Genetic Effects Of Dwarfing Gene Rht12on Improtant Agronomic Traits Of Wheat And Screening Of New Dwarf Mutants

Plant height is an important factor in wheat production, the introduction of dwarf andsemi-dwarfing genes into wheat was significantly increased wheat yield of the world, andbring about the ‘Green Revolution’ which play a key role in solving the food problem.However, there is only a small number of dwarf genes available for wheat breeding to date,the genetic diversity are very narrow and its not good for the sustainable improvement ofyield and quality. Currently, the most widely used wheat dwarf genes are Rht1and Rht2(GAinsensitive), but they also reduced coleoptile length and seedling vigour which may reducecrop water-use efficiency, and performance in some unfavorable environments. So,opportunities exist for replacing Rht1and Rht2in wheat with alternative dwarfing genes, suchas the GA-responsive dwarfing genes (Rht4, Rht5, Rht12, Rht13) which can reduce plantheight without compromising early plant growth. So, a better understanding of theseGA-responsive dwarfing genes is very important for improving the yield potential of wheat.Rht12, a dominant GA-responsive dwarfing gene, can significantly decreased plant height(45％), but its genetic effect on wheat production, and the possible role of Rht12in GAbiosynthesis or signaling are still not clear, and its full potential remains uncertain. So, theobjectives of this work were to evaluate the effect of Rht12comprehensively and to promotethe proper utilization of dwarfing gene Rht12in wheat breeding.Two cross was made using Ningchun45(with Vrn-B1)、Jinmai47(with Ppd-D1) asfemales and Karcagi (with Rht12) as pollen donor respectively, then the homozygousgenotypes was chosen to develop the F2:3and F3:4populations by molecular marker-assistedselection. The phenological development, plant height, grain number per spike,1000-kernalweight and other agronomic traits were evaluated for analyzing the effect of Rht12on wheatand the interaction effects between Rht12and Vrn-B1. Additionally, the Karcagi×Ningchun45population was treated with exogenous GA3for examining the response of Rht12toexogenous GA3on plant development, agronomic traits and yield components and toinvestigate the possible role of Rht12in GA biosynthesis or signaling. Moreover, a wheat EMS induced mutant population (TILLING mutant population) was developed to find moredwarf materials or more dwarf genes. The mutant frequency of the TILLING population wascalculated and the visible mutant phenotype was also analyzed and some dwarf mutants withgood agronomic traits was chosen for further study. The main results of this study were asfollows:1. Genetic effects of Rht12and the interactive effects of Rht12and Vrn-B1Analysis was conducted in the F2:3and F3:4lines derived from the cross betweenNingchun45and Karcagi in three experiments, and found that plant was significantly reducedby Rht12. Plant height of the dwarf lines was reduced by47.6cm (38.3％) compared with thetall lines, and this dwarf effects was expressed from seedling stage. Rht12reduce plant heightthrough reducing the length of internodes without affect the number of internodes, and thepeduncle length was reduced most significantly by22cm (45%) in dwarf lines compared withthat of the tall lines. However, the diameter of the internodes was not affected by Rht12whilethe wall sickness of the internodes was significantly increased and this character was benefitfor improving lodging resistance. Additionally, Rht12can significantly increase the number ofeffective tillers, increase floret fertility (13%) and produced more fertile florets (10%), andfinally increase grain number per spike (4.1;9.7％）compared with the tall genotypes.However, Rht12significantly decreased seed size and1000-kernel weight (20％). Finally,there is no significant difference between the dwarf and tall lines on yield per plant, but due tothe reduced plant biomass, the dwarf lines achieved higher harvest index than the tall lines. Itwas also found that the spike development, especially the double ridge formation phase, wassignificantly delayed by Rht12, and the vegetative growth stage was elongated in the dwarflines. However, the dwarf lines (RRVV、RRvv) was delayed by16days to