| Maize (Zea mays L.) is one of the most important food, forage and energymaterials in the world. It plays a key role in agricultural production, social life, nationaleconomy and resolving the energy crisis. Seed is the basis of the crop production and itsquality affects the efficiency of agricultural production directly. Seed vigor is the mostimportant indicator of seed quality and application value. High vigor is a complex seedproperty that determines its potential for rapid uniform emergence What’s more, it cansave rate, reduce the labor input and the production cost and improve agricultural outputefficiency greatly. QTL mapping and cloning is an important means to analyse thegenetic basis of agronomic traits in maize. In the present study, based on two associationrecombinant inbred line (RIL) populations which were obtained from Yu537A×Shen137and Yu82×Shen137and contain420family lines as materials, two high-densitymolecular linkage maps were constructed by using single nucleotide polymorphism (SNP)molecular marker technology. Through composite interval mapping method, the QTL ofseed vigor-related morphological and physiological traits were detected, including thepercentage of germination, germination energy, germination index, vigor index, seedlinglength, seedling dry weight, root dry weight, simple vigor index, mean germination timeand activity of superoxide dismutase, peroxidase activity, catalase activity,malondialdehyde. By means of the comparative genomics knowledge and bioinformaticsmethods, the function of candidate gene associated with seed vigor therewith is predicted.The related gene, which controls the seed germination capacity at low temperature, wascloned and its function was analysed finally. This study helps to clarify the moleculargenetic mechanism of corn seed vigor, give a theoretical foundation and excellent geneticresources for the creation of high vigor corn germplasm materials and the breeding ofsuperior high yield varieties, and provide beneficial reference for marker-assistedselection. The main results were as follows:1. Three thousand and seventy-two SNP markers, derived from DNA-chip technology,were used to genotype the two RILs and two pairs of parents, that is, Yu82/Shen137andYu537A/Shen137. In total,1397and1371SNP markers showed discrepantpolymorphism, by using the software Jionmap4, with LOD score2.5and the largest genetic marker distance20cM, two genetic linkage maps were conducted, including10linkage groups and1172and1139SNP markers respectively. The lengths totaled1629.61cM and1681.75cM respectively and the average distances between markers were1.39cM and1.48cM in sequence. The distribution of SNP marker was uniform. The twogenetic linkage maps were integrated by using the software Biomercator3.1and theintegration map covered1712SNP markers totally, with the whole genome length of1712.6cM and an average distance of1.00cM between markers.2. One hundred and seventy-two QTLs for seed vigor-related traits were detected in thetwo connected RILs populations under favorable and different artificial aging conditions.Among them, ninety-two QTLs were found in the population based on Yu82×Shen137and they were located on all10chromosomes of maize genomic, with the explanation ofphenotypic variation from5.4%to13.74%of a single QTL. Others eighty QTLs werefound in the population based on Yu537A×Shen137and they were located onchromosomes1,2,3,4,5,7,8,9and10, with the explanation of phenotypic variationfrom5.39%to12.11%of a single QTL. Twenty-five mQTLs were identified on all10chromosomes of maize genomic by meta-analysis and one hundred and forty initialQTLs were integrated; the consolidation ratio was81.39%. Each mQTL contains5.6QTLs on average, involved in the regulation of two to seven traits. Seven mQTLs, that ismQTL2, mQTL3-2, mQTL5-2, mQTL5-3, mQTL3-4, mQTL6-2and mQTL8, includedmultiple QTLs correlated with seed vigor and stable expression under differentconditions. Thirty-three candidate genes were located in18mQTLs corresponding SNPmarker distribution ranges in the regulation of plant development, growth metabolicpathways, signal transduction, withstand adversity, and other functions and so on.3. Eighty-three QTLs for seed vigor-related traits were detected in the two connectedRILs populations in normal and low temperature conditions. Thirty-three QTLs werefound in the population based on Yu82×Shen137and were located on chromosomes1,2,3,4,5,6,7and10, with the explanation of phenotypic variation from5.41%to11.01%of a single QTL. Others fifty QTLs were found in the population based onYu537A×Shen137and located on all10chromosomes of maize genomic, with theexplanation of phenotypic variation from5.39%to11.92%of a single QTL. Six mQTLswere identified on chromosomes3,5,7and10by meta-analysis and thirty-one initialQTLs were integrated, the consolidation ratio was37.5%. Each mQTL contained5.16QTLs on average, involved in the regulation of two to six traits. mQTL3-2included10QTLs correlated with MGT, VI, SDW, RDW, GE, SVI, and embodied multi-effectfully. Thirty-five candidate genes were located in6mQTLs corresponding SNP marker distribution ranges, involved in the regulation of seed germination, plant development,growth metabolic pathways, signal transduction, withstand adversity and so forth.4. Four physiological traits were evaluated by using the RIL population based onYu537A×Shen137.In total, eleven QTLs were detected and located on chromosomes1,2,4,6,7and8, with the explanation of phenotypic variation from5.22%to12.10%of asingle QTL.5. The gene ZmqLTG3-1was cloned through the method of orthologus gene cloning. Thegene had a total coding sequence of606bp and the open reading frame was441bp, whichcontained147amino acids. The protein contained LTP conservative structure of the extraprotein family AAI-LTSS and protein molecular weight and isoelectric point were14.16KDa and8.3respectively，with the similarity of amino acid sequence for ZmqLTG3-1with sorghum and rice being92%and88%. The results of fluorescence quantitativeRT-PCR show that the highest expression level is in the embryo, then in the stem tip,lower is in the leaf and root. With the increase of embryo germination time, quantityincreases gradually, with the highest expression abundance at24h and then regular downalong with germination time. The result of subcell location reveals that the product ofZmqLTG3-1gene encoding was positioned on the cell membrane and it is atrans-membrane protein. Transgenic T3generation, trans-empty vector, wild typeArabidopsis thaliana were used as materials to carry the preliminary validation of itsfunction. Results show that in different abiotic conditions such as drought, hightemperature and salt, compared with control group, the performance and the survival rateof transgenic plant are significantly higher. All these suggest that ZmqLTG3-1gene playsan important role in resisting abiotic stress. |
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