The chromosome number of rye, Secale cereale (Gramineae), is 2n = 2x = 14. The genome formula of diploid rye is given as RcerRcer. When accessory chromosomes are not considered all rye species are diploid showing 14 A chromosomes.
MOLECULAR STRUCTURE OF GENOME
The 1C DNA content of the rye genome is 9.5 pg (Bennett and Smith 1976). The 1C DNA content of individual rye chromosomes ranges from about 1.2 to 1.4 pg considering that chromosome length and DNA content are directly proportional. Only 10-20 % of the genome can be assigned, biochemically, to the major part of the genome, which belongs to the repeated sequence category. The kinetic analysis of genome organization has revealed that repeated sequences are, in general, interspersed among unrepeated sequences. The discovery of a very rapidly reannealing class of DNA constitutes 4-10 % of the genome and although it is believed to be composed largely of sequences capable of renaturation, this class also contains long tandem arrays of simple, repeated sequences (Appels 1982). Ranjekar et al. (1974) were the first to demonstrate several buoyant density components in a fraction of DNA renaturing with a density of 0-0.1 (10-12 % of the genome). However, the predominant component is a well-defined species at 1.702 g/cc in a CsCl-gradient. Smith and Flavell (1977) considered this class of DNA to consist mainly of palindromic sequences, which are distributed, in clusters throughout at least 30 % of the genome. DNA, with a mean fragment length of 500 bp, was fractionated to allow recovery of very rapidly renaturing fraction (Cot 0-0.2). This DNA was shown to contain several families of highly repeated sequence DNA. Two of them were purified which resulted in a fraction renaturing to a density of 1.701 g/cc and comprised 0.1 % of the total genome, and the other polypyrimidin tract DNA that comprised 0.1 % of the genome.
Other hybridization studies between cereals have shown that 22 % of the DNA is species-specific repeated sequences (Rimpau et al. 1978).
PRIMARY TRISOMICS AND TELOTRISOMICS
Spontaneously observed trisomics have been described already in 1935 by Takagy (1935) and Sybenga (1965). A complete series of primary trisomics was established in 1962 (Kamanoi and Jenkins 1962), however got lost later on. Two new sets of trisomics were prepared from the winter rye variety “Danae” by Mettin et al. (1972) and “Heines Hellkorn” by Zeller et al. (1977). The first derived from triploid x diploid crosses and cytological as well as morphological selection (Fig. 4.3.1). The trisomic karyotype is transmitted by about 15 % through the egg cell and almost 0 % through the pollen. Consequently, backcrosses to the female plants are needed in order to maintain the trisomics.
Although the extra chromosome paired quite regular with its homologues (a positive correlation between the relative chromosome length and number of chiasmata per chromosome was evident), in the progeny monotelocentric and other aberrations (Fig. 4.3.2) could be observed for almost all of the 14 chromosome arms (Schlegel and Sturm 1982, Sturm and Melz 1982, Melz and Schlegel 1985).
Until molecular studies became standard in gene mapping during the 1990th, trisomics and telotrisomics have been the most powerful tool for gene localization in rye (Sturm 1978, Melz et al. 1984). Depending on the backcross or selfing progeny different segregation patterns in F2 were used for assignment a morphological or physiological character to a particular (trisomic) chromosome (Tab. 4.3.1).
B chromosomes of rye are very invasive. It is the only plant where the B chromosomes undergo non-disjunction at the postmeiotic mitosis of both female and male gametophyte. It is controlled by a gene or gene complex on the B chromosome itself. By reduced fertility and persistent univalent formation a high degree of chromosomal instability is found, which results in a highly polymorphic population.
The B chromosome of rye is relatively large (Fig. 4.4.1). It amounts about 10 % of the DNA of the haploid A genome. It has a widespread global distribution, and exhibits little structural variation. It is presumed to be derived largely or entirely from the A chromosome set based on similar DNA composition, most recently shown by using in situ hybridization with genomic DNA or the rye specific R173 repeat.
Despite the general similarity the B does not pair with any A chromosomes. However, translocations between B and A chromosomes were observed (Pohler and Schlegel 1990). Chromosome 3R was involved in the rearrangement (Fig. 4.4.2). By the authors it was demonstrated that a terminal segment, including a large terminal heterochromatic block, was transferred to the terminal region of the long arm of B chromosome (Schlegel and Pohler 1994).Although several authors assumed a monophyletic origin of rye B chromosomes in the distant past and a perpetuation in various populations, the results imply similar events also nowadays, which is partially supported by stable fragments occasionally revealed as well as the presence of B chromosomes in numerous plant species.
