A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map.

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones ( approximately 4x genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified approximately 2400 BACs and ordered approximately 700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located approximately 200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in approximately 65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.

Public informatics resources for rice and other grasses.

As an emerging model system, rice will benefit from an informatics infrastructure which organizes genome data and makes it available worldwide. RiceGenes and other Internet-accessible resources are evolving to meet these goals. Grass crops such as rice, maize, millet, sorghum and wheat are closely related but are represented by independent database projects; interlinking these resources would create a broad view of grass genetics and make it easier to compare data across genomes. The future success of grass informatics depends on the development of new comparative mapping displays as well as the participation of the research community in assembling and curating comparative map data.

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family.

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.

Three different macronuclear DNAs in Oxytricha fallax share a common sequence block.

The three members of a cross-hybridizing family of macronuclear DNAs (4,890, 2,780, and 1,640 base pairs) from the protozoan Oxytricha fallax have in common a conserved sequence block 1,300 to 1,550 base pairs long. Adjacent to the common block in the two larger DNAs are sequences which are unique to them, whereas the smallest DNA contains few if any additional sequences. The family reappears when the macronucleus is replaced after conjugation and can be detected in another O. fallax subspecies. In a random collection of cloned macronuclear DNAs, 6 of 15 hybridize to macronuclear DNA families. This high frequency suggests that families sharing common sequence blocks have an important role in macronuclear function.