Genetic diversity and phylogenetic relationships in the rye genus Secale L . ( rye ) based on Secale cereale microsatellite markers

The genetic diversity and phylogenetic relationships in the genus Secale L. (rye) was evaluated using 24 Secale cereale microsatellite (SCM) markers. The average polymorphism information content (PIC) value of each microsatellite locus in 30 Secale accessions evaluated was higher than that in 47 cultivated ryes (Secale cereale ssp. cereale). The mean genetic similarity (GS) index in Secale was lower than that in cultivated rye. The highest within-species GS index was observed for S. sylvestre and the lowest for S. strictum, whereas the highest between-species GS index was found between S. cereale and S. vavilovii and the lowest between S. sylvestre and S. cereale. There was no obvious difference in GS levels in the cultivated rye accessions from Asia, Europe, North America or South America. Cluster analysis indicated that all the Secale accessions could be distinguished by the 24 microsatellite loci. We also found that the S. sylvestre accessions were obviously divergent from the accessions of other species and that the S. vavilovii accessions were closely related to the S. cereale accessions. Our results also showed that S. strictum was heterogeneous and showed great within-species differences. The microsatellite-derived dendrogram faithfully reflected the phylogenetic relationships between Secale species but did not indicate a possible domestication process of the cultivated rye based on the geographical sources of the accessions.


Introduction
The taxonomy of the genus Secale (rye) has for a long time been a matter of disagreement, despite the large number of studies performed.Historically, about 15 different species have been accepted (Roshevitz 1947, Delipavlov 1962), while Frederiksen and Petersen (1998) recognized only three Secale species, i.e. S. sylvestre, S. strictum (plus the subspecies ssp.strictum and ssp.africanum and the varieties var.strictum and var.ciliatoglume) and S. cereale (plus ssp.cereale and ssp.ancestrale).According to taxonomic systems adopted by the American Germplasm Resources Information Network (GRIN, http://www.ars-grin.gov),the genus Secale is presently recognized as containing four species, consisting of the annual outbreeder S. cereale L. , the annual autogamous S. sylvestre Host and S. vavilovii Grossh., and the perennial outbreeder S. strictum (Presl.)Presl.(syn.S. montanum) (Sencers andHawkes 1980, De Bustos andJouve 2002).There are 8 sub-species in S. cereale and 5 in S. strictum, and S. cereale ssp.cereale is the only cultivated rye.
The different recent classifications of the genus Secale have involved phylogenetic analyses based on the use of various technologies, including some biochemical and molecular markers such as isozymes (Vences et al. 1987a,b), random amplified polymorphic (RAPDs) (Del Pozo et al. 1995), rDNA spacer-length (Reddy et al. 1990, Cuadrado andJouve 2002) and internal transcribed spacers (ITS) (De Bustos and Jouve 2002) as well as traditional morphological and cytogenetical methods (De Bustos and Jouve 2002).Simple sequence repeats (SSRs or microsatellites) consisting of tandem repeats of 2 to 6 nucleotides are abundantly distributed throughout the nuclear genomes of all studied plant species (Tautz et al. 1986, 1989, Litt and Luty 1989).Because of their co-dominant inheritance, high polymorphism, good reproducibility and the convenience of the polymerase chain reaction (PCR) microsatellites have become the genetic markers of choice for studies involving plant species (Powell et al. 1996, Zhebentyayeva et al. 2003).Takezake and Nei (1996) concluded that microsatellite DNA seemed to be very useful for clarifying the evolutionary relationships of closely related populations.However, the application of microsatellite markers to plant taxonomy is still very limited because the development of specific SSR primers is time-consuming and expensive plus the fact that genomic microsatellite markers are hard to transfer between the genomes of different species.Fortunately, at least 184 S. cereale microsatellite (SCM) markers have recently been developed (Saal andWricke 1999, Hackauf andWehling 2002).
The objectives of the study described in the present paper were to investigate the phylogenetic relationship of four Secale species using microsatellite markers and to evaluate the genetic diversity and genetic relationships of cultivated rye (S. cereale ssp.cereale) from different countries.

Plant materials
For this study we selected 30 Secale accessions, consisting of 5 cultivated rye and 25 non-cultivated rye, to represent the 4 species and 10 subspecies of the rye genus (Table 1).Meantime we used 47 cultivated rye (S. cereale ssp.cereale) accessions from different countries (Table 1).Accession CN31389 was kindly provided by Dr. D. Kessler of Plant Gene Resources of Canada (PGRC).Accession NGB5073 was kindly provided by Dr. L. Bondo at Nordic Gene Bank, Sweden.Accessions R953/90 and R955/90 were kindly provided by Dr. A. Graner at the Genebank Gatersleben, Institute of Plant Genetic and Crop Plant Research (IPK).Accessions As3045 and As3033 came from the Triticeae Research Institute, Sichuan Agriculture University (TRISAU).Most of the accessions with accession numbers starting with PI were kindly provided by Dr. H. Bockelman of the American Germplasm Resources Information Network (GRIN).

