Isolation And Characterization Of A Pleiotropic Gene, Ghd7, In Rice

Developing high yielding variety is one of the most important goals in crop science.Cloning and characterizing the genes related to yield traits is a major object in rice breeding and rice molecular biology.Yield potential,plant height and heading date are three distinct classes of traits that determine the productivity and adaption of many crop plants.Here we report the story that a quantitative trait locus Ghd7,with major effects on an array of traits in rice including number of grains per panicle, plant height and heading date,has been isolated from an elite rice hybrid by map-based cloning.In our previous study,one QTL here named Ghd7 with pleiotropic effects on heading date,plant height and spikelets per panicle was repeatedly detected in the cross Zhenshan 97 and Minghui 63 derived populations.A recombinant line 50, whose 74%genetic background is from Zhenshan 97 was used to consecutively backcross with the recurrent parent Zhenshan 97.Consequently,near isogenic line for Ghd7(NIL(mh7)) was obtained.As compared with recurrent parent,Zhensan 97,the NIL(mh7) plants delayed heading by 21.2 days,increased plant height by 33.3 cm,and produced 86 more spikelets on the main culm.Further examination showed that NIL(mh7) out-yielded NIL(zs7) by 8.9 g(50.9%) per plant,primarily due to an increase in number of grains per panicle,although NIL(mh7) had fewer tillers per plant than NIL(zs7).Three separate populations derived from NILs/zhenshan 97 were used to fine mapping Ghd7.Ghd7 was located between the markers RM5436 and C39 in the centromere region of chromosome 7 corresponded to a DNA fragment approximately 2284 kb according to Nipponbare sequence.A candidate gene with CCT motif was transformated into rice cultivars Hejiang 19,Mudanjiang 8 and Nipponbare,all of which have relatively small panicle,short stature and early heading.All the transgenic plants showed multivariation of the three target traits and confirmed the candidate gene underlying the genetic basis of trait variation. Meanwhile we found the genetic background also affected the effort of the Ghd7. And Ghd7 could promote cell division in the LD conditions.RNA in situ hybridization and Ghd7 promoter-driven GFP transformation were brought to examine sites of Ghd7 expression.The result indicated that Ghd7 mRNA expressed in the shoot apical meristem,whole young leaves,young panicle primordia,culm,phloem of leaves,and roots.The diurnal expression pattern of Ghd7 was investigated by quantifying the relative abundance of the mRNA in young leaves of NIL(mh7) using quantitative RT-PCR.The transcripts of Ghd7 oscillate with diurnal rhythm.Ghd7 expression was enhanced in the day time under LD conditions and was repressed in SD conditions.Ghd7 can suppress the expression of Ehd1 and Hd3a in the LD conditions,and slightly promote Ehd1 expression in the dark period under SD conditions.The result suggested that Ghd7 together with Ehd1 and Hd3a were involved in a novel rice flowering pathway,which does not exist in Arabidopsis.Phenogenetic analysis of CCT family showed that Ghd7 and VRN2 shared the same clade.They had a very similar expression pattern.These suggests that tropical SD plants that do not use vernalization as a flowering cue,and temperate LD plants that do use vernalization,actually use related genes to repress flowering in LDs. Such similarity in flowering control provides important clues as to how the temperate cereals might have evolved.Five alleles of Ghd7 were identified by comparison of Ghd7 sequences in 19 varieties including a common wild rice,representing a wide geographic range of Asia. The varieties with normal functional alleles like Minghui 63 and common wild rice have a long lifecycle and are distributed in Southern China and Southeast Asia.The allele from Teqing is unique and has a strong function.The varieties with weak functional alleles like Nipponbare have an intermediate lifecycle and are distributed in northern China and Japan.Two types of alleles are nonfunctional,one like Hejiang19 has a premature termination in the first exon;another one has a complete deletion of Ghd7.The varieties carrying these two alleles have a short lifecycle. These results indicate that Ghd7 locus has contributed greatly to both productivity and adaptability of the cultivated rice in a global scale.Diversity and association analysis of Ghd7 was carried out in a natural population containing 90 accessions,74 SNP points were found in the sequence of Ghd7.Some SNPs were highly associated with the phenotype.Microarray analysis shows that Ghd7 was involved in phytohormones, cytokinins metabolism.The OsCKX2 is repressed in the transgenic plants.It is a new finding that CCT family genes related to the plant hormones in rice.The molecular function of Ghd7 will be comprehensive clarified through further studying the relationship between Ghd7 and phytohormones.