The Development Of AIO-seq Method For Next Generation Sequencing,and Research On Genetic Properties And QTL Mapping Of Plant Architecture Using A Maize NAM Population

The rapid development of next-generation sequencing(NGS)technology and decline of expense on sequencing,has enabled the wide application of NGS in whole genome genotyping(WGG).However,compared to the stable growth in sequencing data output,the whole process of library preparation has still been inefficient,thus leading to much more expensive cost on library preparation than subsequent sequencing.Especially for the size selection and library quantification steps,which are time-consuming and labor-intensive,have become the bottleneck for genome sequencing projects involving a large cohort of samples.To tackle this issue,we performed our research and major findings are summarized as following:(1)an all-in-one sequencing(AIO-seq)method for high-throughput sequencing was developed,which condensed the size selection and quantification steps in next generation sequencing library preparation,from an intricate‘one sample one tube'method,to an efficient‘multiple samples all-in-one tube'method;(2)when pooling several libraries to sequence in partial Hiseq X lane,or pooling decades libraries to sequence in a whole lane,expected data output for each library could be obtained after demultiplexing;(3)AIO-seq method could be applied in the library preparation for genomics or transcriptomics to get expectedly even or any expected data output among samples;(4)when conducted WGG on a maize BC₁F₄ population using data output from simplified AIO-seq method,a total of 19quantitative trait locus(QTLs)for plant architectures were identified,of which some QTLs harbor known genes.The successful development of AIO-seq here,dramatically improve the efficiency and reduce the cost of library preparation in whole genome resequencing,which will greatly promote the population genetics-and plant breeding-related projects.Maize(Zea mays),served as an indispensable bioenergy material and staple crop,has been planted worldwide.The variation in leaf angle(LA),plant height(PH),and ear height(EH),as three key components contributing to maize plant architecture,would exert important influence on the final yield.Although previous studies on the genetic mechanism of these three traits using different segregated populations have been conducted,the underlying genetic mechanisms behind plant architecture have not yet been well-characterized.In this study,we utilized a novel maize advanced backcross-nested association mapping population(HNAU-NAM1)and WGG data(including maize 9.4K array and projected one million SNP markers)to comprehensively dissect the genetic basis of LA,PH and EH by implementing three QTL mapping methods,namely separate linkage mapping(SLM),joint linkage mapping(JLM)and genome-wide association studies(GWAS),respectively.The main findings were as following:(1)the results from phylogenetic tree,phenotypic variation range,principal component analysis(PCA),and linkage disequilibrium(LD)patterns on HNAU-NAM1 population which derived from 13 founder parents and comprised 1,625 BC₁F₄/BC₂F₄lines,showed that obviously diversity,weak population structures and low LD distance were existed in HNAU-NAM1 population,indicating that it was suitable to be used for QTL mapping for three traits;(2)on the whole genome,we identified 41,31,and 26 QTLs for LA,PH,and EH,respectively,using SLM method;84,78,and 88 QTLs for LA,PH,and EH,respectively,using JLM method;22,23,and 18 significant SNPs associated with LA,PH,and EH,respectively,using GWAS method;moreover,for each trait,some QTL regions from three mapping methods were overlapped;(3)a total of 10 QTL hot-spot regions which could simultaneously influence three traits were detected,and QTLs controlling each trait in each region could be identified by at least two methods;(4)within 13 major QTLs which could be commonly identified by three methods,underlying candidate genes for plant architecture(including four known genes and eight new genes)were predicted when combining the information of each QTL regions and gene annotations.Collectively,this study evaluated the genetic properties of population structure and LD level in HNAU-NAM1 population,and further systematically dissected the genetic mechanism of maize plant architecture,which has offered new germplasm resource valuable for maize genetics and functional genomics research,and deepened our understanding on the complex regulatory network of plant architecture.These findings have laid a sound theoretical foundation for the development of maize with ideal plant architecture and molecular breeding of dense-tolerant and high-yield elite maize cultivars.