萵苣之轉錄體分析及溫度、日長對其產量之影響

Transcriptome Analysis and the Effects of Temperature and Daylength on Marketable Yield in Lettuce (Lactuca sativa L.)

李阿嬌(Ah-Chiou Lee)

指導教授 ： 羅筱鳳

共同指導教授 ： 許圳塗

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/博士(2015年)

https://doi.org/10.6342/NTU.2015.11034

摘要

萵苣(Lactuca sativa L.)是全球最普遍商業栽培的葉菜類之一，對屬於冷涼季作物的萵苣而言，在亞熱帶臺灣地區周年生產是一大挑戰。本研究以臺灣地區普遍種植9個栽培種為參試材料，其分別代表不結球型葉萵苣、立生型萵苣、奶油萵苣及結球型萵苣亞型Batavia等4種園藝類型，調查各栽培種之生育特性，探討各栽培種自定植至最大可售產量期之天數(days to harvest for maximal marketable yield, DMMY)與溫度和日長間之關係，建立各栽培種DMMY之多元及簡單線性迴歸模型，並應用雜交育種法選育高產品種，及以次世代定序(next generation sequencing)為基礎的RNA-Seq技術，分析莖生長速率不同之兩品種在最大可售產量(maximal marketable yield, MMY)期之轉錄體差異。
於簡易塑膠布溫室內評估萵苣生長量及夏和冬期作產量，參試的9栽培種中，‘福山’(‘Fu San’)有最長的DMMY及最高的MMY，但其全栽培期的生長速度較其它品種慢，而全栽培期之生長速度以‘直立萵’(‘Jhih Li Wo’)及‘圓葉萵’(‘Yuan Yeh Wo’)最高。於冬期作及夏期作栽培，栽培種、栽培季節及其交感作用均極顯著影響萵苣MMY、DMMY及最大可售產量採收日前之生長速率(growth rate before harvest day for maximal marketable yield, GRBHD)。Batavia型萵苣‘福山’(‘Fu San’)、立生型萵苣‘直立萵’(‘Jhih Li Wo’)及不結球型葉萵苣‘白葉萵’(‘Bai Yeh Wo’)對亞熱帶臺灣地區的夏季氣候有較好的適應性。
相同之9個栽培種於連續8生長季栽培，溫度、日長及栽培種對DMMY具有顯著效應，三因子間有交感作用，其中溫度變因解釋了本試驗大部份的變異。各參試栽培種之DMMY對日長的反應在低溫環境條件下較強。依據植株DMMY與溫度及日長間之皮爾森(Pearson’s)相關性係數，參試栽培種可分為2類型，一為DMMY主要由溫度控制，對日長較不敏感，包括‘福山’(‘Fu San’)、‘白葉萵’(‘Bai Yeh Wo’)、‘Bibb’及‘直立萵’(‘Jhih Li Wo’)等，另一類型雖然DMMY主要由溫度控制，但對日長亦很敏感，包括‘黃萵’(‘Huang Wo’)、‘劍萵’(‘Jian Wo’)、‘尖葉萵’(‘Chien Yeh Wo’)、‘圓葉萵’(‘Yuan Yeh Wo’)及‘Grand Rapids TH’等。以單株自定植至採收調查日止所經歷之平均日均溫度(average daily mean temperature)為處理溫度，10、 13及15小時日照為處理日長，進行各栽培種DMMY之多元迴歸分析，其決定係數(R2)為0.72-0.91；由標準化淨迴歸係數得知，溫度對所有栽培種DMMY之相對貢獻程度較日長大。各栽培種於10、13及15小時日長下之DMMY與平均日均溫度之簡單線性迴歸模型顯示，各模型均呈極顯著性差異，決定係數(R2)為0.55 -0.94，溫度每增加1 ℃，縮短0.77-3.11日之DMMY。4栽培種於控制環境條件下驗證溫度及日長對萵苣植株莖生長的影響顯示，高溫促使所有參試地方種莖生長較快，並且日長對莖伸長的效應在低溫環境比在高溫環境強，與簡易塑膠布溫室環境下之試驗結果一致。
