Genetic Diversity And Model Analysis Of Germplasm And Cloning Of BcDREB2 Gene Fragment In Non-heading Chinese Cabbage

The non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino) came of China with plenty, numerous and complex germplasm. However, with the use of minority high yield variety, many farmhouse varieties was eliminated, and variety simplication and genetic frangibility was increasingly serious. In order to exploit the genetic potential, widen genetic foundation and make full use of non-heading Chinese cabbage germplasm, the morphological and molecular marker genetic diversity in non-heading Chinese cabbage germplasm were analyzed, establishment strategy and representative evaluation of selected core collection of non-heading Chinese cabbage were studied, genetic model of plant-height, leafblade-weight and leafstalk-weight traits in SI×Qiu017 combination were analyzed. And the bcDREB2 homologous gene fragment from the leaf of SI×Qiu017 combination was cloned. The expression of bcDREB2 in different tissues was studied by semi-QRT -PCR.1. Morphological Diversity of Non-Heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino)GermplasmThe morphological diversity of 125 non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino)germplasm were studied in the paper. The results showed that the morphological diversity was rich among the accessions assessed. The highest diversity index was 0.972 in Jiangsu province. The coefficient of variance of leaf petiole length(59.71%) was the biggest among all the traits. The first and second principal components totally represented 48.4% of morphological diversity. Based on the morphological data, 125 non-heading Chinese cabbage were clustered into 6 groups. The B. campestris van communis was clustered together with B. campestris var. rosularis, B. campestris var. parachinensis, B. campestris var. multiceps and B. campestris var. tai-tsai, respectively. This indicated there were relative relationships between B. campestris var. communis and other types of non-heading Chinese cabbage. 2. Establishment Strategy and Representative Evaluation of Core Collection of Non-heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino)The strategy of core collection of non-heading Chinese cabbage was studied using 195 accessions cataloged in the Record of Chinese Vegetable and 22 characters including quantitative and qualitative characters as basic data. The adjusted Euclidean distance and UPGMA(Unweighted Pair Group Method with Arithmetic Mean), Flexible-beta method and ward's were used in classification of population. 15%, 20%, 25%, 30% and 35% core subnets were sampled by stepwise clustering with preferred sampling. Twelve parameters, i.e. Shannon index of genetic diversity, Simpson index of genetic diversity, ratio of phenotypic retained, variance of phenotypic frequency, percentage of mean difference, percentage of variance difference, ratio of mean, ratio of variance, range, maximum, minimum, coefficient of variation were used to evaluate the representative between core collection and original. The result showed that it was the optimal sampling strategies that 30% sampled by stepwise clustering with preferred sampling based on UPGMA used in classification of population. 59 partial core germplasm were selected from 195 collection. Indexes of genetic diversity for 22 agronomical characters did not reach significant level between the core collection and the original.3. Analysis of Genetic Diversity of Non-heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino) Germplasm Based on RAPD MarkersThe genetic diversity of 64 accessions of non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino) germplasm was analyzed by RAPD(Random amplified polymorphic DNA). 147 sites were detected by 23 RAPD primers, 71 of which(48.30%) were polymorphic among different accessions. Averagely, 6.39 sites and 3.09 polymorphic sites were amplified by per primer. The Shannon information index of B. campestris var. communis was the highest both among different types and among different ecological regions. The non-heading Chinese cabbage had the richest genetic diversity in Jianghuai drainage area. The genetic differentiation coefficient of non-heading Chinese cabbage was 59.05%, and mainly distributed among populations. The gene flow(0.4031) showed that the gene less flowed among populations.4. Analysis of Genetic Diversity of Non-heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino) Germplasm Based on SRAP MarkersThe genetic diversity were assessed in 64 accessions of non-heading Chinese cabbage all of world by SRAP(Sequence-related amplified polymorphism) markers. 215 sites were detected by 21 SRAP primers, and 112 of which(52.09%) were polymorphic among different accessions. Averagely, 10.24 sites and 5.33 polymorphic sites were amplified by per pair primer. The Shannon information index(0.2161) and Genetic richness((190)88.37%) of B. campestris var. communis were the highest among different types of non-heading Chinese cabbage. The Shannon information index(0.2194) and Genetic richness((185)86.05%) of non-heading Chinese cabbage were the highest in Jianghuai drainage area among different ecological regions. The domestic Shannon information index(0.1949) and Genetic richness((188)87.44%) were the higher than the foreign. The measurement of genetic variation showed that the coefficient of genetic differentiation was 58.22%. Most of the genetic variation existed among populations. The gene flow(0.4031) showed that the gene less flowed among populations, and this indicated that the degree of genetic differentiation was the higher. The cluster analysis showed that six groups can be clustered according to the ecological region or ecological character when genetic similarity coefficient was given as 0.872. The numbers of sites, polymorphic sites and the rate of polymorphic sites amplified by SRAP markers were more 60.25%, 72.94% and 7.85% than those by RAPD marker, respectively. These indicated that the polymorphism of SRAP marker was the higher than RAPD marker.5. Genetic Model Analysis of Plant-height and Leaf-weight Traits in Non-heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino)The mixed major-gene plus polygene inheritance model was used to analyze the inheritance of plant-height, leafblade-weight and leafstalk-weight in the non-heading Chinese cabbage cross between SI and Qiu017. The results showed that the plant-height and leafblade-weight were controlled by one completely negative complete dominant major-gene plus additive-dominant polygenes. For plant-height, the additive effect for major-gene was 5.8. The additive and dominant effect for polygenes was -7.9 and 15.0, respectively. The major-gene heritabilities in B₁, B₂ and F₂ were 33.28%, 37.05% and 51.68%, respectively, and the corresponding polygenic heritabilities were 5.84%, 12.67% and 1.34%, respectively. For leafblade-weight, the additive effect in major gene was 1.9. The additive and dominant effect for polygenes was -1.3 and 1.8, respectively. The major-gene heritabilities in B₁, B₂ and F₂ were 6.98%, 4.33% and 36.08%, and the corresponding polygenic heritabilities were 16.03%, 7.39% and 23.96%. The leafstalk-weight was controlled by one additive major-gene plus additive-dominant polygene(D-2). The additive effect for major-gene was -1.1. The additive and dominant effect for polygene was 1.3 and 2.6, respectively. The major-gene heritabilities in B₁, B₂ and F₂ were 31.72%, 5.27% and 57.94% respectively, and the corresponding polygenic heritabilities were 0.42%, 4.59% and 4.80% in F₂. These results indicated that major gene in F₂ is a key factor and environment factor is also relatively important. This implies that in the genetic improvement of plant-height major-gene was a main factor whereas environmental effect should be taken care.6. Cloning and Expression Analysis of bcDREB2 Homologous Gene Fragment in non-heading Chinese Cabbage(Brassica campestris ssp. chinensis Makino)The bcDREB2(Accession number 495249), which was a DREB2 homologous gene fragment with the length of 429bp, was isolated from the leaf of the non-heading Chinese cabbage cross between SI and Qiu017. The deduced amino acid sequence of bcDREB2 analysis showed that its similarity to Brassica napus DREB2-2, Brassica rapa subsp. pekinensis CBF1, Thellungiella salsuginea DREB1, Arabidopsis thalianar DREB1B, Nicotiana tabacum DREB1a were 99%, 98%, 86% and 78%, respectively. There was an AP2 domain in bcDREB2. The Semi-QRT -PCR analysis of its expression in different tissues revealed that the expression of bcDREB2 in the root and leaf was higher than in bud and tender legume. These showed that bcDREB2 cloned from non-heading Chinese cabbage was the homologous gene fragment of DREB.