reach the doubleridge stage and the dominant Vrn-B1could not compensate this negative effect. The anthesisdate of the dwarf lines was about5～6days later than that of the tall lines which may havenegative effects on grain filling.Additionally, the laboratory test confirmed that Rht12have no negative effects oncoleoptile length. The total number of seminal roots was significantly decreased in the dwarflines and the total root length in the dwarf lines was reduced by6cm (9%) compared to thatof the tall lines. However, the maximum root length of the dwarf group was3cm (15%)longer than that of the tall group, which may be beneficial for water acquisition in deeper soilin dry environments.Moreover, it was found that the tall spring lines (with Vrn-B1) developed faster than thetall winter lines (vrn-B1) before terminal spikelet stage, suggesting that the dominant Vrn-B1 needed less time to undergo vernalization in tall lines. But, in the dwarf lines, the effect of thedominant vernalization gene Vrn-B1was masked, indicating that the dwarfing Rht12allelemight have an epistatic effect on Vrn-B1.2. The interactive effects of Rht12and Ppd-D1The photoperiod insensitivity gene Ppd-D1was introduced to improve the late flowingcharacter of Rht12. Analysis was conducted in the F2:3lines derived from the cross betweenJinmai47and Karcagi, and found that Ppd-D1has no effects on the duration of vegetativegrowth phase (from sowing to double ridge stage), the dwarf lines either with Ppd-D1orppd-D1are need more time (8days) to reach the reproductive growth stage compared with thetall lines. However, the dwarf lines with Ppd-D1development faster during the latereproductive phase, and need56.0days to reach to anthesis date while the dwarf lines withppd-D1need60.5days to reach to anthesis date. Finally, the dwarf lines with Ppd-D1flowingearlier5days than the dwarf lines with ppd-D1and only1～2days later than the tall lines.Ppd-D1was basically overcome the negative of Rht12on flowing time. In the tall lines,Ppd-D1showed the similar effect on spike development as in the dwarf lines. However,though Ppd-D1can not break the negative effect of Rht12on the initiation of double ridgestage, Ppd-D1has significantly reduced the duration time from double ridge stage to anthesis,and promotes earlier flowing. So, it is favorable to use Ppd-D1to improve the flowing time ofthe dwarf lines.Additionally, in the F2:3lines of Jinmai47×Karcagi population, Rht12has the same effecton plant height; plant height was reduced about40cm (38％) by Rht12. Because of Ppd-D1was linked closely to another dwarf gene Rht8, the dwarf lines with ppd-D1showed theshortest plant height in all the four genotypes. Rht12increased the number of effective tillersand grain number per spike (5.8％). Genotypes with Ppd-D1have less grain numbers thangenotypes with ppd-D1which may be caused by the shorter late reproductive phase ofPpd-D1lines that have negative effect on floret development. Moreover, possible due to theearlier flowing of Ppd-D1lines which benefit for grain filling, the dwarf lines with Ppd-D1achiebed bigger1000-grain weight than the dwarf lines with ppd-D1. Finally, the yield of thedwarf lines was slightly shorter than tall lines, but its harvest index was significantly than thetall lines. These results were similar with that in the ningchun45×Karcagi population. So,Ppd-D1can be used to improve grain size in the Rht12dwarf lines.3. The response of Rht12to exogenous GA3The responses of Rht12to exogenous GA3were investigated using F2:3and F3:4linesderived from the cross between Ningchun45and Karcagi in three experiments, and found that exogenous GA3can break the masking effect of Rht12on Vrn-B1and also restore othercharacters of Rht12to normal. GA3shortened the time to double ridge in the dwarf lines andmake the dwarf lines with dominant Vrn-B1displayed a spring-like phenotype. The dwarflines with GA3application flowered7days earlier than the dwarf lines without GA3application and only1day later than the tall lines. However, the GA3-treated tall linesflowered only1.5days earlier than the ones without GA3treatment, its response to GA3wasmuch weaker compared with the dwarf lines. Moreover, after exogenous GA3application, theRht12dwarf plants showed a faster stem elongation