Cultivar Identification And Male Sterile Cytoplasm Analysis In Broccoli (B.oleracea L.var. Italica Planch.)

Broccoli(B.oleracea L.var.italica planch.) is an important vegetable worldwide and belongs to Brassica.Recently,broccoli has become an important vegetable crop for export and domestic consumption in China.The objectives of this study were to identify broccoli cultivars which were widely planted in China,analyze the genetic relationship among broccoli and its relatives,and identify two CMS in broccoli with cytological and molecular analysis.A total of 18 genotypes of broccoli were subjected to random amplified polymorphic DNA(RAPD) and Inter-simple sequence repeat(ISSR) analysis.The 74 RAPD primers generated 614 bands of which 344 show polymorphic;8 ISSR primers produced 84 bands which 67 were polymorphic.Using two selected primers,all broccoli genotypes could be distinguished from each other,indicating that RAPD and ISSR marker are efficient to identify the broccoli cultivars.The broccoli genotypes examined could be separated into two major subgroups.The first major subgroup,subgroup A,included 13 genotypes which arise from Japanese seed company except 'JinL(u｜¨)Wang' and 'US Agriseeds OASI 808F₁'. Subgroup B was comprised of 5 genotypes which belong to early-maturating cultivars.18 broccoli genotypes,6 related Brassica accessions including three genotypes of cauliflower, one genotype of cabbage,non-heading cabbage and Chinese cabbage,and one radish genotype were employed in this genetic diversity analysis.All accessions could be separated into two subgroups(Radish and Brassica) based on UPGMA cluster analysis.The Brassica cluster could be further divided into two sub clusters:â… B.Campestris andâ…¡B.oleracea.Principal coordinates analysis(PCoA) separate the accessions into three groups:â… radish,â…¡non-heading cabbage and Chinese cabbage,â…¢Brassica oleracea.The UPGMA and PCoA indicated that cauliflower has the closest relationship with broccoli then followed with cabbage,Chinese cabbage and non-heading cabbage in Brassica. Radish was independently distant from others.In order to identify the two male sterile materials NAU-LB and NAU-YX in broccoli, the two materials backcrossed with normal line NAU-Q3.The backcrossed progenies NAU-03LB and NAU-04YX showed 100%male sterile which indicated the two materials were cytoplasmic male sterility.According to the cytological analysis of microsporgenesis in NAU-03LB and NAU-04YX,the two CMS has different abortion way.The microspores of NAU-03LB were observed to die at the early free microspore stage which due to the tapetum disappeared prematurely through excessive vacuolization.The result was coincident to the principle observations in radish with Ogura cytoplasm.The inhibition of NAU-04YX anthers development occurred at the stage of archesporial cell with no microsporangium differentiation and consisted of parenchyma and the vascular.The abortion way of NAU-04YX was similar to the Polima CMS.Transmission electron micrographs of NAU-04YX pollen and tapetal cell show the abnormal phenomenon appears in the pollen mother cell stage.Three primer pairs were designed to amplify orf138, a chimeric gene which closely associate with Ogura cytoplasmic male sterility in radish. The amplification patterns of the three pairs of primer are identical in the two CMS broccoli and absent in normal line NAU-Q3.The sequence derived from specific fragment showed highly homologous with orf138.The result of our study provided powerful evidence for indicating that the two CMS in broccoli attribute to Ogura CMS.