Mutation Induction By Gamma Rays In Rice: Quality Assurance Of Induction Mutants And Studies On Several Important Mutant Traits

Mutation techniques have been used for production of genetic variations and breeding new varieties during the past four decades.Recent advances in functional genomic studies have further stimulated great interest in induced mutation.γ-rays irradiation technique has also been used in genomic research as an effective mutation induction technique.In this study,we used ⁶⁰Coγ- rays to irradiate three unique rice genetic lines and selected induced mutants for further research.We addressed the issue of mutant quality control in mutation studies and developed a system by using microsatellite markers.Furthermore,some important mutants with special characteristics,e.g.vivid green leaf color,elongated upper internodes(eui)and maturity were investigated for their genetic basis and potential use in rice breeding.The main results are as follows:1.Three varieties(yellow leaf variety,'Huangyu B',Bt transgenic varieties, Kelong B and BT110)were treated with ⁶⁰Coγ- rays at four doses(100,200,300, and 400Gy,5 Gy per minute).In M₁ generation,dose-dependent negative effect was found on plant height and seed set rate.Plant height was significantly reduced when the dose was above 300Gy,However,Seed set rate was significantly reduced at all doses.The mutation frequency of chlorophyll deficiency and maturity(quantity trait)reached 10^-3but that for gold hull and elongated uppermost internodes(quality trait)was around 10^-4.A 0.81%out-crossing rate was observed in M₂ population of Huangyu B,while 25.0～42.1%genetic variants of maturity selected from Kelong B was proven to be false mutants,implying a high risk of selecting false mutants in mutation program.2.The microsatellite haplotype differences between true induced mutants and their respective parents were investigated using a series of pre-selected and verified true mutants.They included maturity mutants of two transgenic rice lines and mutants induced from lines with a yellow color.While the polymorphism index of SSR markers(up to 353)reached to 5.57%to 68.22%among rice varieties,all induced mutants showed completely identical SSR haplotypes to their respective parent lines in this study.This observation was further proven to be true in the examination of other 8 mutant/parent comparisons.Thus,we proposed that microsatellite assay as a method to exclude any genetic contaminants from induced mutants,with 59 and 90 markers for 95%and 99%levels of power.3.A vivid green leaf color mutant was selected in M₂ plants of Huangyu B treated with 200Gyγ-rays.This mutant line was named Cuiyu B.At the seedling, tillering and heading stage,the total chlorophyll content of Cuiyu B was 87.0,51.6, and 59.1%higher than Huangyu B,respectively,but it was 32.4,38.5,and 32.4% lower compared with Longtepu B(parent of Huangyu B)respectively.The chlorophyll content was consistent with the leaf color phenotype.The F₁(Zaojing R/Cuiyu B)plants had green leaves as Zaojing R and different from Cuiyu B, suggesting that green leaf color(Zaojing R)covered the vivid green leaf color characteristic(Cuiyu B).In the F₂ population of ZaojingR/CuiyuB,there were three leaf color phenotypes,i.e.green,vivid green and yellow.The segregation of these three type plants well fitted into a 12:3:1 ratio,suggesting that the vivid green leaf trait was controlled by two independent and non-linked genes.Seventy four F_2:3lines among 109 F_2:3lines derived from vivid green F₂ plants segregated for vivid green and yellow plants,while other F_2:3lines had plants all with yellow leaves,the segregation ratio again fitted a 2:1 ratio,which further confirmed the control of two independent genes.These results also implied that vivid green gene, tentatively named as VG(t)was a epistatic gene over yellow leaf gene xl(t).In an attempt to mapping the VG(t)gene,52 homologous vivid green F₂ plants(based on the phenotype of F₃ Plants)and 35 yellow plants of the F₂ population of ZaojingR/CuiyuB were used for the development of DNA pools and analyzed for 353 SSR markers.One SSR marker RM492 was initially shown to be polymorphic between parents and DNA pools,but this potential linked marker was not confirmed when analyzed for individual F₂ plants.Further attempts were made to identified linked SSR markers without success.4.Three eui mutants,i.e.XinyuB-100 and Xinyu B-300(mutants of Huangyu B)and Elong B(mutant of Kelong B)were developed.Comparative analysis showed that their uppermost internodes,second and third internodes were significantly elongated while the other internodes were insignificantly different from their parents.Genetic analyses indicated three eui mutants were all controlled by a single recessive gene which were allelic to each other.A GA₃ treatment caused further extension of the third internodes of eui mutants; Observation of germination rate on panicles showed that the eui mutantion might enhance the pre-harvest germination,which is not desired in multiplication and hybrid seed production.The pre-harvest germination rate of eui mutants,wild type parents and their F₁ were 25.0-26.7%,3.89-4.56%,13.2-14.5%,respectively. When eui mutants as recurrent parent cross with CMS line Huangyu A,the eui plants of BC₁ or BC₂ had higher bagged seed set rate(11.39%and 11.41%)than Huangyu A(2.48%).These finding indicated some negative effects of eui gene on hybrid rice breeding. 5.Four early maturing mutants and four late maturing were developed from Kelong B and two early maturing mutants wer developed from BT110.Their agronomic performance and environmental responses were studied.We found that the maturity mutants had significantly reduced performance of most agronomic traits compared with their respective parents.Genetic studies showed four early maturing trait were all Controlled by one recessive gene which were alMic to each other,and so the four late maturing mutants.Two early maturing mutants selected in Jiaxing,i.e.CRD2 and CRD5 from BT110,performed quite differently when they were planted in Lingshui(Hainan,tropic area)and Jiaxing(Zhejiang, temperate region).The CRD2 mutant showed early maturity both in Hainan and Jiaxing,but CRD5 showed early maturity in Jiaxing(about 10 days earlier than BT110)while late maturity in Lingshui(12 days later than BT110).Short-day treatment of CRD2,CRD5 and their parent BT110 resulted in 22.1,19.8%and 38.8%heading acceleration rate,respectively.These findings showed that CRD2 and CRD5 had significant different responding mechanism to photoperiod compared with their parent,implying that the photoperiod gene might be mutated byγ-rays irradiation in the two mutants.The implications of these findings were discussed on their application in hybrid rice seed production.6.Two mutants of Huangyu B,i.e.Cuiyu B and Jinyu B(with golden hull), were directly used as maintain lines to breeding CMS lines.Cuiyu A and Jinyu A were developed by repeated crossing(and selection)of Cuiyu B and Jinyu B to Huangyu A.Another CMS line 'Zijin46 A' was also developed by crossing Huangyu A with a selected F₃ plant of a cross Zixing05B/JinyuB,followed by successively backcrossing to the corresponding selected paternal plants.Cuiyu A and Jinyu A had very similar agronomic traits except the mutant trait compared with Huangyu A.Zijin46 A on the other hand had a complete male sterility with 100%of sterile plants and had characteristics of large panicles,long grain shape, good grain quality and high stigma exertion rate(75.1%).