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We have used the program to design primers for molecular evolutionary studies of the developmental genes KNOTTED1, LEAFY HULL STERILE1 and STMADS11 in grasses, for a phylogenetic study of the plant family Menispermaceae using the chloroplast gene ndhF, and for placing cereal genes on the map of foxtail millet. The primers designed for Menispermaceae have been tested and verified to work well without additional optimization. Tests of other primers are ongoing.


KN1 encodes a homeodomain protein involved in specifying meristem identity (18). The 1040 bp KN1 alignment contained full-length cDNA sequences of barley, corn, rice and wheat downloaded from GENBANK; species that span the major diversification of the grass family. Sequence divergence within the alignment ranged from 0.9-15.6% with the N-terminal region being most divergent. Primaclade only identified suitable amplification primers when at least one degenerate base was allowed (Table 1). In this case 23 primers were identified that amplified a 608 bp fragment (or 58.46%) of the aligned region. Although allowing additional degenerate bases increased the number of primers identified, these primers did not increase the size of the amplified fragment (Table 1).


LHS1 genes are MADS box transcription factors related to SEPALLATA genes in Arabidopsis, that seem to play several roles during grass development (6). The 825 bp LHS1 alignment comprised four published sequences of full-length cDNAs from barley, rice, and maize sequences with 12.1-21.3% sequence divergence (Table 1). When no degenerate bases were allowed, Primaclade identified only two primers, both within the highly conserved MADS region of the genes that would have likely amplified many members of the MADS box gene family. When a maximum of two degenerate bases were specified, the software identified 36 primers that amplified a 696 bp fragment (or 84.36%) of the aligned region, including primers within the variable C-terminal region that make the primers gene specific. As in the KN1 search, allowing additional degenerate bases increased the number of primers identified (to a maximum of 104 primers with 5 degenerate bases) but did not significantly increase the size of the amplified region.

Solanum tuberosum MADS11 (STMADS11)

STMADS11 genes are MADS-box transcription factors involved in the transition to flowering in Arabidopsis, in the formation of floral abscission zones in tomato and likely in determining glume morphology in maize (19). Our original STMADS11 alignment comprised 13 full-length cDNA sequences of rice, wheat, barley and maize from GENBANK with sequence divergence ranging from 0-39.7%. Likely due to the high sequence divergence, Primaclade was only able to identify primers that would amplify 263 bp (or 41.15%) of the aligned region when the maximum five degenerate bases were allowed. To overcome this problem we partitioned the dataset into two smaller, more homogeneous datasets of 10 and 3 sequences and a maximum of 24.1 and 23.5% sequence divergence respectively (STMADS11a and STMADS11b, Table 1). Both datasets contained rice, barley and maize sequences that span the major diversification of the grass family. In the STMADS11a dataset, Primaclade identified 19 primers with a maximum of three degeneracies that amplified a 291 bp fragment (or 46.19%) of the aligned region, whereas in the STMADS11b dataset, Primaclade identified 2 primers when two degenerate bases were allowed that amplified a 556 bp fragment (or 87.01%) of the aligned region (Table 1).

NADH dehydrogenase, subunit F (ndhF)

NdhF is a subunit of the chloroplast encoded NADH dehydrogenase, a protein complex that may protect against photooxidative stress (20), and is widely used as a marker in phylogenetic studies (21). The 2128 bp Menispermaceae ndhF alignment comprised six species with sequence divergence ranging from 1.3-6.7%. Primaclade was very effective in finding primers throughout the aligned region. 105 primers were identified that covered 90.18% of the aligned region when one degenerate base was specified in the Primaclade search (Table 1). Allowing additional degenerate bases increased the number of primers identified to a maximum of 342 with 96.48% coverage when 5 degenerate bases were allowed.

Mapping cereal genes

We have used Primaclade to automate design of PCR-based gene markers for genetic mapping in foxtail millet. 211 predicted genes, including 466 predicted introns, were chosen from an annotated region of rice chromosome 10 and aligned with the maize EST/TUC database available as of mid-May 2004 at the Plant Genome Database ( The pairwise alignments were then submitted to Primaclade to identify primers for use in development of SNP markers for comparative genome mapping in the grasses. Primaclade was able to design pairs of primers for 215 of introns (46% of the total) in 118 (56%) of the predicted genes investigated.

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