Defined A-B translocations were thought to be useful tools for gene amplification, chromosome identification, genetic investigations, and gene transfer experiments.
B chromosomes appear to be transcriptionally inactive, implying either loss of coding sequences or selective amplification of repetitive DNA in its evolution. By AFLP studies it was demonstrated that DNA products specific to B-containing plants were seen at a frequency of about 1 %, significantly less than that predicted on a mass basis. Most of the fragments were found to be highly repetitive and were shown by FISH to be distributed over both A and B genomes. Dispersed fragments fell into two groups: the first showed an equal density of distribution across both A and B genomes, consistent with earlier observations, while the second showed an increased concentration at a pericentric site on the long arm of the B. The redundancy of members of the second group suggests that this region is less complex than the average for the B, and may represent a “hot spot“ of sequence amplification.
The terminal heterochromatin of B chromosome contains both B-specific sequences and sequences also present on the A chromosomes (Houben et al. 1996). The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. The most distinctive region of the B chromosome is a subtelomeric domain that contains an exceptional concentration of B-specific DNA sequences. At metaphase this domain appears to be the physical counterpart of the subtelomeric heterochromatic regions of the standard B chromosome (Langdon et al. 2000).
Until now, several attempts have been made to establish a common chromosome designation (Tab. 4.5.1), a general karyological characterization and a standard karyotype in diploid rye. All efforts, however, did not completely account for the natural variation of chromosome morphology and structural heterozygosity in allogamous rye populations. To overcome the difficulties at least partially, the participants of the 1st and 2nd Workshops on Rye Chromosome Nomenclature and Homoeology Relationships (Sybenga 1983) decided to consider the “Imperial” rye additions to the hexaploid wheat variety “Chinese Spring” as the standard rye chromosome set, although these chromosomes are not completely identical with those of the population of “Imperial“ variety.
Based on the agreements, a preliminary karyotype and its C-banding patterns, a homoeologous designation of added rye chromosomes and a generalized C-banding pattern were proposed. The participants anticipated producing a standard diploid rye homozygous for genetic, biochemical and/or molecular studies, and which should be, in addition, available for cytogenetic testcrosses. Therefore, Schlegel et al. (1987) established from the rye variety “Petka“, a haploid (Fig. 4.5.1), a dihaploid, and, finally, a tetrahaploid plant from the original haploid genome. The variety was chosen because of its spring growth habit, dominantly greenish grains and traits of modern cultivars. “Petka“ was bred in Petkus (Germany) and released in 1961. Its genetic background is related to the genepool of “Petkus“ rye, which has been used worldwide in breeding and research. “Petkus” rye was bred since 1882 by F. von LOCHOW at Petkus, south of Berlin (Germany). The material derived from the landrace “Probsteier Roggen”, marketed by Probsteier land- und forstwirtschaftlicher Verein, Schönberg, near Kiel (Germany). Since only few karyograms in rye are based on chromosome measurements, information on chromosome morphology in a structurally homomorphic rye is given in Table 4.5.2.
The data demonstrate remarkable differences in mean total length as well as relative arm length, which range from 124.24 units in chromosome 1R to 162.46 units in 2R, and from 45.03 units in chromosome arm 5RS to 93.25 units in 5RL, respectively. The arm ratios vary from 1.02 in chromosome 3R to 2.07 in 5R. The small sample standard deviations mean that most of the length differences or arm index variations are statistically significant. This was due to measurements of identified C-banded chromosomes, which reduce mistakes in chromosome determination (Fig. 4.5.1).
The karyogram drawn from the data has been used for detailed description using the C-banding pattern. Prominent blocks of telomeric heterochromatin are stained, which is a common pattern of diploid rye, Secale cereale L. (Fig. 4.5.2). Rye chromosomes 2R, 3R, and 6R show also prominent N-bands near the centromeres (Fig. 4.5.3). Moreover, there is quite a good correlation between the distribution of heavy knobs of chromomeres described by Lima de Faria (1952) and C and/or N bands. Recently, tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions.
The band positions and band sizes are related to the relative arm length, so there are sufficient references for identifying each of the seven chromosomes individually. Applying the standard chromosome band nomenclature taken from Schlegel et al. (1986), a specific reference karyogram was established (Fig. 4.5.4) excluding structural heterozygosity of the genome. This standard karyotype and fully homozygous genotype of the dihaploid and tetrahaploid progeny was proposed as general reference material in genetic and cytogenetic studies.
