Polymerizing Two High Lysine Genes O2, O16 And Waxy Gene Of Maize By Molecular Marker-assisted Selection

Endosperm, an important part of a maize kernel, takes up about 60% to 80% of the total weight of maize kernel, depending on the kinds of the endosperm. Improvement of the nutritional quality of maize kernel is mainly focused on endosperm mutants. Genetic researches find that the content and proportion of endosperm components are controlled not only by endosperm mutant genes but also by the interactions of genes. The latter may even lead to huger change in the endosperm components. Studies on the interactions between endosperm mutant genes suggest that different kinds of double or triple recessive mutants can respectively improve the traits of endosperm and make up for the single recessive mutants'shortages in quality and agronomic traits. To make use of these interactions to improve the nutritional quality of maize has become an important subject in maize quality breeding.Identifications of double and triple recessive mutants of endosperm in the past were mainly based on the phenotype of kernels with the help of testcross experiments. It could identify various mutant genes of endosperm; however, the accuracy was not very high. Molecular marker assisted-selection is a useful tool to obviate interference of environments and select genotypes directly. With its help, to accurately identify endosperm mutants becomes possible and thus the efficiency of crop breeding has been largely improved.The lysine content of the popular quality protein maize, Opaque-2, is only about 0.4%, lower than the nutritional quality standards of food for human and animals. Meanwhile, many waxy maize are of high edible quality but relatively poor nutritional quality due to their low content of lysine. In order to further improve the lysine content of quality protein maizes and their nutritional quality as well as to bring innovative material to maize quality breeding, this study, combined with conventional breeding technologies, carried out a research to polymerizing of o2 and o16 (opaque-16) genes, as well as o2,o16 and wx(waxy) genes of maize by molecular marker assisted-selection.The research chose two inbred lines of high lysine maize, Taixi 19(o2) and QCL3021(o16), as well as QCL5019(wx), to form two backcross populations, namely, the single cross-back cross population(Taixi 19×QCL3021)×Taixi 19, and the three way cross-back cross population[(Taixi 19×QCL3021)×QCL5019]×QCL5019. The foreground selection was performed using simple sequence repeats (SSR) marker umc1066 for o2 gene, phi027 for wx gene and umc1141 for o16 gene in every backcrossing and subsequent inbred generation. Selected plants were then screened for recovery of genetic background using SSR markers covering the whole genome. Grain lysine content was determined by dye-binding lysine (DBL) method. With I2-KI staining method and dual wavelength spectrophotometry method, the waxiness of the kernel was determined and finally diversified polymerization lines of recessive mutant genes were obtained.The results were as follows:1. The MAS results of the polymerizing of o2 and o16 genes through the single cross-back cross population(1) Polymorphism detection of the SSR markers on the target genes loci showed the three SSR markers within o2 gene were could distinguish between donor parent and recurrent parent, the umcl 141 linked marker could help detect o16 gene.(2) The partial correlation coefficient was estimated for the genetic background recovery ratios between each chromosome and the whole genome in BC1F1 generation. The chromosome 3 was excluded due to 100% genetic background recovery ratio. No significant partial correlation (P>0.05) was found for the chromosomes 4 and 8. A positive and significant correlation was identified for the chromosome 10 (0.01 wxwxo16o16O2_>wxwxO16_o2o2>wxwxo16o16o2o2. The total starch content of double and triple recessive polymerization lines containing o2 underwent a significant dropdown.(3) Content determination performed on 169 groups of seeds of four types in the BC2F3 family lines showed:the percentages of amylopectin starch in total content were 96.26%～97.92%, all meeting the standards set by the state on waxy maize.(4) 10 family lines featuring in high lysine content and good waxy quality were identified; their lysine contents were higher than o2 and o16 maizes and much higher than normal ones while their waxyness was equal to that of wx maizes.①7 lines carrying o2 and wx genes were identified. Their percentages of amylopectin starch in total content were at 96.37%～97.06% with an average of 96.67% while the lysine contents were at 0.505%～0.592% with an average of 0.555%. Compared with wx maize, the lysine content was increased by 75.1%.②3 lines carrying o2,o16 and wx genes were identified. Their percentages of amylopectin starch in total content were at 96.26%-96.85% with an average of 96.67% while the lysine contents were at 0.589%～0.639% with an average of 0.616%. Compared with wx maize, the lysine content was increased by 94.3%.(5) With the help of t test, difference analysis was performed on 10 high-lysine polymerization family lines in the BC2F4 generation as well as their recurrent parent QCL5019 in terms of agronomic traits. The results showed only few lines showed significant differences with QCL5019 in some of the traits. among which the line 240 was different significantly in plant height and ear height, line 62 and line 134 in ear length, line 120 and line 134 in ear diameter, The rest of lines had been highly consistent with the recurrent parent in most of the traits.10 lines showed significant extremely difference in 100-kernal weight.The conclusions of this research are as follows:1. The polymerization lines carring o2 and o16 genes can further improve the lysine content.2. Waxy maize with high-lysine content can be achieved through the polymerizing of wx and o2, wx, o2 and o16 genes. The latter has higher lysine content.3. The polymerization lines, including wx and o2, wx, o2 and o16, lead to the decrease of the total starch content.4.3 types of backcrossing polymerization lines, including o2 and o16, wx and o2, wx, o2 and o16, lead to decreased in 100-kernal weight.