Fine Mapping Of A Minor-effect QTL-DTH3b For Heading Date And Map-based Cloning Of A Long Awn Related Gene Awn3 With Near Isogenic Line Of Rice(Oryza Sativa L.)

This paper including the research on fine mapping of a monor-effect QTL-DTH3b for heading date and map-based cloning of a long awn related gene Awn3 with near isogenic line of rice.Flowering or heading time is an important trait for rice to adapt the different cultivation areas and cropping seasons.Here we identified a monor-effect QTL-DTH3b which is a LD-specific regulator of rice heading,and finally delimited it to a 46 kb genomic region.Quantitative real-time PCR analysis of genes involved in rice flowering network indicated DTH3b promoted heading by up-regulating both Hd3a and RFT1 independent of Hdl and Ehdl pathway in LDs.Awn is the specific structure of extending lemma apex in rice,and long awns go against the convenience of harvesting cultivated rice.A major-effect QTL-Awn3,which was detected using advanced backcrossing populations crossed by two awnless parental lines Aso and IR24,was fine mapped in a final 12.3 kb genomic region and molecular cloned.A single nucleotide changed in Awn3 which encodes a protein that contained four functional domain,resulted in long awn in NIL(Awn3).1.A minor-effect QTL,QTL for Days to heading 3b(qDTH-3b,hereafter referred to as DTH3b),was previously identified,but the specific genetic effects of qDTH-3b were not fully unearthed.Here we selected a homologous NIL(viz.NIL(DTH3b))that only carrying a 12 cM homozygous IR24 chromosome segment with DTH3b in Aso background.QTL analysis indicated that DTH3b had a LOD score of 5.46 and contributed 27.35%on days to heading in Aso/NIL(DTH3b)F2 population.Using 600 late-heading F2:3 families and 900 recombinants'progeny,we finally fine mapped DTH3b to a 46 kb genomic region.2.Compared with Aso,NIL(DTH3b)showed 6.9-days delayed flowering in Beijing NLDs and 9.8-days delayed flowering in CLDs while no differences in both Hainan NSDs and CSDs.When seeds were harvested in early October in Beijing,maturity percentage of NIL(DTH3b)was only 29.5%while that of Aso reached to 81.5%.In contrast,both grains of NIL(DTH3b)and Aso were totally matured under SDs in Hainan.Furthermore,NIL(DTH3b)showed a similar leaf emergency to Aso under both CLDs and CSDs,indicating DTH3b is a LD-specific regulator of rice heading and the Aso allele of DTH3b is better adapted to the cool NLD conditions.3.From the qRT-PCR analysis,two florigen genes Hd3a and RFT1 and their downstream genes OsMADS14 and OsMADS15 showed higher expression levels in Aso than in NIL(DTH3b).However,little differences on expression levels of Hd1 and Ehd1 were observed.Meanwhile,we checked six positive regulators(OsGI,RID1/OsID1/Ehd2,Ehd3,Ehd4,OsMADS50 and OsMADS51)and six negative regulators(Ghd7,DTH8/Ghd8,OsMADS56,OsCOL4,PHYB,and SE5)upstream of Hdl and Ehd1,and found expression levels of all these genes were undistinguishable between Aso and NIL(DTH3b),indicating that DTH3b promoting heading by up-regulating Hd3a and RFT1 maybe independent of Hdl and Ehdl pathway.4.We found CSSL23 which was constructed using two awnless parental lines Aso and IR24 showed long awns.Then a major-effect QTL-Awn3 located in the pericentromeric region of chromosome 3 was detected in Aso/CSSL23 BC4F2 population,and a homologous NIL(viz.NIL(Awn3))that carrying a 27.2cM homozygous IR24 chromosome segment with Awn3 in Aso background was selected.QTL analysis showed Awn3 was a single Mendelian factor with a LOD score of 18.97 and contributed 55.17%on percentage of awned spikelets in Aso/NIL(Awn3).5.Compared with Aso,NIL(Awn3)showed longer awns and higher percentage of awned spikelets in different growth conditions.Awns were easily influenced by light time,and longer light times with longer awns.6.Using 1300 extremists with high percentage of awned spikelets in Aso/NIL(Awn3)F2 population,Awn3 was fine mapped in a final 12.3 kb region that only contained one ORF as Os03g0407400.Sequencing of the genome of 0s03g0407400 revealed that there is a single nucleotide substitution(C to A)in a position of 165 bp away from the start codon,causing a premature stop codon and creating a mutant protein with only 54 amino acids.Genetic complementation using both the full genomic sequences and cds of Awn3 completely remove long awn of NIL(Awn3),thus we concluded that Os03g0407400 corresponds to Awn3 gene.7.QRT-PCR result showed Awn3 transcript in various tissues of Aso and NIL(Awn3),including root,stem,leaf,spike and awn,and most abundant in young spike.