Conclusions and future work Transcriptome profiling of genes and pathways associated with apical abortion in broccoli Hsueh-Yu Lu (呂學宇)1,Jhih-Sian Guo1,Bo-Yu Guo1,Li-Ying Shin1,Jyuan-Yu Wei 1,Shih-Feng Fu (傅士峰)1* 1Department of Biology, National Chunghua University of Education, Taiwan (彰化師大生物系) Abstract 2. Anatomy of shoot apical meristems in broccoli lines with apical abortion 5. Transcriptomic analysis of apical abortion by RNA-seq Broccoli (Brassica oleracea var. italica) is a floral vegetable with an important nutritional value due to its content of dietary fiber, antioxidants and anti-carcinogenic compounds. Apical abortion in broccoli is a highly destructive disorder which occurs in transplants. However, little is known about the genetic and environmental factors that affect apical abortion. In this study, we characterized the broccoli cultivars with healthy and aborted shoots by using comparative transcriptome (RNA-seq) at the early vegetative stage. The tolerant line for apical abortion (BT001) and sensitive line (BS001) developed normally at 22/18℃ (day/night). However, the sensitive line showed significant apical abortion under 32/28℃. The result suggested that high temperature leads to apical abortion. Transcriptomic analysis was performed to characterize the differentially expressed genes between the two lines. Gene ontology analysis showed that response to heat and response to high light intensity was enriched in heat-tolerant lines. Genes involved in glucosinolate synthesis was enriched in heat-sensitive lines. Apical abortion in broccoli may result from both environmental and genetic factors. The genetic markers based on the InDel of the differentially expressed genes belonging to high light intensity responses were developed. The DNA molecular markers displayed polymorphisms between the healthy and sensitive lines. The potential of the DNA markers in molecular breeding was examined. Further investigation is in progress to demonstrate the use of these genetic markers in identification of apical abortion varieties. Fig. 2 Microscopic examination of shoot apical meristem in of the apical abortion-tolerant (BT01) and -sensitive lines (BS01) at 22 ℃ and 32 ℃. The two broccoli lines were grown for at different period of time (30, 40 and 60d) post germination. Stereo microscopy analysis of the shoot apex from the two broccoli lines (Upper panel). Light microscopy of shoot apical meristem of the two broccoli lines (Middle panel). Scanning electron micrography of the two lines (Lower panel). BT01-enriched gene annotation (22 ℃) BT01/BS01 fold change>2 BS01-enriched gene annotation (22 ℃) BS01/BT01 fold change>2 3. Formation of lateral shoots after apical abortion in sensitive lines Fig. 3 Analysis of lateral shoot formation after after apical abortion in the broccoli tolerant- (BT01) and sensitive lines (BS01) ) at 22 ℃ and 32 ℃. Results 1. High temperature resulted in apical abortion in sensitive broccoli lines Fig. 5 Transcriptomic analysis of apical abortion in broccoli by RNA-seq. The apical abortion-tolerant (BT01) and –sensitive broccoli lines (BS01) were grown at 22 ℃ for 4 week. RNA samples were extracted from the shoot apex from the two broccoli lines and subjected to high-throughput RNA-seq analysis. Differentially expressed genes with more than 2-fold change from BT01/BS01 and BS01/BT01 were defined as BT01-enriched and BS01 –enriched, respectively. Fig. 1 Growth kinetics and shoot development of the apical abortion-tolerant (BT01) and -sensitive lines (BS01) at 22 ℃ and 32 ℃. The two broccoli lines were grown for at different period of time (30, 40 and 60d) post germination. Apical abortion-tolerant lines (BT01) exhibited normal shoot apex development at both 22℃ and 32 ℃. The sensitive lines (BS01) exhibited normal apex development at 22 ℃. Stunt shoot development was found in apical abortion-sensitive lines (BS01) at 32 ℃. The leaf numbers of the two broccoli lines were analyzed from 30 to 60 days at 22℃ and 32 ℃, respectively. 6. Genetic marker of broccoli for identify sensitive and tolerant line Locus 2541 Locus 2541 4. High-temperature-treated root tissues led to apical abortion Locus 11137 BS01 BT01 BS01 BT01 BS02 BT02 BS02 BT02 BT01 BS01 BT02 BS02 <175 bp Conclusions and future work Differential broccoli varieties may exhibit various apical shoot-forming capacities under high temperature. Apical abortion in broccoli may result from both environmental and genetic factors. Identification of molecular DNA marker by agrobiotechnology in this study will help local seed breeder to obtain healthy broccoli varieties. Fig. 4 Grafting analysis of the apical abortion-tolerant (BT01) and –sensitive lines (BS01) at at 22 ℃ and 32 ℃. The high-temperature –treated (32 ℃) broccoli lines served as root stocks while the sections were collected from the two lines treated with 22 ℃ or 32 ℃.