Rye is known as species maintaining chromosomal interchanges within populations in a more or less high frequency and complexity (Candela et al. 1979). Chromosomal interchanges are of particular importance both for evolutionary studies of rye and for several genetic and/or breeding applications. Until the 1980th the nomenclature of rye chromosomes was confusing (Schlegel and Mettin 1982). Therefore, several efforts were made in order to designate the chromosomes according the homoeologous relationships within the Triticinae (Sybenga 1983). A translocation tester set of rye was established and used for direct crosses with wheat-rye chromosome addition lines. In this way, for the first time a complete series of reciprocal translocations was described by Sybenga et al. (1985). Although several other interchanges have been established before and after those experiments (Sybenga and Wolters 1972, Vries and Sybenga 1976, Augustin and Schlegel 1983), this series became the key tester set of rye involving all seven chromosomes by interchanged chromosomes as follows: 1RS-4RL, 1RS-5RL, 1RS-6RS, 2RS-5RS, 2RL-6RL, 3RS-5RL, 4RL-5RL, and 5RL-7RS. They originated from irradiated pollen grains of “Petkus” rye crossed to several inbred rye lines.
Despite the intensive utilization for gene mapping in rye (Vries and Sybenga 1976), reciprocal translocations were also used for diploidization experiments in tetraploid rye. However, the complex structural heterozygosity of re-constructed tetraploid karyotypes (Fig. 4.6.1) not only did increase the preferential bivalent pairing, but also did decrease the fertility.
Neocentric activity is a newly derived kinetic activity outside the proper centromere. Kattermann (1939) first described the phenomenon in rye. Neocentromeres are rare in plants, but less infrequent in meiosis. Three types of neocentromeres have been described in rye: (1) the neocentromere shows a stable structural differentiation in one end of a given chromosome. It is inherited as a Mendelian gene locus. (2) The neocentromeres are located at terminal regions of some chromosomes. They are mostly associated with distal heterochromatin. They are variable in activity and number among individuals and cells within an individual. There is a polygenic control (Viinikka 1985). They occur in both inbred lines and in allogamous populations. (3) Schlegel (1987) described an additional type in haploid rye. A proximal constriction present on the long arm of chromosome 5R is co-oriented with the ordinary centromere. It behaves like a dicentric chromosome. However, molecular studies showed that the 5RL constriction lacks detectable quantities of two repetitive DNA sequences, CCS1 and the 180 bp knob repeat, present at cereal centromeres and neocentromeres (Manzanero et al. 2000).
Several workers have reported haploids and meiotic studies on haploids in rye (cf. Fig. 4.5.1). Associations of two or more apparently non-homologous chromosomes are often observed at metaphase I. Levan (1942) was the first to demonstrate statistically that chiasma formation between the seven chromosomes was not random. He suggested that one particular chiasma is formed at quite a high frequency while the remaining arises at random. The mean chiasma frequency per pollen mother cell as given by different authors ranged from 0.03 to 0.44 (Fig. 4.8.1). Differentiation between true chiasma formation and secondary end-to-end attachments can be made either by chromosome co-orientation or by proving chromatid bridges and acentric fragments during anaphase I. By testing the random or non-random association of non-homologous chromosomes in the haploid, a statistically significant increase of chromosomes with heavy telomeres on both ends was found, i.e., chromosomes 1R, 2R, 3R, and 7R may show duplicated segments contributing to chromosome pairing in a haploid rye. No chiasma formation was observed in the heterochromatic telomeres. This indicates that this kind of repetitive DNA does not function as a homologous region contributing to crossing over (Schlegel et al. 1987). Obviously inhomologous chromosome pairing in rye was discussed as result of segment duplications of the basic rye genome showing instead of seven only five chromosomes.
It is supposed that cereals within the subtribus Triticinae have a common origin and, likewise, a partial structural homology. It was first concluded by more or less good ability of chromosomes to substitute each other in interspecific hybrids (Gupta 1971, Koller and Zeller 1976, Schlegel 1990). Later, it could be supported by intergeneric hybridization, particularly with wheat, that rye chromosomes can even show chiasmatic pairing with wheat chromosomes (Schlegel and Weryszko 1979). The comparatively high degree of wheat-rye chromosome pairing and, thus, recombination became of interest for plant breeding utilizing the incorporation of useful characteristics of rye species for wheat and triticale improvement. The frequency of wheat-rye pairing ranged from zero in crosses with Secale silvestre to 2.4 % per PMC in S. montanum, respectively.
