Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.     

A taxonomic revision of Secale (Triticeae, Poaceae)

Several taxa have previously been recognized within Secale , but most of them are difficult or even impossible to distinguish morphologically. We recognize only three species: S. sylvestre, S. strictum , and S. cereale. Secale strictum has priority over S. montanum and has two subspecies, ssp. strictum and ssp. africanum , and two varieties within ssp. strictum , van strictum and var. ciliatoglume comb. nov. Secale cereale is also treated as having two subspecies. The cultivated taxa, marked by their tough rachises, are placed in ssp. cereale and the wild or weedy taxa that have more or less fragile rachis, in ssp. ancestrale. A complete synonymy is given for S. cereale , but typification has been omitted because, in many instances, type material does not exist or has been impossible to trace.     

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.     

Isoenzymes of aspartate aminotransferase in perennial and annual rye and their hybrids.

Aspartate aminotransferase patterns were screened in a collection of rye genotypes that included 24 accessions of wild perennial rye (Secale montanum Guss.), 6 accessions of cultivated perennial Derzhavin and Tsitsin rye (Secale cereale x S. montanum), 15 accessions of winter and spring rye cultivars (S. cereale L.), and 9 accessions of perennial and annual rye genotypes bred from S. montanum ssp. kuprijanovii, Derzhavin rye, and winter rye for their resistance to fungal diseases. Aspartate aminotransferase is coded for by four loci. The data fit the model where AAT 1/4 is coded by Aat 1 and Aat 4, two duplicate loci, with null and two active alleles for each locus, alleles 1 and 3 for locus Aat 1 and alleles 2 and 4 for locus Aat 4; dimeric AAT 1/4 enzyme molecules are the products of both intralocus and interloci complementation. Allele 1 of Aat 1 was the most prominent in the isoenzyme patterns of the rye species. Alleles null and 2 of Aat 4 were twice as frequent in the perennial rye accessions, including Derzhavin and Tsitsin rye, than in winter and spring rye. In contrast, allele 4 of Aat 4 was characteristic of S. cereale. Within the screened collection, locus Aat 2 was monomorphic. Among three alleles of Aat 3, allele 2 dominated isoenzyme profiles of both rye species, whereas the other two alleles were species-specific: allele 1 was characteristic of S. montanum and allele 3 was found only in S. cereale. Key words : rye, Secale cereale, Secale derzhavinii, Secale montanum, aspartate aminotransferase, isoenzymes, perennial habit, polymorphism.     

Phylogenetic relationships among Secale species revealed by amplified fragment length polymorphisms.

Amplified fragment length polymorphism (AFLP) data were utilized to analyze the phylogenetic relationships among 29 accessions representing 14 of the most commonly recognized ranked species or subspecies in the genus Secale. We observed 789 AFLP markers of 1130 fragments utilizing 18 P-/M- and E-/M- primer combinations. All polymorphic fragments were used to construct phenetic and phylogenetic trees. The resulting phenogram and cladogram had similar tree topologies. Cluster analysis showed that Secale sylvestre was the most distantly related to all other ryes. Annual forms were grouped together, and the perennial forms appeared more closely related to each other. This suggested that life cycle could have played an important role in determining the relationships among Secale species. Secale sylvestre was considered to be the most ancient species, whereas Secale cereale was the most recently evolved species. Amplified fragment length polymorphism analysis clearly separated all Secale species into only 3 major species groups, within the genus Secale: S. sylvestre, Secale montanum (syn. Secale strictum) for perennial forms, and S. cereale for annual forms. This study demonstrated that the AFLP approach is a useful tool for discriminating species differences, and also gave a much better resolution in discerning genetic relationships among Secale species as compared with previous studies using other approaches.     

非洲黑麦染色体特异性标记的建立与应用

以非洲黑麦、小麦-非洲黑麦双二倍体、安岳排灯麦等为材料筛选100条ISSR引物。其中, 引物UBC815可在非洲黑麦中扩增出1条长561 bp的特异性片段(命名为pSaUBC815₅₆₁), 而小麦对照均未扩出该片段。引物UBC815同样能在黑麦属的瓦维洛夫黑麦(Secale vavilovii Grossh.)、森林黑麦(Secale sylvestre Host.)等5个种扩增出pSaUBC815₅₆₁。根据pSaUBC815₅₆₁设计特异PCR引物U815-F、U815-R, 对小麦族多物种进行扩增, 表明pSaUBC815₅₆₁为黑麦属特有。进而利用一套中国春-Imperial黑麦二体附加系及小麦-黑麦异源材料进行扩增, 结果显示, pSaUBC815₅₆₁分布在黑麦整套染色体上, 并且所有后代材料都能扩增出pSaUBC815₅₆₁, 表明pSaUBC815₅₆₁可作为特异性标记用来检测小麦背景中的黑麦染色质。     

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the Individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aeativum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S.cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattem of Secale vavilovli was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.     

