Research On Leaf Structure, Physiological Features And Markers Of BoCBF Genes In Cold-tolerance Cabbage (Brassica Oleracea Var.capitata L.)

Cabbage （Brassica oleracea L.） is an important cruciferous vegetable crop. In recent years, wintercabbage is grown more and more in the middle and lower reaches of Yangtze River with the changes ofmarket demand. For example, the cultivated area in Jiayu County of Hubei province and its neighboursis up to10thousands hm². The varieties cultivated through the whole winter should be tolerant to below-6℃. At present, the identification of cold tolerance in cabbage mainly depends on the naturally lowtemperature, but this method requires a long period and is susceptible to environmental interference. Inorder to accelerate the breeding progress and improve breeding efficiency, some physiological indexesor molecular markers are needed to assist the cabbage cold-tolerance breeding.Cabbage materials with different cold tolerance were used to study the genetic analysis of coldtolerance in cabbage, the relationship between the chilling injury index （CI） and variation of leafstructure, free amino acid components, chlorophyll fluorescence parameters with or without freezingstress, and the relationship between CI and BoCBF gene variations. The results were as following:1. Genetic analysis for cold tolerance in cabbage.Six generations derived from a combination of10-352with07Q214were used to investigate theinheritance of cold tolerance in cabbage by the model of mixed major gene plus polygene inheritance.The results showed that the best fitted model of cabbage cold tolerance was E₁₀（twoadditive-dominant-epitasis major genes plus additive-dominant-epitasis polygene）. The additive effectvalue for each of two major genes was identical, and both of them had a negative effect. The coldtolerance was mainly controlled by additive effect, so the cold tolerance should be selected at the earlygenerations.2. The relationship between leaf structure, free amino acid components, chlorophyll fluorescenceparameters and cold tolerance.Under freezing stress, the content of aspartate family, glutamate family and alanine familyincreased significantly at the0.05or0.01level compared with the unfreezing condition, while nosignificant change was detected in the content of aromatic family; the content of proline and glycineincreased significantly at0.01level while the content of phosphorus ethanolamine and urea decreasedsignificantly at the0.01level; the content of glycine under freezing stress had a significant negativecorrelation with CI at the0.05level, so the content of glycine can be used as a measurement for cabbagecold tolerance.Compared with the unfreezing condition, the value of Fo increased while the value of Fm, Fv/Fm,PI decreased in all cabbage materials under freezing stress; furthermore, both Fv/Fm and PI had asignificant negative correlation with CI at the0.05level, so Fv/Fm and PI can be used as a measurementfor cabbage cold tolerance. Without freezing stress, the thickness ratio of palisade tissue to spongy tissue, CTR, SR had asignificant correlation with CI at the0.05or0.01level, therefore they can also be used to evaluatecabbage cold tolerance.Comprehensive comparison showed that the thickness ratio of palisade tissue to spongy tissue wasbetter than the other five indexes in evaluating the cabbage cold tolerance.3. The study on markers of BoCBF genes to assist the foundation parent21-3cold-toleranceselection.Two cabbage inbred lines differing in cold-tolerance were used to analyze the effect of CBF genevariations in the freezing stress condition. According to Arabidopsis CBF1gene sequence and thescaffold information of cabbage genome, the full-length coding domain sequence of cabbage BoCBF1and BoCBF2genes were obtained. Based on the sequence variations of BoCBF1and BoCBF2genes,two CAPs markers were successfully developed. Afterwards the CAPs markers were used to screen177individuals of F2population. The analysis showed that the sequence variations of the two genes weresignificantly correlated with cold tolerance （p<0.01）, so the CAPs markers can be used formarker-assisted selection （MAS） in foundation parent21-3cold-tolerance breeding. Real-timequantitative PCR were used to analyze the transcription of BoCBF1and BoCBF2gene, the resultsshowed that both of them expressed a single peak curve in0-24h induced by cold temprature. Moreover,the time of the transcriptional peak in the cold-tolerant materials was earlier than the cold-susceptiblematerials.