The University of Arizona
MSLL
HMPR-MSLL


The University of Arizona (PI Soderlund, Wing), UGA (Bennetzen) and Purdue (San Miguel) were awarded NSF grant #0321724 to construct, sequence and analyze MSLL and HMPR libraries for maize.

Reference: Nelson, W., Luo, M., Ma, J., Haller, K., Bharti, A., Messing, J., Wing, R., San Miguel, P., Bennetzen, J., and Soderlund, C. (2008) Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains. BMC Genomics, 9:621doi:10.1186/1471-2164-9-621. Link

Maize mini-BAC: The MSLL, HMPR, ASM, EST, and FLcDNA maize sequences have been aligned to the BACs, and each mini-BAC links to the genome browser for the BAC.

Overview of HMPR and MSLL

The maize genome comprises approximately 2500 million base-pairs. Over 60% of these base-pairs compose Long Terminal Repeat (LTR)-retrotransposons and 20% compose other repetitive sequences (Whitelaw et al. 2003). In adult tissues, most LTR-retrotransposons appear to be 100% cytosine 5-methylated at all 5'-CG-3' and 5'-CNG-3' sites (Yuan et al. 2003). Recent library construction technologies attempt to take advantage of these facts to (1) make gene-enriched libraries so that sequencing projects can largely exclude highly reiterated segments of a genome and (2) create bridge libraries of clones that span the repetitive segments and therefore anchor and link the gene containing sequence contigs.

Hypo-Methylated Partial Restriction (HMPR, Emberton et al.) clones provide gene-enriched sequences and Methylation-Spanning Linker Library (MSLL, Yuan et al. 2003) clones span large methylated regions; both techniques use methylation sensitive enzymes that will not cut sites containing a 5-methyl cytosine. These two techniques complement the Methyl Filtration (MF, Rabinowicz et al. 1999) and High Cot (HC, Yuan et al. 2003) approaches. The following provides a brief description of the four types of clones:

The HMPR sequences are a good complement to the MF and HC, as (1) they can tolerate methylated bases within the clone; in constrast, MF clones cannot tolerate methylation, (2) they can tolerate repetitve sequences; in contrast, HC clones will under-represent gene families, etc and (3) clones >2kb are easily constructed; in constrast, MF and HC are <2kb. The disadvantage of HMPR is that is requires two restrictions sites within 2-4kb. As can be seen on our Browsers, these 3 types of clones provide a good range of coverage of the gene rich regions.

The MSLL clones range from 35kb to over 100kb. Any clone that is this size resulting from a full digestion is more than likely spanning a region containing a number of methylated sites. That is, few >35 kb segments of a genome will lack SalI or HpaII sites; hence any fragments of this size generated by a complete digest with one of those enzymes will probably contain additional methylated sites. Methylated blocks of DNA will frequently be composed of repetitive DNA. Mate-pairs from MSLL clones can link gene-rich contigs across these long segments of repetitive sequence (such as retrotransposon clusters).

The Consortium for Maize Genomics generated over 450k MF sequences and over 455k HC sequences, which were assembled into AZMs (Assembled Zea Mays, Whitelaw et al. 2003, see details.). This grant has generated HMPR and MSLL sequences, as described below. The results of both sets of sequences can be viewed on the Maize Mini-BAC Browser pages.

HMPR and MSLL libraries

HMPR: A total of 40,299 end sequences from clones with size range of 2-4kb. The following libraries are available from the AGI BAC/EST Center.
The reads are available from genbank, query ZMMBH.

AGI Enzyme Digestion Reads
ZMMBHa HpyCHIV 10U/ug 171
ZMMBHb HpyCHIV 0.1U/ug 181
ZMMBHc HpaII 10U/ug 734
ZMMBHd HpaII 0.5U/ug 1482
ZMMBHe HpaII 0.25U/ug 5189
ZMMBHf HpaII 0.125U/ug 10428
ZMMBHg HpaII 0.05U/ug 1488
ZMMBHh HpaII 0.025U/ug 177
ZMMBHi HpaII 0.0125U/ug 174
ZMMBHj HpaII 0.0005U/ug 177
ZMMBHk HpaII 0.00025U/ug 183
ZMMBHl HpyCHIV 0.00025U/ug 1441
ZMMBHm HpyCHIV 0.00025U/ug 2239
ZMMBHn HpyCHIV 0.00025U/ug 1492
ZMMBHo HpyCHIV 0.00025U/ug 2550
ZMMBHp HpyCHIV 0.00025U/ug 8218
ZMMBHq HpyCHIV 0.00025U/ug 1782
ZMMBHr HpyCHIV 0.00025U/ug 1544
ZMMBHs HpyCHIV 0.00025U/ug 649

MSLL Libraries A total of 80732 end sequences from clones with size ranges listed below. Libraries a-d,f,h,i,j are currently available from the AGI BAC/EST Center.
Sequences are available from Genbank, query the library name, e.g. "ZMMBLa".

AGI Enzyme Length Sequences
ZMMBLa SalI 35-60 9984
ZMMBLb HpaII 35-60 9984
ZMMBLc HpaII 20-35 9984
ZMMBLd SalI 20-35 13824
ZMMBLe HpaII 12-20 9206
ZMMBLf SalI 60-100 5436
ZMMBLh SalI >100 6158
ZMMBLi HpaII 60-100 8495
ZMMBLj HpaII >100 7715
ZMMBLz HpaII 7-12 8448

The libraries are BAC libraries with the exception of z, which is plasmid.

References

Emberton, J., Ma, J., Yuan, Y., SanMiguel, P., and Bennetzen, J. Gene Enrichment in Maize with Hypomethylated Partial Restriction (HMPR) libraries. Genome Research 15, 1441-1446.

Rabinowicz, P.D., Schutz, K., Dedhia, N., Yordan, C., Parnell, L.D., Stein, L., McCombie, W.R., and Martienssen, R.A. (1999). Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat Genet 23, 305-308.

Whitelaw, C.A., Barbazuk, W.B., Pertea, G., Chan, A.P., Cheung, F., Lee, Y., Zheng, L., van Heering en, S., Karamycheva, S., Bennetzen, J.L., SanMiguel, P., Lakey, N., Bedell, J., Yuan, Y., Budiman, M.A., Resnick, A., Van Aken, S., Utterback, T., Riedmuller, S., Williams, M., Feldblyum, T., Schubert, K., Beachy, R., Fraser, C.M., and Quackenbush, J. (2003). Enrichment of gene-coding sequences in maize by genome filtration. Science 302, 2118-2120.

Yuan, Y., SanMiguel, P.J., and Bennetzen, J.L. (2002). Methylation-spanning linker libraries link gene-rich regions and identify epigenetic boundaries in Zea mays. Genome Res 12, 1345-1349.

Yuan, Y., SanMiguel, P.J., and Bennetzen, J.L. (2003). High-Cot sequence analysis of the maize genome. Plant J 34, 249-255.


Email comments to will@agcol.arizona.edu

Last Modified Thursday December 10, 2009 14:58 PM and 22 seconds