Genetic Dissection Of Zn Accumulation And Response To Zn Stress In Brassica Rapa L. Vegetable Varieties

Zn is an essential micronutrient required by all organisms for its functions in many physiological processes as a structural or catalytic component of proteins. Unfortunately Zn deficiency is a widespread problem, affecting humans in case of Zn shortage in food, but also affecting crops in case of poor Zn availability in soil. Zn deficiency in food can be improved by Zn supplementation to the daily diet. Soil can be fertilized with extra zinc. Both approaches are not always cost-efficient and in the case of soil fertilization sometimes ineffective because of zinc binding in the topsoil Breeding and growing crops for biofortified Zn content and Zn efficiency are promising and sustainable approaches to solve the Zn deficiency problems in human and soil. On the other hand heavy metal pollution of biosphere has accelerated rapidly since the onset of the industrial revolution and heavy metal toxicity poses major environmental problems. Phyremediation is a relatively new approach to removing contaminants from the environment. It maybe defined as the use of heavy metal hyperaccumulator to remove, destroy or sequester hazardous substances from environment. It has become a topical research field in the last fifteen years since it was proposed in 1991 as it is safe and potentially cheap compared to traditional techniques. However this technology has not been used in a practical way in large scale, mainly due to the shortcomings of hyperaccumulators which are long cycle and low biomass. Brassica rapa vegetable are the most important vegetable in China with the largest consumption. It also included several cultivar groups with relatively short cycling and large biomass. Genetic analysis of Zn accumulation and homeostasis in B. rapa vegetables will provid a genetic bases for the improvement Zn content and Zn efficiency in B. rapa vegetables. The genes related to Zn accumulation cloned from hyperaccumulator T. caerulescens could be used in a GMO approach to construct of materials for phytoremediation.This thesis deals with the genetic characterization of Zn accumulation and homeostasis of B. rapa vegetables and to find out if genes related to Zn accumulation in T. caerulescens can be used in GMO approach to increase Zn content in B. rapa leaves.Chapter 1 gives an overview of progress of genetic mechanism in plant Zn accumulation and QTL analysis in B. rapa.Chapter 2 describes the cloning of candidate genes related with Zn accumulation from T. caerulescens and functional analysis of some of these genes. Totally 7 full-length cDNAs were isolated by T. caerulescen cDNA library screening. Two of seven genes, TcNRAMP3 and TcNRAMP4, were confirmed for their functions in mineral transportion.Chapter 3 describes the construction of genetic linkage map and QTL analysis of leaf mineral and phosphate content in B. rapa. QTL analysis also carried out for excessive Zn toxicity in B. rapa. An AFLP map with 222 AFLPs and 10 linkage group covering 1066 cM was constructed using aheading Chinese cabbage DH population. The average distance of intervals is 4.81 cM. By using the map, 22 QTLs for mineral content and 2 QTLs for phosphate content were detected. The percentages of variation explained were varied between 6.8-18.8%. One QTL controlling tolerance to toxic Zn stress was detected in both 100 uM and 300uM Zn treatment, while one QTL was only detected in 300uM Zn treatment.Chapter 4 describes the characterization of the genotypic variation for Zn accumulation and Zn homeostasis in B. rapa. In total 188 genotypes belonging to 9 cultivar groups, covering the geographical distribution of B. rapa vegetables in China, have been screened for shoot Zn, Fe and Mn content and for Zn tolerance. Leaf Zn content varied between 23.2-155.9(j.g g''d. wt. Both relative shoot and root dry biomass are suitable indices for evaluation of tolerance to high or deficient Zn stress. Two relative tolerant genotypes to excessive Zn stress and one Zn efficient genotype were selected from the germplasm collection.Chapter 5 describes the establishment of Ecotilling for discovery of DNA polymorphisms in Brassica rapa natural population. We used mung bean nuclease(MBN) instead of routinely used CEL I to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we adopted a strategy universal to all genes by adding Ml3 adapter to 5'end of gene specific primers to make IRD dye labeled Ml3 forward and reverse primers. A Brassica rapa ZIP gene homologue was subjected to the analysis to practice the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.