Molecular Mapping Of A High-lysine Mutant Gene And Analyses Of Heterofertilization Of O2 Endosperm And Allelic Variation At O2 Locus In Maize

A large proportion of endosperm proteins in maize consist of storage proteins and are deficient of some essential amino acids, particularlly lysine and tryptophan. Thus, maize, a staple crop for food and feed in the world, is of inferior of nutrient quality for monogastric animals and man. Protein supplementation to correct such deficiencies is costly and wasteful of energy in animal nutrition, and is not feasible for most developing countries, which rely on cereal for human consumption. Therefore, the effective way for the improvement of nutritional quality in maize is genetically increasing the amounts of essential amino acids of endosperm proteins.Efforts to improve the protein quality of maize seed have focused on mutants with reduced zein synthesis and higher lysine content. In the period of 1994 to 1999, YANG Wen-Peng isolated a higher lysine mutant from Robertson's Mutator (Mu) stocks, which contain mutations induced by insertion of Mu transposons. The gene controlling this high-lysine mutation was temporarily termed opaque-16(o16) due to its opaque endosperm.The main purposes in this study are analyzing the effect of o16 gene on lysine content of maize kernel, locating it with SSR markers and studying on the efficacy of pyramiding it with o2 gene in lysine content. In order that these analyses are reliable, it is necessary to screen out an applicable method for assaying lysine content in maize seed. In addition, the phenomenon of o2 endosperm hetero-fertilization, and the allelic variations of SSR sites within O2 gene and association between the variations and their lysine level were investigated because the heterofertilization of o2 endosperm can affect exact molecular tagging of o2 gene and its main cause is allelic variation of O2 locus.Using inbred lines of high-lysine mutant and wild type, analyses with statstic were performed for 4 colorimetric methods of ninhydrin, acid orange-12 dye binding lysine, 2-Chloro-3,5-dinitropyridine and 2,4,6-Trinitrobenzene sulfonic acid.Utilizing two o16 mutant lines of QCL3021 and QCL3024, wildtype line of QCL3010 and o2 line of Qi205, two F_2:3 populations were created. One population derived from a cross between QCL3024 and QCL3010; another population derived from a combination of Qi205 × QCL3021. Genetic linkage maps of SSR markers were constructed by MAPMAKER software. The molecular genetic mapping of o16 gene was performed by composite interval mapping. The effect of pyramiding o2 and o16 genes onlysine content was analysed by using simple statistical method.Using three back-cross populations of o2 line as recurrent parent, the o2 endosperm heterofertilization was investigated by means of molecular marker detection and endosperm phenotype identification.Using 14 o2 lines and a wildtype line, recessive allelic variations at three microsatellite (SSR) sites within the 02 gene and the association between the variations and lysine content were analysed by means of SSR marker detection, nucleotide sequencing of amplified fragments and determination of lysine content.The chief results obtained through above analyses are as follows:A. Among the four colorimetric methods of assaying lysine concentration, the results assayed by colorimetric methods of acid orange-12 dye binding lysine, 2-Chloro-3,5-dinitropyridine and 2,4,6-Trinitrobenzene sulfonic acid were accurate. However, the result assayed by ninhydrin colorimetry was not veracious.B. The genetic variance of lysine content in the F2:3 population from the cross of QCL3024 x QCL3010 was regulated by 0I6 gene. Based on two data sets of the linkage maps of the F2 plant marker genotypes and the lysine content of F3 seeds originated from the two F2:3 populations, the 0I6 gene was located within 5 cM, at either 3 cM or 2.2 cM from umcll41 in the interval between umcll21 and umcll41 on the long arm of chromosome 8, depending on the recombination rate in the two populations.C. According to the data of the F23 population constructed from the o2 and 0I6 line, the double recessive mutant effect was analyzed. The average lysine content of the F3 "o2o2ol6ol6" families identified by the umclO66 and umcll41 markers was approximately 30% higher than that of the F3 o2o2 and "0I60I6" families, respectively. The lysine content of seven F3 families among nine F3 double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line.D. In the backcross populations using o2 as recurrent parent, there was a phenomenon of endosperm heterofertiliztion. The percentage of heterofertilization was about 0-4.5% and higher than the mutation rate of SSRs (10"3¹0'2 per locus per gamete of each generation).E. At the umclO66 site, there were two alleles, a recessive allele with two perfect GCCAGA repeats and a dominant allele with three perfect repeats. At the phiO57 site,there were three alleles, two recessive alleles with three and five perfect GCC repeats, respectively, and another with four perfect repeats consistent with dominant allele. At the phill2 site, at least four alleles existed, among which one recessive allele had lbp deletion, another had a 15bp deletion, and other had no PCR products compared to the dominant allele; and all alleles had unchanged AG repeats. The lysine content of kernel in the 02 and o2 lines correlates, to a considerable extent, with nucleotide variations at the umclO66, phiO57 and phill2 sites.F. The phiO57 site in exon 6 in 02 gene was identified to be a hyper-variable region in the coding sequence of 02 gene.Following conclusions can be drawn in this study:I. The colorimetry of 2-Chloro-3,5-dinitropyridine is relatively accurate, easy operating, middle costing and less time consuming. It is especially applicable to an assay of lysine content of families in segregating population from a combination of wild type line and high-lysine line (particularly one with modifer genes).II. The umcll41, an SSR marker, can be used in marker assisted selection for ol6 gene in maize.III. The pyramiding of o2 and ol6 can further increase lysine content of maize seed.IV. The phenomenon of endosperm heterofertilization can be used for obtaining heterofertilized endosperm mutant.V. It may be usefull for selecting a material with larger variation and higher lysine content, if the three markers, umclO66, phiO57 and phill2, are used together in molecular marker-assisted selection for o2 gene in maize.The aforementioned results and conclusions may be of scientific significance and practical value in the research work of molecular marker aided selection and gene pyramiding for quality protein maize.