Identification And Genetic Analysis Of A White Stem Tobacco Mutant

Chlorophyll (Chl) can be regard as the most important photosynthetic pigments in plants that playindispensable roles in performing the photosynthesis and maintaining the global ecosystem. Chl has aclose relationship with leaf color, the quality and processing characteristics of the plant. When thecontent of Chl is changed, plant leaves will exhibit various mutant phenotypes, such as albino, yellowgreen, stay green and so on. The burley tobacco is a white stem mutant possessing excellent quality as aresult of reduction in Chl. In this article, we described an EMS induced white steme mutant ws1incommon tobacco (Zhongyan100, ZY) and accomplished the identification and genetic analysis of thismutant. The main results are as follows:(1) Compared with the wild type ZY, the ws1mutant plants showed a light color throughout thedevelopmental stage, especially in stems. Each part of the stem in ws1was lighter than the equivalent inZY. The color of ws1had a gradual change from the yellow green on the top to pale white at the bottomof the stem, whereas ZY showed a light green color at the bottom and normal green on the top of thestem. Although no significant difference was observed between the upper young leaves of ws1and ZY,there was obvious color difference between the lower old leaves of ws1and ZY. The visual discernibledifferences between ZY and ws1appear in the cross period of seedlings.(2) Each part of leaves in ws1has lower Chl content than the equivalent in ZY. The Chl content ofws1has a larger decline rate than that of ZY from upper leaves to lower leaves. Fluctuating between3.41and3.87, no significant differences in Chla/b ratios were detected between ws1and ZY.(3) To analyze the genetic mechanisms underlying the ws1mutant phenotype, two crosses weremade between Honghuadajinyuan (HD) and ws1. All the F1and RF1individuals displayed the samegreen-colored phenotypes as HD. In the F2and RF2populations,13and12white-colored individualswere observed from the polulations of234and285, respectively, conforming to the segregation ratio of15:1. The segregations of the BC1and RBC1populations fit the expected ratio of3:1. The above resultsindicated that the ws1mutant phenotype is controlled by two recessive nuclear genes, and neither genealone could give rise to the abnormal light color phenotype.(4) The highly similar phenotypes and the same inheritances between ws1and burley tobaccoimplied that they might contain the same mutant alleles. To check this hypothesis, allelism test wascarried out by reciprocally crossing Burley21with ws1. Both F1and RF1exhibited the typicalphenotypes of burley tobacco that did not segregate in F2populations. The results indicated that thesame two recessive genes governed the burley charater in common tobacco.(5) To segregate the WS1A and WS1B genes, the green-colored BC1F1plants were selfed toproduce BC1F2populations. About2/3of them showed the expected segregation ration of3:1statistically, one of which was used to map ws1a, and another one was used to map ws1b. According tothe different recombinant between ws1a and markers, the ws1a locus was mapped between PT54006 and PT51778with a genetic distance of8.04cM and3.96cM, respectively. The ws1b locus was finallymapped between PT53716and TM11187, each with a genetic distance of8.56cM from ws1b.(6) With the ws1as tester and the equivalent ZY as driver, the subtractive cDNA library betweenws1and ZY was constructed by suppression subtractive hybridization (SSH).152unigenes with averagelength of437.32bp, were assembled from750High Quality ESTs. About145genes were blasted bythese unigenes, and12nuclear genes relevant to chloroplast proteins were obtained. The chloroplastproteins are involved in light system â… , transcription and translation of chloroplast genome, depletingenergy metabolism of pigment complex and chloroplast and other unknown function, etc., which maybe associated with the mutant trait.