Rye (Secale cereale L.) is a temperate cereal belonging to the tribe Triticeae, which is grown mainly in Europe and Northern America. Its uses include grain, hay, pasture, cover crop, green fodder, and green manure. More than 50% of the annual rye harvest is used for bread making, resulting in rich, dark bread that holds its freshness for about a week. Despite its relatively low acreage compared to other cereals, rye is of great importance due to its broad tolerance to biotic and abiotic stress, a feature generally lacking in other temperate cereals. Thus, rye remains an important grain crop species for cool temperate zones.
Besides its importance as a crop, rye is one of the parents of a man-made species Triticale and the short arm of rye chromosome 1 (1RS) has been introgressed into several hundreds of wheat cultivars [1
]. In fact, some of the most successful wheat varieties carry the 1BL.1RS translocation as the presence of 1RS in the wheat genome increases both yield and protein content in grains [3
]. Moreover, 1RS carries a cluster of genes encoding resistance to stem, leaf and yellow rust – Sr31, Lr26 and Yr9, respectively [4
] and a self-incompatibility locus [5
]. On the down side, 1RS carries the Sec-1
locus coding for ε-secalin, which negatively affects bread making quality [6
]. Thus, it would be of great advantage to isolate those genes individually through map-based cloning and develop markers for marker assisted selection in rye and wheat.
Despite the economic importance of rye, little is known about its genetic make up at the DNA sequence level. To our knowledge, there is no ongoing sequencing project in rye, and there are no plans to target gene-rich fractions of its genome. Rye is underrepresented in the sequence databases compared to wheat and barley for which 1,104,002 and 529,839 sequences respectively, are deposited in GenBank. There are only 9,807 rye sequences (about 5 Mbp) available, of which about 90% are expressed sequence tags (ESTs). Updated list of rye genes, markers and linkage data was created by Schlegel and Korzun [7
]. The lack of sequence information is a major limitation for marker development and gene cloning in this species.
The monoploid genome size of rye (1Cx = 7,917 Mbp) is the largest among temperate cereals, almost 40% larger than that of bread wheat (Table ). This is due to the presence of a large amount of highly repetitive sequences. Flavell et al. [8
] estimated the repetitive DNA content of rye to be 92%. Despite the progress in sequencing technology and bioinformatics, sequencing the whole rye genome remains a very difficult and expensive task. In particular, genome shotgun sequencing of such a large and repetitive genome seems currently impossible. On the other hand, the short arm of rye chromosome 1 represents only 5.6% of the rye genome and with the molecular size of 441 Mbp, 1RS is comparable in size to the whole rice genome, which was recently sequenced [9
]. Recently, a method has been developed to dissect large plant genomes into individual chromosomes using flow cytometric sorting (reviewed in [12
]). A protocol for sorting individual rye chromosomes has been set up by Kubaláková et al. [14
], and Šimková et al. [15
] created two BAC libraries from flow sorted 1RS arms. The library represents a valuable tool for map-based cloning, targeted sequencing and marker development.
Genome size of major Triticeae species
End sequencing of BAC clones enables generating random sequence information distributed across the whole genome. Kelley et al. [16
] developed a protocol for high throughput BAC end sequence (BES) generation using automated sequencers. This protocol is now a routine in large sequencing centers, reducing cost and enabling the creation of large data sets. Nevertheless, the number of BESs from the Triticeae tribe is currently limited with only 37,609 hexaploid wheat BESs and 32 Triticum monococcum
BESs in GenBank, representing the whole tribe. Beyond the sequence information itself, BESs are a valuable source of molecular markers. Shultz et al. [17
] used BESs derived from BACs, representing minimum tiling path of soybean, to develop new microsatellite markers. Among the first 135 primer pairs tested, more than 60% were polymorphic. Paux et al. [18
] took advantage of a BAC library specific for wheat chromosome 3B [19
], and sequenced BAC ends to isolate chromosome-specific molecular markers based on inserted transposable elements (ISBP – Insertion Site Based Polymorphism). Paux et al. [18
] succeeded in developing thirty-nine 3B-specific markers to anchor BAC contigs to the genetic/deletion map and have since then developed several hundreds of ISBP markers from 3B (unpublished). According to the authors' estimate, about 5% of BESs are suitable for ISBP development.
Here, we report on DNA sequence composition of the short arm of rye chromosome 1 (1RS) and on the development of new molecular markers for this chromosome arm using 2 Mb of BAC end sequences. We demonstrate that the combination of chromosome arm-specific BAC library with BAC end sequencing technology offers a cost efficient strategy to survey the composition of the rye genome and saturate chromosome 1RS with molecular markers.