應用栽培種中在各季具有最高MMY及DMMY、但初期生長速率最低之Batavia型萵苣‘福山’(‘Fu San’)為母本，分別與相同園藝類型Batavia型萵苣‘TOT423’雜交，並經各級試驗，育成具早熟性之新品種Batavia型萵苣‘桃園1號’(‘Taoyuan No. 1’)；以及與不同園藝類型立生型萵苣‘直立萵’(‘Jhih Li Wo’)雜交，並經各級試驗，育成全期生長速率較高之新品種Batavia型萵苣‘桃園3號-綠寶’(‘Taoyuan No. 3-Lyubao’)。比較兩新品種與其共同母本品種之產量，前者春及秋期作之產量指數分別較母本增加12%及23%，後者冬及春期作之產量指數分別較母本增加41%及45%，顯示對於共同母本品種而言，以與不同園藝類型萵苣雜交所產生的後裔族群中選拔，選育到較高產量新品種的可能性較高。同樣地，以初期生長速率較高的不結球型葉萵苣‘劍萵’(‘Jian Wo’)為母本，與具高MMY、DMMY及GRBHD特性之Batavia型萵苣‘福山’(‘Fu San’)雜交，育成之不結球型葉萵苣新品系‘TYLT09H436’，因為具有較母本高之GRBHD而有高MMY特性。
以莖生長速率不同之‘直立萵’(‘Jhih Li Wo’)及‘福山’(‘Fu San’)兩品種為材料，其於高溫且長日條件下，分別於定植後16及31天達莖長5 cm之MMY期，利用RNA-Seq技術分別於定植後4、16及31天分析比較莖頂轉錄體。‘直立萵’(‘Jhih Li Wo’)及‘福山’(‘Fu San’)分別在定植後16天及16和31天之不同生長期中，檢測到865及729和845個具有差異表現轉錄子(differentially expressed transcript, DET)。萵苣莖快速生長期為營養生長到生殖生長的轉換期，故進一步檢測MMY之DET中與生殖生長相關之DET。‘直立萵’(‘Jhih Li Wo’)及‘福山’(‘Fu San’)於MMY期之DET依GO功能註釋獲得具顯著性、且與生殖生長有關的GO term分別有8及5項，參與的DET數量各有57及51個。‘直立萵’(‘Jhih Li Wo’)及‘福山’(‘Fu San’)兩品種與生殖生長有關之共同調節(co-regulated) DET共有13個，其中經由GO分析獲得Batavia9495/COS10074、Batavia11963/COS26758、Batavia18270/COS2098、Batavia13982/ COS793及Batavia4093/COS3321等5個DET，在‘直立萵’(‘Jhih Li Wo’) MMY期的表現量增幅較高，經由檢索前人研究報告獲得8個DET，其中Batavia34365/COS34232、Batavia7703/COS7895及Batavia9486/COS29989與花發育階段之GA調節機制相關，在‘直立萵’(‘Jhih Li Wo’)的表現量較高，均可解釋‘直立萵’(‘Jhih Li Wo’)莖伸長較‘福山’(‘Fu San’)快的原因。將‘直立萵’(‘Jhih Li Wo’)及‘福山’(‘Fu San’)個別品種專一的DET於KEGG資料庫檢索，分別獲得2及1個DET參與植物荷爾蒙訊息傳導項目；此外，‘直立萵’(‘Jhih Li Wo’)在生物體系統(organismal systems)之環境適應性項目下比對到植物晝夜節律(Circadian rhythm-plant)途徑，而‘福山’(‘Fu San’)則無，此與‘直立萵’(‘Jhih Li Wo’)對日長敏感性較‘福山’(‘Fu San’)高的生育特性相符合。

