國立臺灣大學 國立臺灣大學 國立臺灣大學
國立臺灣大學生物資源暨農 生物資源暨農 生物資源暨農學院 生物資源暨農 學院 學院園藝 學院 園藝 園藝學系 園藝 學系 學系 學系 碩士論文
碩士論文 碩士論文 碩士論文
Department of Horticulture
College of Bio-Resources and Agriculture National Taiwan University
Master Thesis
台灣草苺 台灣草苺
台灣草苺 台灣草苺(Fragaria hayatae Makino)之植物性狀及其與 之植物性狀及其與 之植物性狀及其與 之植物性狀及其與
‘桃園三號 桃園三號 桃園三號’草苺 桃園三號 草苺 草苺(Fragaria ×ananassa Duch.)之種間雜交 草苺 之種間雜交 之種間雜交 之種間雜交 Fragaria hayatae Makino: Characteristics and
Artificial Hybridization with
‘Taoyuan No. 3’ Strawberry (Fragaria ×ananassa Duch.)
李慈慧 李慈慧 李慈慧 李慈慧 Tzu-Hui Lee
指導教授 指導教授 指導教授
指導教授： ： ： ：陳右人 陳右人 陳右人 陳右人 博士 博士 博士 博士
A d v i s o r ： ： ： ：Iou-Zen Chen, Ph.D.
中華民國 中華民國 中華民國
中華民國九十八 九十八 九十八年 九十八 年 年六 年 六 六月 六 月 月 月
June, 2009
誌謝
人生中的第一本書得以完成，有太多人要感謝。首先感謝恩師 陳右人博士三 年來的指導，關心與鼓勵，您親力親為陪同學生採集，改裝生長箱，訓練開車以 及培養學生的信心，這樣師徒制的精神與做人處事的風範學生會永遠記住。感謝 論文輔導委員 李金龍博士的指導、鼓勵並提供大方向的建議。感謝大學導師 鄭 正勇博士的指導與對學生的關心。感謝口試委員 阮素芬博士與 彭鏡毅博士細心 批閱論文，並給予寶貴建議，使本論文更加完善。謝謝張祖亮老師、葉德銘老師 以及陳凱儀老師曾於課堂上給與的建議與幫助。謝謝各位曾經教過我的老師。
感謝江秀真小姐、楊美珠學姊、蔡昆展學長及陳盟松學長於台灣草苺蒐集之 協助。感謝紅豆學姊、彩雲學姊、桂端學姊、嫣薇學姊及貞余學姊於大學時期引 領我進入實驗之門。感謝繼中學長、香霖學姊、淯茱學姊及春賓學長於試驗上的 提點。
感謝曾阿姨與美玲姊在日常生活的關心。感謝 Window 學長提供第一批植物 材料並於試驗初期的指導。感謝書妍學姊於分生實驗、統計分析及各樣問題上的 幫助。感謝晉瑋學長陪同野外採集，解決電腦上的問題並指導開車。感謝我親愛 的同學們：具有男人野性直覺的荔枝王子銘至、有義氣有力氣的梨接班人世宗、
對實驗室管理有異常熱心的 HPLC 強者毓翔，SAS 及 Sigmaplot 達人懷恩，莉安及 毓慧，謝謝你們陪我度過三年超棒的研究生生活。于瑩、君瑋、徐瑾、芃函、培 碩等學弟妹們於試驗期間的貼心幫忙，我衷心感激。能進入常綠果樹研究室認識 各位實在相當幸運，謝謝你們。
感謝精溫幫的好伙伴 Lulu 學姊、婷雁學姊、耐吉學姐與宛玲，你們豐富了我 的研究生生活。感謝毓婕、幼玫、思綺、子瑄、家燕與喬琪你們陪我度過了或快 樂或傷心的時光；感謝大同生研的景耀、奕瑋、菁平及芳弘，你們是我最瘋狂與 麻吉的好朋友。謝謝廣仲，你的音樂陪我度過寫論文的漫漫長夜，恭喜你得新人 獎，yeah！謝謝所有曾經幫助、關心或為我禱告的朋友，祝福你們平安健康！
感謝我的父親 李惠樹先生，您對園藝的喜愛無疑對我走上園藝之路有莫大影 響，感謝我的母親 張彩娜女士，您教我認識路邊可食用的野菜、野花與野果，是 領我入園藝門的啟蒙老師。謝謝老爸、老媽、弟弟與妹妹，你們對我的愛與包容，
支持我完成學業，I love you！感謝天上的父，從小到大罩我至今，有您在我船上 我就不怕風浪。
謹以此論文獻給我的父親、母親、弟弟與妹妹。
李慈慧 謹誌於士林家中 2009.7.3
中文摘要
台灣草苺(Fragaria hayatae Makino)是台灣之特有種，部份文獻認為是黃毛草 苺(F. nilgerrensis Schlecht.)的一個亞種。但台灣草苺果實紅色且富含花青素，形態 上明顯異於黃毛草苺。本研究室曾於小雪山採集得一群形態異於典型台灣草苺，
但與黃毛草苺較相近之白果草苺，此群草苺在台灣尚無文獻紀錄。本試驗調查台 灣草苺、白果草苺、黃毛草苺及其他草苺屬植物形態特徵及 RAPD 分子標誌之差 異，以釐清其間之親緣關係與分類地位。性狀調查結果顯示白果草苺之葉柄及走 莖為綠色，花瓣及果實均為白色；台灣草苺之葉柄及走莖為紅色，花瓣為白色基 部帶有紅紫色條斑，果實為紅色；黃毛草苺之葉柄為綠色，走莖為紅色，花瓣及 果實為白色。白果草苺與台灣草苺之葉片與花之大小以及果實高無顯著差異，而 黃毛草苺之葉片、葉柄、花之大小及果高則顯著大於台灣草苺及白果草苺。RAPD 分子標誌分析之結果顯示，白果草苺與台灣草苺的親緣關係非常接近(相似距離 = 0.94)，這兩者與黃毛草苺則有相當程度之差異(相似距離 = 0.43)。綜合形態特徵調 查及 RAPD 分子標誌分析結果，台灣草苺應非黃毛草苺之一個亞種，且白果草苺 應為台灣草苺之變種而非來自中國的黃毛草苺。
