| Maize is one of the most important feedstuffs, industrial raw materials and model crops for the research of molecular biology. In the past thirty years, molecular maker technology plays an important role in plant genetics, especially in gene fine mapping and cloning, molecular marker-assistant breeding and cluster breeding as well as genetic map construction, origin and evolution in maize. However, the complex genome of maize with abundant repetitive and transposon sequences presents challenges to dissect the genetic bases of economically important traits. With the development of sequencing B73genome and next-generation sequencing technology, scientists can effectively perform genetic mapping and genome analysis of diverse genetic resources, assessment of structural genome variation, and identification of new target genes of microRNA, which was recognized as a new type of regulator at the beginning of this century. The regulator could cause degradation of RNA and suppression of translation via negative regulating the gene expression after transcription by complementary paired with target mRNA. In this study, we genome wide developed a high density of genetic linkage markers by using high throughput resequencing technology as well as degradome and microRNA sequencing in IBM syn10population and B73inbred lines in four ear development stage. After we called the genotype data, we used the flowering time, plant height and cob related traits to verify the bin marker data, and predicted the target genes of microRNAs in B73Lines. The results indicated that by combining the QTL data and microRNA data, it would be a foundation resource in identification and classification of the key functional candidate genes in the future. The main results from this study are as follows:(1) Genome-wide genetic marker discovery and genotyping using next-generation sequencing in the intermated B73×Mo17Syn10doubled haploid (IBM Syn10) mapping population. In this study, we deep re-resequenced an important inbred line, Mo17, and conducted ultra-high density genetic mapping in a ten generations intermated B73×Mo17doubled haploid population (IBM Syn10DH population) based on a sliding window approach. With26.65-fold coverage of Mo17genome,2,200,187homozygous SNPs and897,651heterozygous SNPs were detected out of3,097,838parental SNPs in Mo17. According to Phytozome7.0database, we annotated169,016SNPs in coding regions,104,311non-synonymous and64,705synonymous SNPs as well as180,587indels ranging from1bp to5bp using SOAPindel software.Subsequently, we re-sequenced280lines of the IBM Syn10population with0.31×coverage to construct an ultra-high density genetic bin map including6,618bin makers based on the parental SNPs. A bin map was constructed with35,128recombination breakpoints for all280lines in total, with the average physical length per recombination bin ranging from50kb to18.8Mb.Then the280individuals were merged into bin maps, consisting of6,618recombination bins as bin markers. The average ratio of genetic-to-physical distance was6.95cM/Mb for the whole genome, ranging from0-0.4cM/Mb.3597markers in Syn10showed distorted segregation where2,474markers were partial to B73and1,123markers were partial to Mo17. For survey of genetic composition,176lines of Syn10were bias to genetic background of B73and38lines of Syn10were bias to Mo17. The expected expansion factor was6.5and the map genetics length was adjusted by expected expansion factor directly, the length of adjusted map was1722.9cM in Syn10.We detected135QTL for flowering time and plant height traits in IBM syn4and Syn10populations. The results showed that we have done fine mapping of cloned genes such as TFL2and PhyA2to1Mb physical region, where we obtained25candidate genes related to plant height and flowering time. Furthermore, we constructed an updated integrating map with1,151,856high quality parental SNPs. Thus, our findings provided the base genetic data for QTL fine mapping and cloning in the future.(2) QTL mappling of maize cob traits at normal and limited nitrogen management. Based on high-density bin marker obtained from resequencing IBM Syn10population,117QTL were found for cob traits and23QTL for grain yield (GY). Among those QTL for cob traits,22QTL were found for cob weight (WEI),24QTL for cob volume (VOL),25QTL for cob density (DEN),26QTL for cob length (LEN) and20QTL for cob diameter (DIA). Two candidate genes were found in QTL regions, ral encodes an transcription factor to regulate the maize inflorescence branches elongation in chromosome7, the physical coordinate is110-114Mb; ba2also encodes an transcription factor to regulate the maize inflorescence branches elongation in chromosome2, the physical coordinate is31-34Mb.(3) Identification of microRNAs and target genes related to maize ear traits by high throughput sequencing. In the chapter, we used deep-sequencing, miRNA microarray assays, computational and bioinformatic methods to identify, profile, and describe conserved and non-conserved miRNAs during ear development of B73, which can be divided into four stages:growth point elongation, spikelet differentiation, the floret primordium differentiation and floret organ differentiation phase. The results showed that7,981,459(3,436,342distinct) reads were perfectly matched to the maize genome by small RNA sequencing, representing74.85%(66.64%distinct) of the total reads. Meanwhile, the degradation sequencing of these libraries resulted in average12,441,777(1,941,522distinct)20-nt sequences for the four developmental stages of ear. We used MIREAP software to predict known and new miRNAs including their pre-miRNAs, and identified385candidate miRNA genes. Meantime we performed miRAlign to identify these miRNA genes that are paralogs or orthologs to known miRNAs, and detected99known miRNA genes encoding96mature miRNAs out of which61miRNAs star sequences (miRNA*) were detected. In addition, maize-specific miRNA genes had51loci that encode23non-redundant mature miRNAs, which belonged to new families. Herein, we temporarily designated them in the form of zma-miRsl-zma-miRs23. If several maize-specific miRNAs belong to the same family, we named them like known mature miRNAs (eg. zma-miRs6a, b, c, d etc.). Six miRNA*out of23new miRNAs were also detected in this study, which further provided enough proof for the existence of miRNAs in maize.To elucidate the potential regulatory roles of miRNAs during the process of ear development in maize, we detected the expression of all the conserved and newly identified miRNAs by microarray. A total of53miRNAs (8.4%) on the microarray, were identified as putative differentially expressed miRNAs at the1%significance level with different expression pattern. In total,127target mRNAs of known and13target mRNAs of new maize-specific miRNAs were identified. These results indicated that the small RNA (sRNA)-guided post-transcriptional regulatory mechanisms play important roles in ear development.Finally, by using the high resolution bin markers and degradome sequencing, we find six miRNA target genes overlap with QTL for cob WEI, VOL and DEN in chromosome2and3. These results benefit for better understanding both forward and reverse genetics architecture of cob related traits in the future. |
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