Development Of Molecular Markers Anchoring Pink-leaved Gene Pi In Kale

Ornamental kale (Brassica oleracea var. acephala DC) is biennial which has rich and varied leaf color and shapes.Due to it seems to peony which has gorgeous appearance,as well as it prefers cold and cool climate,so it is talent showing itself in the field of virescence in northern China. Leaf colour is one of the most important agronomic traits in kale. When the temperature is below 15℃, the inner leaves change from green to brilliant color, with a high quality of ornamental.In this study, the parents respectively as pink leaf and white leaf kale.Screening 1359 large F2 population by flanking markers of preliminary positioning,68 recombinants are obtained,and kale pink-leaved gene Pi is located again.A genetic linkage map was constructed. The anthocyanin biosynthesis genes as well as the Scaffold of Ni2C12 and their sequences were provided by the genome database of Brassica and Arabidopsis thaliana, then the new markers are designed in the ascertain region of Pi.The main results are as follows:1. In the previous research,Ni2C 12 and Boac04 are the flanking markers obtained from the preliminary mapping of the gene Pi.Using them to screen the large F2 populations one by one, constructed the recombination populations of the gene Pi successfully,which contain 68 individuals.2. Due to initial location of Pi gene is use 184 F2 populations, the positioning result is not accurate enough. In this study,relocation of the pink-leaved gene by using 1359 large F2 populations constracted combining with the initial position flanking markers. As a result, the initial location area is located in the range of 0-11 Mb within C03 chromosome correctly.The gene Pi is located in the top of the C03 chromosome and genetic distance is 4.5 cM between Ni2C12 and the gene Pi, as well as 8.5 cM between Boac04 and the gene Pi.The marker Ni2C12 is the closest flanking marker to the pink-leaved gene Pi.3.76 anthocyanin biosynthesis regulatory genes are excavated successfully in Brassica chromosome 03 range from 0 to 16 Mb,by refering gene annotation information based on the genome database of Brassica and Arabidopsis thaliana.80 pairs of primers are successfully designed. The results show that 74 pairs of SSR primers could amplify the product, and the amplification efficiency is 91%. Among them, there are 4 markers with polymorphism among the parents, and all of them are codominant markers. Screening recombinants further,the result shows that compare with Boac04 the recombinants of 4 codominant markers, which are reduce by 16,10,16,13,respectively.The molecular markers of Pi gene are more closely linked.4. Compared Ni2C12 sequencing results to the Brassica genome database, the results show that Ni2C12 is located on the Scaffold000123 of Brassica genome,where the specific physical location is 555793-556023 bp. In order to search SSR loci accuratly near Ni2C12, the interval 0-1 Mb of Scaffold123 is determined.A total 824 SSR loci are obtained by using SSRHunter to search the SSR loci, which dinucleotide and trinucleotide repeat motif of SSRs are on the total number for 811, accounting for 98.42%. There are 6 tetranucleotide repeat motif of SSRs and 5 pentanucleotide repeat motif of SSRs.But hexanucleotide repeat motif of SSRs appeared only two times.39 SSR sequences are get which are designed SSR primers by homology screening. The 35 pairs of SSR can amplify the ideal product, among them 14 primers are polymorphic between the parents and the gene pools.They can be used to the research of fine mapping in the pink-leaved gene Pi in kale.