為配合種間雜交之需求，本試驗亦研究溫度與光週對台灣草苺生育之影響。
結果顯示低溫會抑制台灣草苺之營養生長，低溫處理結束植株移至溫暖環境數週 後，低溫處理者之營養生長反而較旺盛。以 15/10℃或 10/5℃配合 10 小時日長處 理 6 至 10 週後，25%-50%之植株可開花。以 15/10℃配合 14 小時日長或 10 小時 日長以及 15/5℃配合 10 小時日長處理 6 週後，8.3%之台灣草苺可開花。由於開花 植株百分比低於 50%，台灣草苺之最合適開花誘導條件仍待進一步研究。
台灣草苺(2x)與栽培種草苺(F. ×ananassa)‘桃園三號’(8x)進行互交，以台灣草 苺為母本時，雜交結果率為 73%，而以栽培種草苺‘桃園三號’為母本，雜交結果率 為 41%。以栽培種草苺‘桃園三號’作為母本得到的雜交種子之發芽率為 34%，另一
組合之種子無法發芽。成功存活的 21 個雜交後代中，7 株可開花結果，但果實多 為畸形。畸形果可能是授粉不完全之結果，或可能是成功雜交得到 5 倍體之證據。
Abstract
Fragaria hayatae Makino is endemic to Taiwan. It was considered as a subspecies of F. nilgerrensis Schlecht. in some literature, but F. hayatae is characterized with red fruit and anthocyanin in all parts of the plant, and it is significantaly distinct from F.
nilgerrensis in morphological characteristics. A white-fruited strawberry population that is morphologically distinct from typical F. hayatae but similar to F. nilgerrensis was found in Shiaoshueshan by our lab, this white-fruited form was never reported in Taiwan. Morphological characteristics and RAPD markers were studied to clarify the relationship among F. hayatae, the white-fruited strawberry, F. nilgerrensis, and some other Fragaria species. The white-fruited strawberry had green petiole, green runner, white petal, and cream-white colored fruit which were distinct from the red petiole, red runner, white petal with purplish-red blush at base, and red fruit of F. hayatae. Fragaria nilgerrensis and the white-fruited strawberry both lack of anthocyanin coloration in petiole, petal, and fruit, but F. nilgerrensis had red runners whereas the white-fruited strawberry had green runners. Despite the color of the plants, the white-fruited strawberry and F. hayatae was similar in size of leaf, petiole, flower, and fruit height.
Fragaria nilgerrensis was significantly larger than F. hayatae and the white-fruited strawberry in size of leaf, petiole diameter, and size of flower. RAPD marker analysis indicated closer relationship between the white-fruited strawberry with F. hayatae (similarity index = 0.94) rather than F. nilgerrensis (similarity index = 0.43). We suggested that the white-fruited strawberry should be a mutant of F. hayatae and not F.
nilgerrensis from China.