DNA isolation and PCR amplification
Genomic DNA was extracted from young leaves using the cetyltrimethylammonium bromide (CTAB) procedure of Doyle and Doyle (1987).The samples for each accession consisted of DNA bulked from 25-30 individual plants.
We used 24 microsatellite markers (Table 2), consisting of 13 markers described by Saal and Wricke (1999) and 11 markers described by Hackauf and Wehling (2002).All the primers were synthesized by Shenergy Biocolor, Biological Science and Technology Co., China.The final reaction volume was 25 mL, containing approximately 50-100 ng template DNA, 1 unit of Takara rTaq polymerase (Takara Bio, Inc., Kyoto, Japan), 0.2 mM of each primer, 200 mM of each deoxynucleotide triphosphates (dNTP) (Takara Bio, Inc., Japan), 1.5 mM MgCl 2 , and 1xPCR buffer.The PCR amplifications were carried out in a PTC-240 thermocycler (Genetic Technologies, MJ Re-search, USA) under the conditions described by Saal and Wricke (1999) and Hackauf and Wehling (2002) with minor modification.The PCR amplification products were separated on a 6% (w/v) denatured polyacrylamide gel and visualized by silver staining.

Data scoring and analysis
Polymorphism information content (PIC) values were calculated for each microsatellite locus according to the formula: where p ij is the frequency of the jth allele for the ith marker summed over n alleles (Anderson et al. 1993).For each genotype x marker combination, the presence (1) or absence (0) of a microsatellite allele was treated as an independent character.The data matrix was then used to calculate the genetic similarity (GS) index (Nei and Li 1979) as , where N ij is the number of microsatellite alleles common to genotypes i and j, while N i and N j are the total numbers of microsatellite alleles observed for genotypes i and j, respectively.Genetic relationships among Secale accessions were estimated using the unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis of the GS matrix (Rohlf, 2000)

