Study Of Imprinted Genes In Early Embryo And The Mechanism Of Haploid Induction In Maize

Maize is one of the most important crops in the world and is also an essential raw material for the food,fuel,and fodder industries.Maize hybrids are widely used in more than 97%of maize planting area.The traits of hybrids were co-determinated by the allelic genes from two parents,in theory,the gene expression level from two parents was 1:1 or 1:2(endosperm).In previous studies,allelic gene expression bias was observed in the hybrids development stage,and gene imprinting is a typical phenomenon which showed bias in allelic gene expression.While,this phenomenon was widely reported in maize endosperm,whether gene imprinting existed in maize embryo was still controversial.On the other hand,gain of elite inbred lines is a crucial step for hybrid breeding.In traditional breeding process,gain of inbred lines needs long cycle but low efficiency which could not suitable for the drastic competition demand of breeding industrialization nowadays.The haploid technology based on the haploid inducer lines has the advantages of low cost,high efficiency and accelerate isozygoty,which could also gain the DH lines rapidly and short the breeding cycle.Although haploid technology was widely used in production,the mechanism underlying haploid formation,though,remains unclear.Based on the above two questions,we applied high throughput RNA sequencing with laser capture microdissection and manual dissection of embryo samples,we analyzed the allelic gene expression patterns and gene imprinting in maize early embryos from reciprocal crosses between inbred lines B73 and Mo17.In addition,we applied the cytological assay and single pollen sequencing technology to compare the differences of pollen development process between haploid inducer lines and inbred lines,and discussed the induction mechanism of haploid inducer lines.The main contents were listed below:1)we separated the maize embryo samples by manual dissection from reciprocal crosses between inbred lines B73 and Mo 17 at six time points(3,5,7,9,11,13 days after pollination),using high throughput RNA sequencing,we detected 9532 differential expressed genes.Among them,6020 genes were not affected by cross-direction.These genes functions showed that the high expressed gene functions in 5-13DAP were water deprivation,regulation of transcription,CTP synthase activity,nutrient reservoir activity,starch metabolic process in turn.Co-expression analysis of the genes in early maize embryo revealed sequential gene activation during maize embryo development,and help us deeply understand the gene expression pattern in early maize embryo.2)Through the analysis of the study on parent-of-origin gene expression bias,we detected 64 genes imprinted in 5-13DAP embryo,among them,5DAP embryo hold the maximum number of imprinting genes(57 genes).Meanwhile,among the 64 imprinting genes,20 genes showed continuously imprinted in at least two adjacent stages.Furthermore,with the help of laser micro dissection technology,we analyzed the embryo samples of 3DAP and 5DAP,and detected 41 imprinted genes in total.Among them,32 genes were imprinted in 3DAP embryo and 16 genes were imprinted in 5DAP embryo.The imprinting phenomenon was detected under both dissection samples and showed more genes imprinted at the earlier embryo stages.3)To further analyse the relationship between gene imprinting and kernel development,we randomly selected six imprinting genes for in situ hybridization,the results demonstrated that they were all relatively high expressed in maize embryotic tissue,especially in the top of the embryo.Furthermore,in the research on the four imprinted genes mutants,we found the embryo and kernel of two gene's mutants inbred ears were smaller than that of background lines.So we inferred that imprinted genes might play important roles in early embryo development.4)Through the study on the phenotype of inbred and hybrid progeny of inducer CAU5,we found that no abortion kernel were detected in the hybrid ears of CAU5 as female,while CAU5 as male,10%abortion kernels were detected in the hybrid ears and more than 50%abortion kernels were existed in the inbred ears of inducer CAU5.So we speculated that the pollen of haploid inducer lines may be abnormal.5)In the TTC staining experiment,we found that in three inbred lines B73,Jing24 and Qi319,the proportion of pollen grains with high viability(40%)and middle viability(40%)was much higher than other three classes,while in the three inducer lines CAU5,CAUHOI and CAUB73,the proportion of pollen grains with low viability(30%),no viability(20%)and middle viability(25%)was higher,and the high viability class(8%)was much less abundant than in the regular lines.The pollen competitive analysis with different pollination interval showed that pollen competitive of inducer lines were lower than that of inbred lines.6)We tested the chromosome abnormal behavior of diakinesis(N=4585),metaphase I(N=4286),anaphase I(N=3983)and dyad(N=2429)in inbred pollen(Qi319)and inducer pollen(CAU5 and'CAUHOI)by fluorescence in situ hybridization(FISH).Although the highest percentage of abnormal chromosome behavior in two lines were both happened in anaphase I(the value of CAU5 was 0.91%and the value of Qi319 was 0.07%),both of these two values were low and showed no obviously difference between two lines,so we speculated that the pollen of inbred and inducer lines may not exist obvious difference in the meiosis stage.7)We compared the chromosome integrity of tetrad stage and mature pollen between inbred and inducer lines by using the single pollen sequencing technology.We found that the degree of chromosome fragmentation in inducer lines were more serious than that of inbred lines.The chromosome fragmentation value of CAUB73 was 26.9%,CAU5 was 27.3%,CHOI3 was 8.75%,while the ratio in inbred lines were generally low,the value of B73 was 6%and Chang 7-2 was 4.5%.So we speculated that the larger scale of chromosome fragmentation occurring in the gametophyte may directly cause haploid in maize.8)We used two transgene lines:HiIIA×BCENH3(coix+maize)-GFP,B73CENH3-YFP and their corresponding non-transgene lines to cross with the haploid inducer lines CAU5 and CAUHOI to evaluate their induction rate in the four combinations.The results indicated that,the genetic background had a great influence on the induction rate in the lower generation populations,with the backcross and selfing progress,the transgene line of HiIIAxBCENH3(coix+maize)-GFP showed a significant positive effect on the induction rate of inducer lines.Our study of the gene expression pattern and confirmation of the gene imprinting phenomenon in early embryo,shed new lights on the relationship between gene expression and kernel development in maize.On the other hand,our study on the induction mechanism of haploid inducer lines will provides a theoretical basis for selecting new haploid inducer lines with higher induction rate.