Development And Application Of BSA-seq-based Methods For Mapping Genes And QTLs Related To Panicle Traits In Rice

Rice is one of the most important food crops in the world.It is a major source of food for more than half of the world's population.Rice is grown throughout the world including tropics,subtropics,semi-arid areas and temperate regions.Improvement of plant architecture is one of the most important objectives in many breeding programs.Plant architecture is key to improvement of productivity as it was shown during the era of Green Revolution in the 1960s.Inflorescence architecture in particular is of major agronomic importance as it determines a plant's harvest index as well as yield.To increase productivity,it is necessary to identify the genes and understand the mechanisms involved in regulation of panicle architecture.A huge number of genetic factors regulate the development of panicle traits in rice.Those factors coupled with environmental influences complicate the development of an ideal form of panicle architecture to greatly boost yield and ensure food security.The combination of bulked segregant analysis with next-generation sequencing(BSA-seq)has led to the development of a new powerful genetic research tool that allows rapid discovery of genes and quantitative trait loci(QTLs).Many different BSA-seq methods have been developed and successfully applied in plants.However,more studies are needed to increase the efficiency and reliability of BSA-seq.This study aimed to develop novel methods of BSA-seq and employ them to identify genes or QTLs related to panicle traits in rice.The main results are as follows:1.MutFind:modification of MutMap.A new BSA-seq method coupled with mutagenesis named MutFind was developed and its feasibility was demonstrated in rice for identification of genes.MutFind is a modification of MutMap,but it has an advantage that it can directly find the target genes from mutants generated by irradiation without needing the help of multiple markers.The general procedure of MutFind is:Rice seeds are irradiated.M1 panicles are collected individually and the M2 individuals are grown in panicle lines.The M2 lines containing mutants are harvested and used to generate M3 plant lines.Mutants(MT)and homozygous wild-type(WT)plants are selected from the M3 lines to make a pair of distinct DNA pools(M-pool vs.W-pool).The two pools are deep sequenced and mutant sites showing opposite homozygous genotypes in the two pools are searched.By referring to the genome annotations,the causal mutant genes are identified.Using MutFind,we obtained three mutants of panicle traits from an Indica variety Minghui-86(MH-86)mutagenized with gamma rays and successfully identified the mutant genes.The first mutant was floret aberrant and sterile(fas).The mutant site was found in the LOC_Os04g43050 gene on chromosome 4,where a single nucleotide change from A to G change caused an amino acid change from Isoleucine to Threonine.The gene encodes a Dicer Like-4 protein.The second mutant was reduced panicle length(rpl).The mutant site was found in the LOC_Os11g12740 gene on chromosome 11,where a single nucleotide change from C to A at the intron splicing recognition site resulted in abnormal splicing.The gene encodes a PTR2 protein.The third mutant was short and roundish grain(srg).The mutant site was found in the LOC_Os05g06280 gene on chromosome 5,where a 5-bp deletion caused a frameshift mutation.The gene encodes a Kinesin-13 A protein.These three genes have been reported before as regulators of similar mutant phenotypes.Comparison indicated that the reported mutant phenotypes of these three genes were the same as those observed in this study.This confirmed that our results were correct,suggesting that the MutFind method developed in this study is feasible and efficient.2.Modification of QTL-seq.A modified QTL-seq method was developed to map QTLs underlying male sterility-conditioned glume split(mscGS)using an F2 population.The main merit of our method is that it can map QTLs only based on the two pools without the need of sequencing the two parents.mscGS is a serious problem in hybrid rice production,which severely affects the quality of hybrid seeds.We mapped two major QTLs,named qmscGS5 and qmscGS6.The former was located on chromosome 5,spanning 27.40-30.00 Mb with the most probable interval being 28.00-30.00 Mb.The latter was located on chromosome 6,spanning 27.50-30.90 Mb with the most probable interval being 28.00-30.75 Mb.3.BSA-seq with random pools for QTL mapping.A new method of QTL mapping using BSA-seq was developed,in which two random DNA pools are set as internal controls.The two random pools allow the threshold for QTL detection to be determined experimentally without the need of statistics and computer simulation.We employed our method to map QTLs underlying grain length and grain width using an F2 population,in comparison with the method of QTL-seq.While QTL-seq could not detect any QTL in the population,our method identified a QTL for grain length on chromosome 3.The results indicated that our new method can increase the power of QTL detection.