Recent molecular maps show remarkable conservation of gene order among wheat, rye, barley, millet, rice etc., disrupted only by a few gross interchromosomal rearrangements and the emergence of genome-specific non-coding sequences, particularly at the physical ends of chromosomes. Rye has diverged from wheat by at least seven to thirteen translocation events (Fig. 4.9.1) after only 6 million years of divergence, while, for example, barley appears to reflect precise synteny with the basic wheat genome (Devos et al. 1993). Other authors provide evidence that some genomes fix rearrangements more readily than others. These different rates of species divergence through chromosomal rearrangement do not appear to correlate to the breeding system, because high levels of evolutionary translocations are found both in rye, an outbreeder, and, e.g. Aegilops umbellulata, a predominantly self-pollinated species.
TAXONOMY, CYTOTAXONOMY AND ORIGIN
Apart from artificial polyploids, all rye species are diploid (2n = 2x = 14). However, numerous reports about accessory or B chromosomes are available in both wild and cultivated Secale montanum and S. cereale. Roshevitz (1947) recognized 14 species but it is questionable whether all these have to be given specific rank. Two groups of species can readily be separated as being important in the evolution of the cultivated rye. First, there is a group of annual weeds, such as S. ancestrale, S. dighoricum, S. segetale and S. afghanicum, which cytologically resemble each other and cultivated rye (Schlegel and Weryszko 1979). They could be included as subspecies of S. cereale. This group is confined to agricultural areas, the weedy types being widespread in cereal crops in Iran, Afghanistan and Transcaspia (Zohary 1971).
Second, there is an aggregate of wild perennial forms widely distributed from Marocco through the Mediterranean area, Anatolia to Iraq and Iran. These have been separated into distinct species but are most probably described as variants of a single species, S. montanum. Members of this group are cytologically similar and interfertile. However, they differ from the S. cereale complex by two major chromosomal reciprocal translocations involving three pairs of chromosomes (Fig. 4.10.1). Stutz (1972) proposed a stepwise evolution of S. cereale from S. montanum. The annual species are supposed to be derived from the introgression of S. montanum into S. vavilovii. This annual selffertile species was in turn derived from the annual S. silvestre as a consequence of chromosome translocation (Fig. 4.10.2). The latest revision of rye taxonomy was given by Frederiksen and Petersen (1998). The recognize only three species of of Secale, i.e. S. sylvestre, S. strictum, and S. cereale. Secale strictum has priority over S. montanum. It includes two subspecies, i.e. ssp. strictum and ssp. africanum. Secale cereale also includes two subspecies, i.e. the cultivated taxa, marked by their tough rachises of ssp. cereale and the wild or weedy taxa showing fragile rachis of ssp. ancestrale.
Comparison of chloroplast DNA variation confirmed the particular distinctness of S. silvestre from the remaining taxa. This basic differentiation between S. silvestre and S. montanum and/or S. strictum took place during Pliocene or later. In this context it became clear that rye shows a purely maternal inheritance of chloroplasts (Corriveau and Coleman 1988), although Fröst et al. (1970) postulated a biparental mode of inheritance.
The most likely place of origin of the weedy rye is in the area central and eastern Turkey, northwest Iran and Armenia. It is the area of maximum genetic diversity and coincides with the high degree of variability of the perennial S. montanum. The cold and harsh climate of this area possibly favored rye instead of the two main cereals, barley and wheat, which also spread into this region. It is no doubt that under such conditions rye could have become established as a cornfield weed. Weedy annual races with brittle and semi-brittle rachises were and still are colonizers.
In many taxa introgression is a more or less frequent phenomenon, depending on the spontaneous tendency to interspecific hybridization. As hybridization played an important role in the evolution of plant speciation, it has been suggested that interspecific crosses will offer better results in taxa in which such evolutionary mechanisms have been common. Only few experiments have been successful with diploid crop plants, although the improvement of their performance is as important or even more valuable than in polyploids.
By crossing of hexaploid wheat and diploid rye and subsequent backcrossing several monosomic and monotelosomic rye-wheat chromosome additions were produced (Schlegel 1982, Schlegel et al. 1986) using rye cytoplasm as background (Tab. 4.11.1). However, during the first experiments also alloplasmic rye-wheat additions were available, when, in crosses with rye, the hexaploid spring wheat “Chinese Spring“ was used as female.
Monosomic additions were maintained by backcrossing to the euploid rye parent and subsequent microscopic screening (Fig. 4.11.1). Because of missing male transmission of the extra chromosome self-pollination did not result in disomic addition lines. Characteristic morphological and physiological distinctness could be detected depending on the alien chromosome added to rye. Since the population size was to small no agronomic performance testing was possible.