and dry weight accumulation rate, similarto that of tall plants, compared with the untreated Rht12dwarf plants. GA3applicationincreased the lengths of each internodes especially the peduncle length (19.3cm;81.5％).However, the diameter and wall thickness of those internodes were significantly reduced inthe GA3-treated dwarf lines, especially the first and the second, which significantly decreasedthe lodging resistance. Finally, compared with the untreated Rht12dwarf plants, plant heightwas increased by40.7cm (53%) in the GA3-treated ones and was only5～10cm shorter thanthe tall lines. For the tall lines, plant height was only increased6.1cm (4.8％) under GA3application, suggesting that the tall plants were not as sensitive as the Rht12dwarf plants toexogenous GA3. Additionally, exogenous GA3increased plant biomass but it reduced thenumber of fertile florets, which resulted in a lower grain number per spike, lower plant yieldand lower harvest index. Exogenous GA3increasing seed size in the Rht12dwarf lines and the1000-grain weight of the GA3-treated Rht12dwarf plants was significantly increasedcompared with untreated dwarf plants, indicating that GA3could partially compensate for thesubstantial negative effect of Rht12on yield components. Thus, it is clear that exogenous GA3can restore the ‘dwarfing characters’ of Rht12to normal. It suggested that Rht12mutants maybe deficient in GA biosynthesis rather than in GA signal transduction.4. Screening of new wheat dwarf mutantsTo develop more dwarf mutants, a wheat EMS induced mutant population (TILLINGmutant population) was developed using wheat cultivar Jinmai47, and the variation ofphenotypes and the mutation of candidate genes were analyzed. Firstly, altered morphologicaltraits were record, dwarf, albinism, late heading and other mutant phenotypes were found inM2individuals, and about4.2%of the surveyed lines displayed noticeable phenotypes(distinctive from Jinmai47). Moreover, variants in plant height comprised32%of all visiblephenotypes noted and mainly yielded different kinds of dwarfism. Additionally, the mutationfrequency at DNA level was1/35kb, which means that one mutation per34kb. It is suggestedthat the TILLING population not only has rich phenotypic mutations, but also has high mutation density in the genome which would be a valuable resource for screening desiredwheat mutants needed in forward and reverse genetic researches. Then, the target fragment ofphotoperiod gene Ppd-D1and the dwarf gene Rht-D1was screened for SNPs by TILLING(Targeting Induced Local Lessions In Genomes) method. The detection method was alsomodified by using agarose gel, non-denaturing polyacrylamide gel and HRM technology.Finally,15and3mutations were obtained in Ppd-D1and Rht-D1, and the average mutationfrequency is1/48kb. Sequencing confirmed that all of the mutations were C to T or G to Atransitions, and some mutations led to amino acid changes or have negative effects on proteinfunction. Mutants of Ppd-D1and Rht-D1were characterized and not found noticeable alteredphenotypes in these mutants. Furthermore, to select dwarf mutants with good agronomic traits,backcross was conducted using the mutants and the wild type. Now, some dwarf mutant withstable heredity, earlier flowing time and good agronomic traits were chosen for furtheranalysis and the screening of SNPs on dwarf related genes (genes involved in gibberellinsbiosynthesis or signaling) was ongoing.Conclusively, Rht12significantly reduced plant height, increased the number of effectivetillers, grain number and also the harvest index, and has no negative effects on seedling vigour;Rht12significantly delayed the spike development phase and it should be introduce otherdevelopment-promoting genes, like Ppd-D1, to promote spike development, and compensatefor the delay in spike development in Rht12lines. Additionally, Rht12is sensitive toexogenous GA, and exogenous GA3can restore the characters of Rht12to normal, suggestingthat Rht12mutants may be deficient in GA biosynthesis rather than in GA signal transduction.Moreover, many dwarf mutants and also some mutants of the candidate genes were found inthe TILLING population, and this TILLING population will be a useful resource for bothmutation breeding and functional genomics.