Exploration Of The Regulatory Mechanism Controlling The Frequency And Distribution Of Meiotic Recombination In Maize And Arabidopsis

Recombination is initiated by formation of programmed double-strand breaks(DSBs)in chromosomal DNA regulated by a complex of proteins that includes topoisomerase-like protein SPO11,MTOPVIB and several accessory proteins.And then,meiotic DSB results in the formation of two recombination products,crossovers(COs)and non-crossovers(NCOs)by the different repair pathways.CO numbers are much less then DSB numbers in most species being studied.In Arabidopsis,there are around 250 DSBs,based on various estimates,formed per meiosis.However,their repair only results in the fo:rmation of about 10 COs,which constitute about 85%of COs in Arabidopsis are sensitive to interference(Class I COs),In contrast,the remaining 15%are interference-insensitive(class II COs).Until now,there are three mechanisms(CO assurance;CO interference;CO homeostasis)that control the number as well as distribution of COs.And the CO homeostasis mechanism was only confirmed in yeast and mice.In this study,we will explore whether the CO homeostasis mechanism also exists in plant,and whether efficiency in di:fferent plants are the same,and whether there are any other mechanisms existed in regulating the CO by a variety of molecular biology methods.The main results are as follows:1.We generated a transgenic line of hypomorphic allele of SO11-1.The T2 lines displayed a broad range of male fertility defects,ranging from producing 20%to 100%of viable pollen relative to wild type.The numbers of DSBs quantified in the lines with reduced fertility were significantly lower and proportional to the decrease in pollen viability.The DSB numbers were proportional to the levels of SPO11-1.2.We selected a transgenic line,named spoll-l-w2,that showed a～33%reduction in DSB formation and only a moderate pollen viability defect.Progression of meiosis in the spo11-1-w2 line was not obviously different from the wild type throughout meiosis.And we did not find any obvious abnormalities when examining the progression of synapsis.3.Approximately 13%reduction in chiasmata was observed in the spoll-l-w2,and a 14.9%reduction in type I CO formation among them(chromosomal foci of MLH1).These results provided evidence that the reducing DSB numbers had an effect on class I CO formation.4.By examining the CO rates in several chromosomal intervals genome-wide in male meiosis of the BC1 progeny,a roughly 8.5%reduced in CO numbers between spo11-1-w2 and wild type.The decrease was not uniform across all chromosomes and amounted to 11.4%for chromosome 1,2.3%for chromosome 2,7.0%for chromosome 4,and 11.6%for chromosome 5.These differences appeared to be related to genetic length of the chromosomes.-33%reduction in DSB formation resulted in only～14%reduction in CO,indicating that CO homeostasis mechanism operated in Arabidopsis meiosis.5.The more intresting thing was that we discovered that the remaining COs in the BC1 progeny exhibited dramatic redistribution along chromosomes,with more COs being formed along chromosome arms at the expense of the centromeric and pericentromeric regions.This result confirmed that the number of DSBs can influence the CO distribution,suggesting a new regulatory mechanism in CO formation6.There were two copy of PRD3 in maize(ZmPRD3a and ZmPRD3b).Their amino acid sequence similarity reached-77%.Loss-of-function of ZmPRD3a caused a dramatic reduction in the number of DSB.In constrat,no any detectable change in DSB formation was found in the knockout mutant of ZmPRD3b,indicating the ZmPRD3a played a major role in DSB formation.Most importantly,95%reduction in DSB formation resulted in only～50%reduction in CO,indicating that CO homeostasis mechanism also operated in maize meiosis.7.In addition,as one of my subproject,we proved that the distinct pattern of histone modifiations was associated with and/or contributes to the divergence of the recruitment of methionine chain-elongation pathway from leucine biosynthesis.In general,genes involved in leucine biosynthesis were robustly associated with H3k4me2 and H3K4me3.In contrast,despite the considerable abundances of H3K4me2 observed in some of genes involved in methionine chain elongation,H3K4me3 was completely missing,and the H3K4m3-depleted pattern had no effect on gene transcription.