In order to transfer wheat chromosomal segments and/or single genes into the rye complement experiments were designed to take advantage of gametic selection after spike irradiation, which would facilitate to recovery of desirable translocations. Thus, premeiotic spikes of several addition lines were exposed to X-rays of 1,000 r dosage.
The resulting pollen grains were used to fertilize emasculated euplasmic spring rye plants. The resulting F1 seedlings were screened by chromosomal banding techniques for the presence of alien chromatin and further characterized by meiotic analysis. A successful example of gene transfer could be demonstrated involving chromosome 6B of hexaploid wheat, var. Chinese Spring. 36 treated spikes used for pollination gave 364 seeds for cytological screening. Since male transmission of the intact extra chromosome could not be fully excluded, all of the progenies were checked for somatic chromosome number. Among 188 plants analyzed 169 (90 %) individuals had 14 chromosomes, one (0.5 %) 14+trye, one (0.5 %) 13, one (0.5 %) 28, and 16 (8.5 %) 14+1wheat. Thus, the added wheat chromosome can be transmitted, at least, to some extent through the pollen cells although previous studies of selfed alloplasmic 6B additions never yielded disomic progeny. By application of differential chromosome staining all 15-chromosome plants were classified as rye plus one wheat chromosome 6B. However, among the 14-chromosome progeny, two types were found to have novel sites of N-bands as compared to rye. One of the two plantlets showed in all somatic cells, in addition to the 13 normal rye chromosomes, one prominent chromosome with a heavy N-band situated near the centromere. This band did not match any of the rye-specific N-bands. Its location and size resembles the heterochromatic block of chromosome 6B. More detailed studies revealed a dicentric structure of the rye-wheat translocation (Fig. 4.11.2). The results provide clear evidence of transfer of alien genetic material to diploid rye. It is tolerated not only in the embryo but also by the adult plants. It also demonstrates that wheat genes can compensate for the loss of rye genetic information (Schlegel and Kynast 1988).
Compared to other cereal and crop plants rye was less accessible for DNA and/or gene transfer experiments. In addition, rye is known as one of the most recalcitrant species in tissue culture. Neither the tissue culture ability nor the regeneration ability is as good as in wheat, barley, rice or other grasses. Merely a genotype of Secale vavilovii (Grosse et al. 1996) showed a higher in vitro utilization as compared to average of the S. cereale genotypes tested (Flehinghaus et al. 1991). This strongly hindered the application the co-cultivation approach using Agrobacterium tumefaciens. The first break-through obtained Pena et al. (1987). Work on the development of the male germ line of rye showed that 14 days before first meiotic metaphase the archesporial cells are highly sensitive to caffeine and colchicine injected into the developing floral tillers. The authors considered that at this stage the archesporial cells might also be permeable to other molecules, such as DNA. Therefore they injected DNA carrying a dominant selectable marker gene into rye plants. It was the plasmid pLGVneo1103 including the aminoglycoside phosphotransferase II gene (APH 3’) under the control of the nopaline synthetase promoter. The plants were injected two weeks before meiosis. Because of the high degree of self-incompatibility the "JNK" rye, seeds were produced by pair-wise crossing of infected tillers.
From 3,023 seeds screened for kanamycin resistance and derived from 98 plants, seven seedlings remained green after 10 days growth on kanamycin-containing medium. These apparently kanamycin resistant plantlets were assayed for the presence of APH(3’)II enzymatic activity. The two of the seven resistant seedlings, each resulting from an independent injection experiment, showed APH(3’)II activity. This was the first unequivocal report on a gene transfer experiment in rye.
Recently, a reproducible transformation system for rye was established, using inbred lines with superior regeneration potential from tissue cultures. Biolistic parameters, such as tissue age during bombardment and micro-projectile density, were compared in a multi-factorial experiment. By using the selectable marker genes bar or nptII transformation efficiency between 2 to 4 % of the bombarded explants was found. A total of 37 independent transgenic rye plants were produced by biolistic gene delivery. For Agrobacterium tumefaciens-mediated gene delivery, different influencing factors were combined and led to morphologically normal and fertile transgenic plants at a frequency of 3.9 % of the inoculated explants. Moreover, a selection system was also developed for "direct" production of transgenic plants. The high-molecular-weight glutenin subunit genes Ax1, Dx5 and Dy10 from wheat were introduced into rye and their stable expression in endosperm of primary transformants of rye and in their segregating progeny demonstrated (Herzfeld 2002).