Developing SSR And InDel Markers And Mapping Of Lobed Leaf Mutant In Non-heading Chinese Cabbage

Leaf was the major organ that used in photosynthesis and respiration and transpiration ofplant and played a very important role in the development of plant. The shape of leaf marginhad smooth, serrated, lobed and so on, its diversity was a very important factor that form thediversity of leaf shape. Large number of genes involed in the leaf marginal shape pathway hadbeen cloned from model plants, but the genetic factor(s) conferring leaf lobed phenotype inChinese cabbage were unknown. In this study, SSR and InDel markers in A10(16899818-17299817) were developed using the method of SSRHunter software andhomologous sequence alignment, the polymorphisms and transferability of these markerswere analyzed firstly. Then, we used the F8near-isogenic line of a mutnat plant of "aikangqing" as our experimental material and analysed the rule of inheritance of the lobed leafand screened the linked molecular marker for it and mapping it eventually. The main resultswere as follows:1.394SSRs were mined with an average distance of0.98kb in400kb DNA sequence.Dinucleotide and trinucleotide repeats SSRs were dominant, accounting for79.44%and18.78%respectively. In order to improve the accuracy and transferability of the SSR, themined SSRs were blasted by homology and15suitable SSRs were choosed by the conditionand used to design primer. Morever, some differences of InDels but not SSRs were found insome SSRs sequences and19suitable InDels were choosed by the condition and used todesign primer. The polymorphic analysis of34new primer pairs were detected in6genotypesof Chinese cabbage.28primer pairs showed the amplification, accounting for82.35%ofdesigned primers, and27primer pairs showed polymorphisms, accounting for79.41%ofdesigned primers. PCR products from4random SSR primer pairs were selected for sequenceanalysis. The results showed that100%of the fragments contained SSRs.2. The transferabiliable analysis of28new primer pairs were detected in cabbage, rapeand radish samples, the percentage of available SSRs and InDels amplified are85.71%,100%and77.78%, respectively. It showed that these SSRs and InDels had good transferability among species of cabbage,rape and radish.3. The anlysis of morphological characteristics of the wild plant and the mutant showedthat the first leaf of the mutant were palm-lobed and the lobe were main focus on the leaf baseand there were almost not secondary lobes on primary lobes. The averagy depth of the BrcLL2lobe was0.865and the averagy number of the lobe of BrcLL2plant was6and the leaf arearatio of BrcLL2plant was70%of the wild plant. The analysis of variance showed that themutational plant of BrcLL2had significant differences in the depth of lobe, the number oflobe and the leaf area ratio compared with the wild plant.4. The F1that come from the reciprocal cross between the wild plant7HK16B-2and themutant7HK16H-6had the lobed leaf, so the lobed leaf trait was controlled by nuclear genesand was dominant to the smoothed leaf trait. The segregation population of F2were countedas the number of the lobes of the third leaf, the analysis of variance showed that the lobed traitof leaf accorded with the proportion of1:2:1and was controlled by a dominant nuclear gene,named it BrcLL2and minor genes could also influence the lobed leaf.5. The NIL line population of F8was used as the mapping population. Using the SRAP,SCAR,SSR and InDel techniques and bulked segregant analysis (BSA), We found ninemarkers that linked to the BrcLL2gene controlled the lobed leaf trait and initially mapped theBrcLL2gene between the SSR11and FC1-FE46on A10, with the genetic distance of0.194cM and6.913cM. The result may be useful for the isolation of the BrcLL2gene.