Results
Five cultivated rye (S. cereale ssp.cereale) accessions and 25 accessions of other Secale species or subspecies were selected to detect the genetic variations and relationships within and between Secale species.For the 30 accessions selected a total of 113 microsatellite alleles were amplified, of which 107 (94.7%) were polymorphic with an average of 4.7 alleles per locus and a range of from 2 to 8. The average PIC value of the 13 markers from Saal and Wricke (1999) was 0.589 while that of the 11 markers from Hackauf and Wehling (2002) was 0.620.The average PIC value of the 24 microsatellite loci was 0.604 and ranged from 0.315 to 0.799.In these accessions the highest PIC value was for the SCM101 marker.These results indicated a high level of the microsatellite polymorphism in the 30 accessions The genetic similarity and range of variation within and between species were calculated based on the GS matrix (Table 3).The GS index for the 30 accessions ranged from 0.326 to 0.932 with a mean of 0.633.The highest genetic similarity occurred between S. sylvestre accessions R953/90 and R955/90 while the lowest was between S. segetale accession PI61867 and S. sylvestre accession CN31389.The highest within-species GS index (0.884) was for S. sylvestre and the lowest (0.649) was for S. strictum.The lowest between-species GS index (0.444) was for S. sylvestre and S. cereale, and the GS indices be- tween S. sylvestre and S. vavilovii and between S. sylvestre and S. strictum are also lower.The results indicated that the S. sylvestre accessions studied were more divergent from the accessions of the other species.The GS index between S. vavilovii and S. cereale was the highest at 0.783, indicating that S. vavilovii was closely related to S. cereale.
The genetic relationships in Secale as estimated using UPGMA cluster analysis of the genetic distance 1-GS matrix are shown in Figure 1.All 30 accessions could be distinguished by 24 microsatellite markers.The six S. sylvestre accessions were divergent from the other accessions and all closely clustered into group V.The S. strictum ssp.africanum accession As3033 and S. strictum accession PI401400 were divergent from the other S. strictum accessions and clustered into group IV, while S. strictum accessions (PI531829 and PI568257) were clustered into group III.The S. strictum ssp.kuprijanovii accession PI326282 and two S. strictum ssp.anatolicum accessions (PI445973 and PI445974) were clustered into group II.The three S. vavilovii accessions and all S. cereale subspecies were genetically related and clustered into group I.
For the 47 S. cereale ssp.cereale (cultivated rye) accessions (Table 2) a total of 82 microsatellite alleles were amplified, of which 69 (84%) were polymorphic with an   average of 3.3 alleles per locus and a range of from 1 to 7. We also found that 22 out of the 24 (91.67%)microsatellite markers used were polymorphic in the 47 cultivated rye accessions studied, the remaining two microsatellite markers (SCM 180 and SCM304) being monomorphic.The average PIC value of the 13 markers from Saal and Wricke (1999) was 0.383 while that of the 11 markers from Hackauf and Wehling (2002) was 0.576.In cultivated rye the average PIC value of the 24 microsatellite loci was 0.471 and ranged from zero for SCM180 and SCM304 to 0.797 for SCM5.These results indicate that the microsatellite polymorphism in the cultivated rye was lower.
The mean GS index for the 47 cultivated rye accessions was 0.773 and ranged from 0.622 to 0.921.The highest GS index (0.921) was observed between the Australian accession PI346416 and accession PI372118 from the Russian Federation, while the lowest (0.622) was between accessions PI542468 from Mexico and PI345001 from Macedonia.The genetic similarity and range of variation between cultivated rye accessions from different continents were calculated based on the GS matrix (Table 4).The GS index for the European accessions was the highest (0.794) while that for the North and South American accessions was the lowest (0.731).The highest GS value (0.777) was observed between Asia and Europe, and the lowest (0.742) between Europe and North American.Our microsatellite-derived data showed no obvious differences in GS index between the cultivated ryes from different continents.
The genetic relationships in cultivated rye (Figure 2) were estimated by UPGMA cluster analysis of the genetic distance (1-GS) matrix.Accessions PI323449 and Secale representatives and 0.576 for 47 cultivated ryes, respectively.Saal and Wricke (1999) developed the microsatellite markers from a genomic library, whereas the markers developed by Hackauf and Wehling (2002) were derived from expressed sequence tags (EST) sequences.Our results suggest that the EST-derived microsatellite markers were superior to the microsatellite markers from genomic library, and could reveal a relatively higher level of polymorphism in the genus Secale, especially in cultivated rye.
The phylogenetic relationships of Secale at the species level has been investigated using diverse techniques (Venses et al. 1987a, Reddy et al. 1990, Del Pozo et al. 1995, De Bustos and Jouve 2002).Our study showed that the S. sylvestre accessions were very divergent from the ac-cessions of the other species investigated, agreeing with the majority of previous studies (Reddy et al. 1990, Del Pozo et al. 1995, De Bustos and Jouve 2002).Our S. strictum accessions were heterogeneous, with large differences between subspecies.For example, we found that S. strictum ssp.africanum, once considered to be a separate species (Khush 1962), was indeed clustered in a separate group, while S. strictum ssp.kuprijonovii, the hypothetical ancestor of the other taxa (Hammer, 1990), was clearly similar to S. strictum ssp.Anotolicum but divergent from the other S. strictum subspecies, supporting the results published by Hammer (1990) and De Bustos and Jouve (2002).Traditionally, S. vavilovii has been considered to be a separate species (Khush 1962, Del Pozo et al. 1995, Cuadrado and Jouve 1997, De Bustos and Jouve 2002) but we found that it was closely related to S. cereale.No obvious differences were found between the S. cereale subspecies, supporting the results of Secale rDNA ITS analysis reported by De Bustos and Jouve (2002).
It is thought that cultivated rye originated in the Mount Ararat and Lake Van area of eastern Turkey, linguistic evidence suggesting that the introduction of cultivated rye to southern and western Europe and Central Asia were independent of each other (Sencer and Hawkes 1980).Khush (1962) concluded that cultivated rye probably entered Europe by two routes, one being through the northern Caucuses and the other through central Asia.Bushuk (1976) proposed that cultivated rye was probably distributed from south-western Asia to Russia, and thence into Poland and Germany from where it gradually spread throughout most of Europe and eventually to North America and western South America.Rye was introduced into China from Turkey and later the species was into Japan.Ma et al. (2004) found that American cultivars were more closely related to Chinese cultivars than to European cultivars and that temporal isolation had influenced the genetic diversity of rye more than geographical isolation.
In their book concerning the origin of isolating mechanisms in flowing plants, Max et al. (1978) observed that geographical, ecological and reproductive isolation should be taken into account when studying plant evolution.In our study we analyzed the genetic similarities of cultivated rye accessions from Asia, Europe, North America and South America, but could not make any deductions regarding the domestication process of cultivated rye, indicating that further studies are needed to detect the phylogenetic relationships and evolution process of cultivated rye.

Figure 2 -
Figure 2 -The genetic relationships of cultivated rye accessions based on microsatellite markers.

Table 1 -
The 72 Secale accessions used in this study.
**Means the representative accessions selected for the subspecies S. cereale ssp.cereale.

Table 2 -
Hackauf and Wehling (2002)ize range of alleles, polymorphic information content (PIC) value and chromosome location of the Secale microsatellite markers used in this study.The markers came from the studies ofSaal and Wricke (1999)andHackauf and Wehling (2002).
*Grouped by citing author (see reference list).

Table 3 -
Genetic similarities (GS) and range of variations within and between Secale species.