Development And Application Of Mustard Structural Variation Based On High-throughput Sequencing

Mustard(Brassica juncea (L.) Czern.) is an economically important vegetable crop in world-wide, traditionally divided into root mustard, stem mustard, leaf mustard, sprouts mustard, seed mustard and oil mustard according to edible parts. Brassica juncea(Brassica juncea Coss. var. tumida Tsen et Lee,2n=36, AABB) is an allotetraploid derived from interspecific hybridization between B. rapa (2n=20, AA) and B. nigra (2n=16, BB) and followed by spontaneous chromosome doubling (U,1935).Structure variations (SV) were developed in current study. They are variations in structure of chromosome consisting of insertions, deletions, duplications, inversions and translocations, widely spreaded in plant genomes and are very important causes of phenotype variations. The recently development of next generation sequencing technology enables rapid development of SV molecular markers and make the utilization of SV markers in plant genetic diversity research and quantitative traits analysis easier.In this study, we scanned the structural variation polymorphism based on30×coverage of the genome sequencing and re-sequencing of two inbred lines in Chinese mustard to detect SV polymorphisms, and developed PCR-based SV markers aiming at QTL analysis of swollen stem in Brassica juncea and the genetic diversity analysis among mustard germplasms. The results are as follows:①Sequencing and re-sequencing of two inbred lines in B. Juncea.29.90Gb data was obtained including19.57Gb170bp-insert library and10.33Gb500bp-insert library. Sequencing data generated a total of304,440,194reads with59.8×coverage.28.35Gb high quality data was utilized for17-mer analysis, expecting978Mb genome size. Assembled results were mapped to the reference sequence. Total100997SVs were detected, among which49.61%,47.45%,0.13%,0.3%,2.52%data accounted for insertion, deletion, inversion, intra-chromosomal translocation and inter-chromosomal translocation respectively. SV sequences with500-800bp size were selected and used to design forward and reverse PCR primers with20 oligonucleotides according to300bp upstream and downstream structure variation regions. A total of833PCR-based SV markers were developed in B.juncea.②QTL analysis of tumor-like stem swelling. Two associated regions were detected on LG1. QTL region for stem diameter and weight was between BJsvl737and BJsvl774. LOD value reached a peak in the vicinity of BJsvl362. QTL region for stem length was between AT4G27490and AT4G33920. LOD value reached a peak in the vicinity of AT4G27490. BJsv1362and AT4G27490were blasted among Brassica Rapa and Arabidopisis thaliana genome. The results indicated that the forward and reverse sequences of BJsvl362primer were located on different chromosomes in Brassica Rapa and Arabidopisis thaliana genome. While AT4G27490was located on A01of B. rapa genome, similar to its position on LG1drawn on this experiment.③SV markers were employed to analyse genetic diversity among47mustard germplasms. Molecular-marker evolutionary phylogenetic trees in these variaties were constructed with the application of unweighted pair-group (UPGMA), neighbor-joining (NJ) and minimum evolution method (ME) methods based on4selected polymorphic SV primers. Cluster analysis results of the three methods are basically the same and partially in accordance with the traditional organ-based and geographic origin classification of mustard. WT6, TN02, IN46, IN209and03B0109were separated from other species in UPGMA method, the rest42varieties were clustered into three main groups and six subgroups. While in NJ, AU201and IN02were separated from other germplasms, the remaining mustard were divided into five large groups and eight subgroups. Results in ME were approximately the same as NJ, except the difference that AU201and IN46were distant from other germplasms in ME.