Map-Based Cloning And Related Genes Analysis Of The Unilateral Cross-Incompatibility Ga2-S Locus In Maize

Maize is a monoecious plant,possessing self seed-setting and outcrossing seed-setting.It was found that with gametophytic incompatibility,certain genotypes of popcorn when used as female were cross incompatible to dent and flint corns.However,using the popcorn as male,the reciprocal cross was fully successful.Similarly,plants of some annual teosinte populations can fertilize maize but do not accept its pollen.So far,a few cross-incompatibility(CI)locus have been found and distributed different chromosomes.The strong locus of Ga1-s,Ga2-S,Tcb1-S confers nearly complete non-reciprocal cross-sterility organs on the same plant.A wide range of molecular biology and breeding application value enabled the potentially unique mechanism of action in the fertilization.A maize inbred line 511L carrying the strongest allele of the Ga2-S locus was chosen for genetic studies.The main results were as follows:1.In the present study,the pollen viability and process of pollen tube growth in vivo were compared between 511L and W22.In vitro pollen germination can be useful to detect alterations in germination or tube growth performance,but no significant difference in the two inbred lines was found.By scanning electron microscope(SEM)pollen germination in stigma observation 1.0 HAP(hour after pollination)of 511L/511L(Ga2/Ga2)and 511L/W22(Ga2/ga2),the results indicated no obstacle in alien pollen germinated on silks.We also compared pollen tube growth among 511L selfing,511L pollinated with W22 pollen and W22 pollinated with 511L pollen at five stages.There was no difference of pollen grain germination and pollen tube growth at 2.0 HAP time interval between compatible and incompatible crosses.Significant difference in pollen tube growth occurred 2.0 HAP after pollination between the two crosses.At 20.0 HAP interval,pollen tubes reached ovule in compatible crosses,while pollen tubes were found in the silk segment 0.5024cm distal to the pollination area in the incompatible cross and no pollen tube ever reached the ovule area.The growth of ga2 pollen tube was inhibited and only increased slowly around 2.0-20.0 h after pollination.We also found ga2 pollen tube tip presented abnormal enlargement,accumulation of callose plug.Transverse section analysis of the top half of pollinated Ga2/ga2 silks showed few pollen tubes being invaded into transmitting tract(TT),thus indicating that pollen tube was obstructed in TT.Our results clearly showed that the cross incompatibility of 511L was due to pollen tube growth inhibition around 2.0 h after pollination and cannot reach silk bottom toward ovule.2.Ga2-S locus is governed by two factors,Male-factor P and Female-factor S.Male-factor P controls the pollen competence while the Female-factor S governs the pistil barrier.Male-factor of the Ga2-S locus is dominant and gametophytic.P genotype pollen can invade into 511L silks seed-setting.But Female-factor of the Ga2-S locus is recessive and sporophytic.Only ss genotype silks gained ability to reject ga2 pollen.Thousands of inbred lines were crossing as male with 511L in search of Ga2-M allele inbred lines,twenty-six strains own pollen compatible ability without silk incompatible ability.We performed the law of Ga2 Cross-incompatibility.3.Male-factor cross incompatibility gene was mapped between markers GL6 and GS.The physical distance is about 1.308 Mb based on the B73 RefGen_v3 sequence through marker assisted selecting recombinants from 45,574(511L//W22/511L)homogeneous population plants.4.Female-factor cross incompatibility gene is recessive and sporophytic.F1 from 511L(donor of a strong allele of ga2,Ga2-S)and Zheng58(carrying the null allele ga2)was backcrossed to 511L to map the female-factor(W22/511L//511L).Female-factor was mapped between markers GL and GS in an about 1.703 Mb slightly larger interval than that of the male-factor.5.511L BAC library consists of 207,360 clones,and contain maize nuclear DNA inserts with an average size of 121.7-130.5kb,covering about 10.6×haploid genome equivalents.Seventeen positive recombinant BAC clones covering whole 1.7Mb interval were sequenced by PacBio RS II after screening pools by line and column pools monoclone.The total 2.496G data was assembled by CANU and output 11 contigs(scaffolds),which one of the maximum scaffold was 587,798 bp and contigs average length was 220,565 bp.Scaffolds and contigs were compared with screening BAC markers by BLASTN,and 7 contigs were mapped with BAC markers.In addition,compared with B73 RefGen_v4 reference sequence,the 511L BAC sequence presented significant differently.6.Based on the sequences of the recombinant BAC clones and B73 RefGen_v3 sequence,12 genes with complete structure were predicted by FGENESH in the interval.In addition to unannotated gene and pseudogene gene,SP-STK(LRR receptor-like serine/threonine-protein kinase),Daple-like gene,Endoplasmin homolog,RBP47A(Polyadenylate-binding protein RBP47A-like),chaperone protein dnaJ 49-like were also predicted.There were 5 other predicted genes only in BAC clones.Those were pectin esterase inhibitor Pectinesterase/Pectinesterase inhibitor,PME/PMEI)and four pectinesterase gene,in which its sequence similarity was more than 93%.7.We used RNA sequencing(RNA-Seq)to measure changes in maize pollen and silks between 511L and B73,of which 2,067 exhibited differential expression in pollen.Analysis of gene ontology found that DEGs were significantly for binding,small molecule,ligase_activity and gene expression.Based on their enrichment,they may paticipate in pollen development and germination.The profiling analysis silks between 511L and B73 with different treatment revealed that 2,385 differentially expressed genes were identified in before and after pollination.Fourteen differentially expressed genes were identified and down-regulated with cross W22 after pollination in 511L,including expanisn B2,MYB3,hydrolyze enzyme,and small molecule.Seventy-eight differentially expressed genes were specially expressed in 511L/W22,involved in enzyme metabolic and protein binding process,and related to Ca2+signal transmission.A few differentially expressed genes were located in the Ga-S locus region where they are candidate genes underlying cross-incompatibility.