Origin and genetic structure of feral rye in the western United States

Feral rye (Secale cereale) is a serious, introduced weed of dry land agricultural regions of the western United States. It closely resembles cultivated cereal rye (Secale cereale cereale L.) with the exception of having a shattering seed head. Feral rye may have originated from hybridization of cultivated rye with mountain rye, Secale strictum, as past studies of northern Californian populations suggest, or directly from volunteer cultivated rye. We characterized the genetic structure of feral rye populations across a broad geographical range and reexamined evidence for hybrid origin versus direct evolution from domesticated cultivars. Eighteen feral populations were examined from three climatically distinct regions in the western United States. Seven cultivars, four mountain rye accessions, and one wild annual relative (Secale cereale ancestrale) were included in our analysis as possible progenitors of feral rye. Individual plants were scored for 14 allozyme and three microsatellite loci. Estimates of genetic diversity in feral populations were relatively high compared to those of the possible progenitors, suggesting that the weed had not undergone a genetic bottleneck. Weed populations had no geographical structure at either a broad or a local scale, suggesting idiosyncratic colonization and gene-flow histories at each site. Feral rye populations were no more closely related to mountain rye than cultivars were. They were, however, weakly clustered as a distinct lineage relative to cultivars. Our results do not support an interspecific hybrid origin for feral rye, but do suggest that the sampled populations of feral rye share a common ancestry that may explain its weedy nature.     

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.     

An empirical test of the treatment of indels during optimization alignment based on the phylogeny of the genus Secale (Poaceae)

The ability of the program POY, implementing optimization alignment, to deal with major indels is explored and discussed in connection with a phylogenetic analysis of the genus Secale based on partial Adh1 sequences. The Adh1 sequences used span exon 2-4. Nearly all variation is found in intron 2 and intron 3, which form the basis for the phylogenetic analyses. Both in some ingroup and outgroup taxa intron 3 has a major duplication. Previous phylogenetic analyses have repeatedly confirmed monophyly of both Secale and Hordeum, the latter being part of the outgroup. However, optimization alignment only recovers both genera as monophyletic when knowledge of the duplication is incorporated in the analysis. The phylogenetic relationships within Secale are not clearly resolved. Subspecific taxa of Secale strictum have identical sequences and they are confined to a monophyletic group. However, the two subspecific taxa of Secale cereale do not form a monophyletic group, and the position of Secale sylvestre is uncertain.     

ON THE ORIGIN OF CULTIVATED RYE

From extensive cytological, ecological, and morphological studies, it appears that cultivated rye (Secale cereale L.) originated from weedy products derived from introgression of S. montanum into S. vavilovii. Secale vavilovii appears to have been derived from S. silvestre as a consequence of chromosomal translocations. Secale silvestre was, in turn, derived from S. montanum or a common ancestor. Secale africanum, S. dalmaticum, S. ciliatoglume, and S. kuprijanovii appear to be only slightly modified isolated populations of S. montanum. Populations of S. anatolicum are weedy forms of S. montanum, genealogically and chromosomally distinct from the weedy annual forms from which S. cereale arose.     

Evolutionary relationships in the genus <Emphasis Type="Italic">Secale</Emphasis> revealed by DArTseq DNA polymorphism

