids Aegilops kotschyi × Secale cereale and their parental forms (Ae. kotschyi and S. cereale) were analyzed using two-dimensional gel electrophoresis in non-denaturing conditions. In the amphiploid isozymess were detected, which were not detected in leaves of parental plants. In contrast, some parental isozymes were not detected in refer to the gels of the amphiploids. Detection of new isoforms in the amphiploids and no detection of some parental isoforms is discussed considering the recombination, gene suppression, acting of inhibitors, chromosome translocation and also refer to the previous results of the electrophoretic analysis of proteins and enzymes of Aegilops sp. × S. cereale amphiploids.     

The order of the <Emphasis Type="Italic">bs,Skdh</Emphasis>, and <Emphasis Type="Italic">Aadh1</Emphasis> genes in chromosome 5R of rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Segregation analysis was performed in the progenies obtained in analyzing crosses of hybrids of spring and winter accessions of rye Secale cereale L. and wild S. montanum subsp. anatolicum (Grossh.) Tzvel. (syn. S. strictum (J. Presl) J. Presl). The test genes controlled the brittle stem (bs), the allelic variants of aromatic alcohol dehydrogenase (Aadh1) and shikimate dehydrogenase (Skdh), and the growth habit (Vrn1). A linkage was observed in the inheritance of the brittle stem and the aromatic alcohol dehydrogenase and shikimate dehydrogenase alloenzymes. The order of genes was established to be bs-Skdh-Aadh1, and the genetic distances were estimated to be bs-(9.0%)-Skdh, bs-(10.8%)-Aadh1, and Skdh-(5.3%)-Aadh1. The recombination coefficient between the Skdh and Aadh1 genes varied from 2.2 to 18.2%, averaging 5.3%. The growth habit was inherited independently of the bs-Skdh-Aadh1 linkage group.     

Pollen proteins after two-dimensional gel electrophoresis and pollen morphology of the amphiploids Aegilops kotschyi and Ae. variabilis with Secale cereale

Proteins from pollen of parent forms and amphiploids Aegilops variabilis ×Secale cereale and Ae. kotschyi×S. cereale, obtained by in vitro propagation or colchicine treatment of F₁ hybrids, were subjected to a study by two-dimensional (2-D) electrophoresis. Qualitative and quantitative diversities of protein patterns were revealed for the amphiploid pollen. The majority of peptides found in the parent forms were also present in the patterns of the amphiploid pollen; however, some of the parent-form-peptides were not expressed and proteins characteristic only of the amphiploids appeared. In the 2-D combined protein pattern obtained for the parent forms, amphiploids Ae. variabilis × S. cereale produced pollen with a poorer spectrum of proteins. In amphiploid 408B, obtained from treated the F₁ generation with colchicine, the 2-D pattern revealed the presence of less than 50% of the proteins recorded for the parent forms. Pollen grain morphology was studied under a scanning microscope. The structure and shape of exines differed from those of the parents. In the parent forms the pollen grains had only one pore, while in amphiploid pollen, one, two or three pores were observed. Possible explanations for the differences in the 2-D patterns of amphiploids and their parent forms (impoverishment of the protein spectrum and appearance of new peptides) are (1) somaclonal variation and mutagenic activity of colchicine, (2) suppression of structural genes, (3) activity of regulators and (4) translocations. Pollen grains with two or even three pores could appear as a result of the independent activity of the genes from three amphiploidal genomes. Received: 6 March 2001 / Accepted: 26 June 2001     

Putative MADS-Box Retropseudogenes in Rye (Secale L.)

The fragments of MADS-box genes belonging to the agamous and agamous-like structural classes were isolated by direct amplification of genomic DNA from annual rye (Secale cereale L.) and perennial rye (Secale montanum Guss.). The characterized fragments (deposited in the Genbank as the accession nos. AF332885–AF332887 and AF346894) comprise the complete sequences of exons 1 to 5 and lack corresponding introns. Their nearest homologs are the maize genes zag1 and zag5 (the Genbank accession nos. L18924 and L46398). One more agamous-like fragment isolated from annual rye (AF362364) is similar to theTaMADS12 wheat gene (AB007505). The fragment comprises exons 3–5 and contains a 105-bp insert between the exons 3 and 4; this insert does not resemble any MADS-box introns presently known. We assume that all these fragments of MADS-box genes are retropseudogenes.     

Comparisons of the hybrids Hordeum chilensexH. vulgare,H. chilensexH. bulbosum,H. chilensexSecale cereale and the amphidiploid of H. chilensexH. vulgare

Summary Diploid hybrids between Hordeum chilense and three other species, namely H. vulgare, H. bulbosum and Secale cereale, are described together with the amphidiploid of H. chilensexH. vulgare. Both the diploid hybrid and the amphidiploid of H. chilensexH. vulgare were chromosomally unstable, H. chilensexH. bulbosum was less so, while H. chilensexS. cereale was stable. Differential amphiplasty was found in all combinations. No homoeologous pairing was found in the Hordeum hybrids but in H. chilensexS. cereale there was chromosome pairing both within the two genomes and between the genomes.     

Molecular cytogenetic characterization of a new wheat <Emphasis Type="Italic">Secale africanum</Emphasis> 2R<Superscript>a</Superscript>(2D) substitution line for resistance to stripe rust

A stable, highly fertile wheat Secale africanum substitution line LF24, derived from the F₇ generation of a cross between Mianyang11 (MY11) and Triticum durum, S. africanum amphiploid (YF) was identified through molecular cytogenetic analysis. Application of C-banding, in situ hybridization and molecular markers analysis showed that LF24 was a wheat S. africanum 2R^a(2D) substitution line. When inoculated with stripe rust isolates, T. durum and MY11 were highly susceptible, while S. africanum, YF and LF24 were immune. It is confirmed through molecular cytogenetic analysis that the stripe rust resistance of LF24 was derived from S. africanum chromosome 2R^a. We compared the banding patterns and disease resistance of reported chromosomes 2R from different S. cereale introduced into wheat background, and found that there was new stripe rust resistance gene(s) on S. africanum 2R^a. LF24 is a new substitution line which can be used as stripe rust resistant source in wheat improvement.     

A Fragment of the rpoC2 Chloroplast RNA Polymerase Gene in Perennial and Annual Rye

A 1397-bp fragment corresponding to the rpoC2 chloroplast RNA polymerase gene was obtained by direct rye DNA amplification. Two rye species, Secale montanum Guss. and S. cereale L., did not practically differ in the structure of this DNA fragment (the nucleotide sequences were 99% identical). The corresponding nucleotide sequences in rye and wheat (Triticum aestivum L., Genbank accession no. AB027572) were 97–98% similar. The extent of the homology of various stretches of the rpoC2 rye gene with the corresponding sequences in maize and rice was 81–95%, whereas the deduced amino acid sequences of rpoC2 in rye, wheat, maize, and rice were considerably identical (96–97% of homology). The rye fragment of the rpoC2 gene differed from the corresponding sequences in three other grass species primarily by a short (49 bp) insert into the region of numerous short repeats corresponding to nucleotides 15750/15751, 28728/28729, and 27472/27473 in wheat, maize, and rice, respectively.