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The waxy (wx) gene in maize is associated with higher content of amylopectin in the endosperm and better flavor. The opaque-16 (o16) gene is associated with higher lysine content in the endosperm and better nutritional value. To pyramid the wx and o16 genes, cross and backcross populations were constructed using the o16 line QCL3024 and the two waxy lines, QCL5019 and QCL5008, as parents. The linkage marker umc1141 for the o16 gene and the internal marker phi027 for the wx gene were used to select the target genes. Simple sequence repeat markers covering the whole genome were used for background selection in individual progenies of the backcross population. The grain lysine content was determined using the Acid Orange-12 Dye Binding Lysine method. Qualitative and quantitative analyses of the grain content of amylopectin were performed using the I2-KI procedure and double-wavelength spectrophotometry, respectively. Four lines of the double recessive genotype wxwxo16o16 were obtained from the F4 generation of the cross popula- tion and three lines of the same genotype were obtained from the BC2F4 generation of the backcross population. The lysine content of the pyramid lines was 16-27 and 18-28 % higher than the waxy parents QCL5019 and QCL5008, respectively. The pyramid lines had 61-63 % more amylopectin than the high- lysine parent QCL3024. The three pyramid lines from the backcross population had similar genetic back- ground to the waxy parent QCL5008. Our results are of significance for the improvement of maize quality.

Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of mio16. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and mio16 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.