Identification Of Imprinted Genes And Dissection Of Genetic Basis Of Heading Date In An 8-parent MAGIC Population In Rice

Genomic imprinting is an epigenetic phenomenon,in which two alleles at the same locus are expressed preferentially.The expression difference between them depends on the origin of the parent.At present,hundreds of imprinted genes have been identified in plants,but their conservation is very low both within and among species.Cultivated rice(Oryza sativa L.)is divided into two subspecies,indica and japonica.There is reproductive isolation between them,which hinders the full exchange of genetic materials.Whether imprinted genes are conserved among subspecies is uncertain.In this study,RNA sequencing was carried out in the embryo and endosperm of three groups of reciprocal hybrids including inter-subspecies(9311 and Nipponbare,R9N),indica intra-subspecies(9311 and Zhenshan 97,R9Z)and japonica intra-subspecie(Zhonghua 11 and Nipponbare,RZN)after 5-7 days of pollination.The main results are as follows:1.Imprinted genes were mainly detected in endosperm.Four imprinted genes were identified in the embryo,one of which was a paternally expressed gene(PEG),and the rest was maternally expressed genes(MEGs),while a total of 913 imprinted genes were identified in the endosperm,most of which were MEGs.2.The varied number of imprinted genes were identified in three combinations.546,286 and 211 imprinted genes were identified in R9 N,R9Z and RZN,respectively.72,46 and 16 imprinted genes were commonly identified in R9 N and R9 Z,R9N and RZN,R9 Z and RZN.Five imprinted genes(os01g0151700,os07g0103100,os10g0340600,os11g0679700 and os12g0632800)were identified in all three combinations.RT-PCR sequencing confirmed that these five genes only expressed maternal alleles in these combinations.3.Some imprinted genes are clustered in two-gene tandem on chromosomes.The cluster imprinted genes in R9 N,R9Z and RZN accounted for 26.7%,10.5% and 38.9%,respectively.4.There are not many conserved imprinted genes among rice,maize and Arabidopsis.Through homologous alignment,we found that there were 15 and 27 conserved imprinted genes between rice and Arabidopsis,and between rice and maize,respectively.Their functions are various.5.Most of the identified MEGs are involved in energy metabolism and seed development.GO analysis showed that MEGs were mainly enriched in carbohydrate metabolism and biosynthesis,nitrogen metabolism and transmembrane transport.Two genes of the five MEGs commonly identified in all three combinations were edited.Compared with the wild type,the garin filling rate of the mutant was slower.As a result,the TGW(thousand grain weight)was decreased.This study provides useful resources for functional analysis of imprinted genes in rice and even cereal crops.Multi-parent advanced generation inter-cross(MAGIC)population has the advantages of strong power of biparent populations and high accuracy of natural populations in QTL mapping.However,in previous studies,GWAS(genome wide association study)based at SNP(single nucleotide polymorphism)level was often used for QTL(quantitative trait loci)mapping,which did not make full use of the advantages of MAGIC population.In this study,we constructed a QTL mapping method based on bin-GWAS to scan QTLs of heading date in 8-parent MAGIC population.The main results are as follows:1.An 8-parent MAGIC population was constructed in rice.Eight varieties with high genetic diversity were selected for three rounds of crossing in pairs to obtain 8-way F1 families.Then 560 RILs(recombinant inbred lines)were obtained by single seed descent(SSD)to F7 generation.2.The contribution of 8 parents to the genome constitution of the population was similar ranging from 11.4% to 12.7%except MH63 contributed 15%.Segregation distortion was identified in a few genomic segments,of whichfoursegments were closed to the know fertility genes Os JAG,Os CP1,S5 and TMS2,and one contained afertility gene RMS.3.Phylogenetic tree and principal component analysis showed that MAGIC population did not have an obvious population structure.4.The bin map of MAGIC population was constructedwith 12405 bin.The variation range of bin length ranged from 10 kb to 900 kb,and 84.1% of bin was about 10~50kb,with an average length of 29.2kb.The average number of bin in each family was 282.1.5.GWAS at SNP level detected 4 and 5 QTLs in 2017 and 2018 respectively.Three QTLs were detected repeatedly in two years,and four QTLs were located nearby Hd3 a,Ghd7.1,Ghd8 and Ehd1.GWAS at bin level(bin-GWAS)detected 6 and 5 QTLs respectively in 2017 and 2018.Four QTLs were detected in two years,four QTLs were located nearby Hd3 a,Ghd7.1,Ghd8 and Ehd1,and one QTL was located in a 30-kb bin containing Hd1.6.The 8 parentalalleles of each QTL were divided into different types according to their genetic effects on heading date by multiple comparison.These eight parental Hd1 alleles were divided into five types.Compared with Hd1-1,the other alleles promoted heading by 3 days,8 days,11 days and 14 days.Hd3 a and Ghd7.1were divided into two types.The heading date of Hd3a-2 is 10 days earlier than Hd3a-1;and the heading of Ghd7.1-2 is delayed by 10 days than Ghd7.1-1.Ghd8 can be divided into four types.Compared with Ghd8-1,the other alleles delayed heading by 3 days,6 days and 10 days respectively.Ehd1 can be divided into three types.Ehd1-2 and Ehd1-3 headed 5 days and 7 days earlier than Ehd1-1.In conclusion,bin-GWAS has more QTL mapping power,and identifiesfavorable alleles for breeding in a MAGIC population.