Till this day, there is not much known about the phylogeny of the Secale genus; therefore, in our research, we tried to shed some lights on the issue of rye (Secale genus) taxonomy. The genetic diversity and phylogenetic relationships were evaluated using 13,842 DArTseq? polymorphic markers. The model-based clustering (STRUCTURE software) separated our 84 samples into three main clusters: perennial cluster, annual cluster, and S. sylvestre cluster. The same result was obtained using Neighbour Joining tree and self-organizing maps. Secale sylvestre, S. strictum, and S. cereale are the three main species of the Secale genus. Three samples of rye are in basal positions in phylogenetic trees. These accessions share ancient morphological characters and are probably the ancestors of different lineages within Secale. Annual Secale taxa, with the exception of S. sylvestre, create one mutual taxon. We have found out that the semi-perennial taxa of S. cereale var. multicaule and S. strictum subsp. ciliatoglume are genetically closest to the annual species of S. cereale. Phylogenetic signals for semi-perennial and annual taxa are also present in S. strictum subsp. africanum. SNP-based analysis revealed that evolution of annual S. cereale has already begun in S. strictum subsp. africanum. The results showed that S. vavilovii cannot be considered as a separate species but a subspecies of S. cereale Secale cereale subsp. dighoricum is a hybrid. It is still not clear whether we can consider S. strictum subsp. strictum and S. strictum subsp. kuprijanovii as two separate species.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

Isolation and chromosomal localization of new MITE-like sequences from <Emphasis Type="Italic">Secale</Emphasis>

A species-specific DNA sequence (marker) that can detect the presence of Secale cereale chromatin in common wheat background was developed by using wheat microsatellite primer Xgwm614. One rye-specific DNA amplified fragment of 416bp (pSa614₄₁₆) was obtained from Secale africanum and a wheat — S. africanum amphiploid. The primer Xgwm614 also gave rise to specific bands in all Chinese Spring-Imperial rye addition lines 1R to 7R. Sequence analysis revealed that pSa614₄₁₆ was strongly homologous to a miniature inverted transposable element (MITE) stowaway-like element. Results of fluorescence in situ hybridization showed that the signal of pSa614₄₁₆ was distributed along all S. cereale. cv Jingzhou chromosomes, but the signal strengths were unbalanced on the seven rye genome chromosomes. This repetitive element may be useful as a molecular marker for the introgression of rye germplasm into the wheat genome.     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

In vitro synthesis of lectins in cell-free extracts from dry wheat and rye embryos

Cell-free extracts from dry wheat (Triticum vulgare L.) and rye (Secale cereale L.) embryos do not synthesize their corresponding lectins when incubated under conditions optimalized for translation of either exogenous or endogenous mRNA. Only when the extracts are prepared and incubated in the complete absence of sulphydryl containing compounds lectins are synthesized in vitro. Since immunoprecipitation techniques could not demonstrate unequivocally the presence of lectin among the cell-free translation products a specific affinity purification procedure has been developed in order to proof the presence of stored lectin mRNAs in dry wheat and rye embryos.Abbreviations DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCA trichloracetic acid     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

Actinomycete complexes in the rhizosphere of winter rye on soddy podzolic soil

The structure of actinomycete complexes in the rhizosphere of winter rye (Secale cereale L.) varieties originating from different ecosystems and geographical zones was studied in field experiments on soddy podzolic soil. In addition to streptomycetes, the complexes studied contained actinomycetes of the genera Micromonospora and Streptosporangium, represented at high occurrence and comparable abundance rates. The rhizosphere of most of the studied rye varieties was dominated by micromonosporas. The antifungal potential of mycelial prokaryotes associated with winter rye was assessed. The taxonomic and functional structure of actinomycete complexes was shown to be similar in rye varieties originating from the nonchernozem zone of Russia. However, the actinomycete complex proved to be much different in the rye variety that developed in the steppe zone of the lower Volga region.     

黑麦基因组特异DNA片段的分离与SCAR标记的建立

以2个栽培黑麦、2个野生黑麦和4个普通小麦为材料,从200条10碱基RAPD随机引物中筛选出1条引物H11。H11在小麦中有1条低拷贝扩增,而在黑麦中却有极高拷贝的扩增。对H11在黑麦中的高拷贝片段进行克隆、测序,得其全长679 bp,记作OPH11679。根据OPH11679设计特异PCR引物H11-F和H11-R,对小麦族其它物种和含黑麦染色质的物种进行验证,结果发现仅含黑麦染色质的物种能扩增出长为643 bp的片段(命名为pScH643),这表明该片段为黑麦所特有。用H11-F和H11-R对1套中国春-Imperial黑麦附加系等进行扩增,结果显示pScH643片段分布在黑麦整套染色体上,这一结果在小麦-黑麦异源材料的分析中得到进一步验证。即表明pScH643片段可作为SCAR标记用于含黑麦染色质材料的检测。