青花菜開花基因之探勘與應用

Abstract

青花菜內含抗癌營養成分，而被視為重要的蔬菜作物，受限於源自於地中海西北部及歐洲溫帶地區，高溫逆境影響青花菜結球以及花球球況，進而影響青花菜的產量。在臺灣地區，青花菜種植季節僅限於九月至隔年四月，夏季進口需求大，因此育種家致力於早生且具有耐熱特性的青花菜研究。透過結球期較早的青花菜GWAS-208及結球期較晚的GWAS-140作為兩親本，建立F2族群，透過連鎖群分析及結球期相關數量性狀基因座定位，效應最大的兩個QTLs非別座落在第三及第六條染色體上，外表型解釋率達44.05%。透過分子標幟輔助選拔，透過QTLs側翼分子標幟作為前景選拔，將早結球特性的基因導入具有優良親GWAS-140，經過三個世代的回交，BC3F1 91% 之遺傳背景與GWAS-140相同，且比GWAS-140提早結球。在兩個QTLs區間內，分別找到三個與結球期相關的候選基因，FLC3、VRN2、PAN，以及一個與熱逆境反應相關的候選基因HSFA8，這些基因在早晚生親本GWAS-208、GWAS-140之間有非同義點突變。這些基因在大白菜、甘藍型油菜、阿拉伯芥等十字花科植物中，具有高度的保守性。透過real-time PCR分析早晚生親本GWAS-208、GWAS-140在四葉齡、始球期、適收期等三個時期，FLC3、VRN2、HSFA8，之基因表現在早晚生有差異，透過定序113個青花菜品系FLC3、VRN2、PAN、HSFA8，進一步分析候選基因序列變異與結球時間之間的關聯性，發現青花菜族群結球時間與FLC3、PAN、HSFA8 之單體型分群相關，推測FLC3、PAN、HSFA8 參與青花菜結球途徑。利用所探勘到之候選基因設計功能性分子標幟，運用於BC3F2世代，期望在分子標幟輔助選種下，更精準地協助早生青花菜選種，以推進青花菜育種策略及臺灣青花菜種苗繁殖。

Broccoli (Brassica oleracea L. var. italica) has been considered a valuable vegetable for its anti-cancer properties. Since Broccoli originated in the northern and western coasts of the Mediterranean, the European temperate regions, it prefers growing in cool temperature. High temperature in summer is unfavorable for broccoli to bolt, resulting irregular shape and uneven size of heads consequently. In Taiwan, Broccoli only can be cultivated from September to the following April. Therefore, great demands of broccoli have to be relied on massive imports in summer. To breed new broccoli cultivars with early heading date to meet market requirement is the final goal. According to the identification of the genes or quantitative trait loci (QTLs) conferring heading date with the F2 population, derived F1 cross between GWAS-208 with early heading date and GWAS-140 with late heading date. Linkage analysis and interval mapping, two major QTLs, mapped on chromosomes 3 and 6, could explain 44.05% of phenotypic variance. The flanking markers of these two QTLs were used as foreground selection to introgress heading date genes, and then applied the result to broccoli breeding program by marker-assisted selection. Three generations of backcross which were applied foreground and backgroud selection with markers, BC3F1 individuals contain more than 91％ of GWAS-140 genome and show the early heading characteristic. Between the interval of the two major QTLs, we identified three heading date candidate genes, FLC3, VRN2, PAN, and one heat stress response gene, HSFA8. Most of the SNPs mutants lead to nonsynonymous mutations between GWAS-208 and GWAS-140. Amino acid sequences of FLC3, VRN2, PAN, and HSFA8 were highly conserved among Brassicaceae species such as Brassica napus, Brassica rapa, and Arabidopsis thaliana. In addition, real-time PCR analysis of FLC3, VRN2, HSFA8 showed the differences between GWAS-208 and GWAS-140 in three stages. To determine the correlation between the candidate genes and heading date, there were 113 more accessions sequenced and FLC3, PAN and HSFA8 haplotypes were associated the heading date. The Result indicated the possibility role of FLC3, PAN, and HSFA8 in regulating the heading date pathway genes mentioned above. Until now, four selected BC3F1 individuals were selfed to establish BC3F2, and one more round of MAS will be executed to have accessions with early heading date and good horticultural traits. Furthermore, the optimized strategy of broccoli breeding by MAs will facilitate crop seed enterprise in Taiwan.