關鍵字

萵苣 ； 栽培種 ； 可售產量 ； 迴歸分析 ； 品種選育 ； 轉錄體分析 ； 次世代定序

並列摘要

Lettuce (Lactuca sativa L.) is one of the most commonly commercial cultivated leafy vegetables. Year around production of lettuce, a cool-season leafy vegetable, in high tunnels is a challenge for growers in subtropical regions. The aims of this research were to characterize the growth of nine locally grown lettuce cultivars, determine the relationships between days to harvest for maximal marketable yield (DMMY) and climatic variables, temperature and daylength, in individual cultivars in high tunnels, develop new high-yielding cultivars by hybridization, and describe the differential transcriptional events occurred at maximal marketable yield (MMY) stage in cultivars ‘Fu San’ and ‘Jhih Li Wo’ by RNA-Seq analysis.
The assessments of lettuce growth in high tunnels indicated that Batavia lettuce ‘Fu San’ exhibited the highest DMMY and MMY, but slower growth rate among nine cultivars. ‘Jhih Li Wo’ and ‘Yuan Yeh Wo’ showed the highest growth rate during entire cultivation period. MMY, DMMY and growth rate before harvest for the MMY (GRBHD) during Winter 2008 and Summer 2009 were significantly different among planting seasons, cultivars and their interactions. The results also revealed that Batavia lettuce ‘Fu San’, romaine lettuce ‘Jhih Li Wo’, and leaf lettuce ‘Bai Yeh Wo’ were more adaptable to the summer hot climate in Taiwan.
The evaluation of DMMY for the same nine cultivars during eight successive seasons cultivation revealed that lettuce DMMY was significantly affected by temperature, daylength, cultivar, and their interactions. Most variation could be explained by temperature. The effect of daylength on DMMY for 9 cultivars was stronger under low temperature regimes than that under high temperature regimes. All cultivars were classified into 2 types according to Pearson’s correlation coefficients between DMMY and temperature/daylength. One type was controlled mainly by temperature and insensitive to daylength, including ‘Fu San’, ‘Bai Yeh Wo’, ‘Bibb’ and ‘Jhih Li Wo’. The other was controlled mainly by temperature and sensitive to daylength, including ‘Huang Wo’, ‘Jian Wo’, ‘Chien Yeh Wo’, ‘Yuan Yeh Wo’ and ‘Grand Rapids TH’. The mean daily temperature from planting to harvest for single plant was considered as the temperature treatment, and 10 h, 13 h and 5 h were set as photoperiod treatments. Multiple regression equations were solved to explain the correlations of temperature and daylength to DMMY. The coefficients of multiple determinant (R2) for each cultivar were ranged 0.72-0.91. As revealed by the standardized partial regression coefficients, the contribution of temperature to DMMY in lettuce plants was higher than that of daylength in all cultivars. The coefficients of determinant of the proposed simple linear regression models for each cultivar under 10,13 and 15 h respectively were ranged 0.55-0.94, with every 1 ℃ decrease in daily mean temperature , DMMY decreased by 0.77-3.11 days. Four cultivars were grown in growth chambers to verify the effects of temperature and daylength on lettuce stem growth. Stem growth was faster at higher temperatures. The effect of daylength on stem elongation under low temperature regimes was stronger than that under high temperature. The growth chamber experiments confirmed the results of the high tunnel experiments.
Two cultivars of same horticultural type, Batavia lettuce ‘Fu San’ and ‘TOT423’ were crossed to integrate high vigor from ‘TOT423’ to ‘Fu San’, and ‘Taoyuan No. 1’ with early maturity was developed. Inter-type cross between romaine lettuce ‘Jhih Li Wo’ and Batavia lettuce ‘Fu San’ was made, and ‘Taoyuan No. 3-Lyubao’ with high growth rate was developed. In comparison with their maternal, the yield index of ‘Taoyuan No. 1’ increased by 12% and 23% in spring and autumn respectively, and those of ‘Taoyuan No. 3-Lyubao’ increased by 41% and 45% respectively. In terms of improving yield of the same maternal, the efficiency of integrating plant growth characteristics from different horticultural type of lettuce cultivar was possibly higher than from the same type. Similarly, through integrating high MMY, DMMY and GRBHD in Batavia lettuce ‘Fu San’ to leaf lettuce ‘Jian Wo’ with higher growth rate during growth initiation, superior line leaf lettuce ‘TYLT09H436’ with high GRBHD and MMY was developed.
‘Jhih Li Wo’ and ‘Fu San’ with different stem growth rate were used to compare transcriptome at different growth stages using RNA-Seq technology. Shoot tips were sampled from ‘Jhih Li Wo’ at 4, 16 d after transplanting (DAT) (MMY stage),‘Fu San’ at 4, 16 and 31 DAT (MMY stage). Of the annotated contigs, 865, 729 and 845 differentially expressed transcripts (DET) were identified respectively. In the libraries of ‘Jhih Li Wo’ and ‘Fu San’ at MMY stage, 57 and 51 DETs were involved in 8 and 5 significant gene ontology (GO) terms related to reproduction, respectively. There were 13 co-regulated DETs in both cultivars. 5 of them, Batavia9495/COS10074, Batavia11963/COS26758, Batavia18270/COS2098, Batavia13982/COS793 and Batavia4093/COS3321 were identified based on GO analysis and showed more increased expression in ‘Jhih Li Wo’. Based on the public published literatures, 8 identical DETs were obtained and Batavia34365/COS34232, Batavia7703/COS7895 and Batavia9486/COS29989 were involved in GA regulation, and the expression was higher in ‘Jhih Li Wo’. The results could explain its faster stem growth. Through the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of cultivar specific DETs reproduction related reavealed that 2 and 1 DET involved in plant hormone signal transduction were identified in ‘Jhih Li Wo’ and ‘Fu San’, respectively. Moreover, 2 DETs associated with the circadian rhythm-plant pathway were identified in ‘Jhih Li Wo’, while none in ‘Fu San’. The result was consistent with previous trials revealing the former was more sensitive to daylength.

並列關鍵字

Lactuca sativa L. ； cultivar, marketable yield ； regression analysis ； breeding ； transcriptome ； RNA-Seq

參考文獻

Rappaport, L. and S.H. Wittwer. 1956a. Flowering in head lettuce as influenced by seed vernalization, temperature, and photoperiod. Proc. Amer. Soc. Hort. Sci. 67:429-437.

Rappaport, L. and S.H. Wittwer. 1956. Flowering in head lettuce as influenced by seed vernalization, temperature, and photoperiod. Proc. Amer. Soc. Hort. Sci. 67:429-437.

Piao, S., P. Ciais, Y. Huang, Z. Shen, S. Peng, J. Li, L. Zhou, H. Liu, Y. Ma, Y. Ding, P. Friedlingstein, C. Liu, K. Tan, Y. Yu, T. Zhang, and J. Fang. 2010. The impacts of climate change on water resources and agriculture in China. Nature 467(7311):43-51.

周佳、劉紹臣. 2012. 全球氣候變遷觀測. 大氣科學 40(3):185-213.

Howden, S.M., J.F. Soussana, F.N. Tubiello, N. Chhetri, M. Dunlop, and H. Meinke. 2007. Adapting agriculture to climate change. Proc. Nat. Acad. Sci. 104:19691-19696.