For the need of interspecific hybridization, the effects of temperature and photoperiod on growth and flowering in F. hayatae were studied. Cool temperature
slowed down the vegetative growth rate during treatment, but accelerated the growth after transferring the plants into warm temperature condition for several weeks. Flowers were initiated in plants treated under 15/10℃ or 10/5℃ under 10 hour day length for 6-10 weeks, and plants treated under 15/10℃ 14 hour day length, 15/10℃ 10 hour day length, and 15/5℃ 10 hour day length for 6 weeks. The rate of plants flowering was 8.3% to 50% under the above condition, and the optimal inductive condition for flower initiation in F. hayatae awaits further investigation.
Reciprocal cross was made between F. hayatae (diploid) and the cultivated strawberry ‘Taoyuan No. 3’ (octoploid) to study the interspecific hybridization compatibility. 34% of seeds from F. ×ananassa ‘Taoyuan No. 3’ × F. hayatae germinated while no seed germinated in the other combination. Seven out of the 21 survived hybrid seedlings bloomed and bore fruits. The fruits were usually misshaped, which should result from partial pollination, or might be the evidence of obtaining pentaploids from successful hybridization.
Content
誌謝 ... i
中文摘要 ... ii
Abstract... iv
Content ...- 1 -
List of Figures...- 3 -
List of Tables ...- 5 -
Introduction ... 1
Literature Review ... 3
1. Taxonomy and Distribution of the Strawberry Species... 3
2. Brief History of Strawberry Domestication ... 7
3. Strawberry Species in Taiwan ... 8
4. Breeding Potential of the Wild Strawberries ... 9
5. Application of Morphological Traits in Fragaria Species ... 16
6. Application of RAPD Markers in Fragaria Species ... 18
7. Growth and Development of Strawberry... 20
Materials and Methods ... 24
1. Morphological Variations among Fragaria hayatae, White-fruited Strawberry and other Fragaria Species ... 24
2. Molecular Variations among Fragaria hayatae, White-fruited Strawberry, and Other Fragaria Species ... 25
3. Effects of Temperature and Photoperiod on Growth and Flowering of Fragaria hayatae... 29
4. Interspecific Hybridization between Fragaria hayatae and F. ×ananassa... 31
Results and Discussion ... 33
1. Native Habitat of Fragaria hayatae and Discovery of the White-fruited Strawberry ... 33
2. Morphological Characteristics of Fragaria hayatae, White-fruited Strawberry and Other Fragaria Species ... 34
3. RAPD Characteristics of Fragaria hayatae, White-fruited Strawberry and Other Fragaria Species ... 43
4. Relationship between terminal leaflet and actual leaf area ... 45
5. Effects of Temperature and Photoperiod on Fragaria hayatae ... 46
6. Interspecific Hybridization between Fragaria ×ananassa and F. hayatae... 51
Conclusion ... 54
Reference ... 55
Appendix: Monthly mean temperature during experiment in Taipei………99
List of Figures
Fig. 1. World distribution of Fragaria species...61 Fig. 2. Distribution of Fragaria hayatae in Taiwan ...62 Fig. 3. Native habitat of Fragaria hayatae...63 Fig. 4. Plants of Fragaria hayatae, white-fruited strawberry, and F. nilgerrensis ..64 Fig. 5. Flowers of Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F.
vesca, and F. ×ananassa ‘Taoyuan No. 3’ ...65 Fig. 6. Fruit of Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F. vesca,
and F. ×ananassa ‘Taoyuan No. 3’...66 Fig. 7. Principal component analysis 2D scatter plot based on the correlation of 19
vegetative morphological characteristics in Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F. vesca and F. ×ananassa ‘Taoyuan No.
3’………67 Fig. 8. Dendrogram of Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F.
vesca, and F. ×ananassa ‘Taoyuan No. 3’ clustering by UPGMA method based on Euclidean coefficient correlation derived from 19 vegetative morphological characteristics...68 Fig. 9. Principal component analysis 3D scatter plot based on the correlation of 19
vegetative and 22 reproductive morphological characteristics in Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F. vesca, and F. ×ananassa
‘Taoyuan No. 3’ ...69 Fig. 10. Dendrogram of Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F.
vesca, and F. ×ananassa ‘Taoyuan No. 3’ clustering by UPGMA method based on Euclidean coefficient correlation derived from 19 vegetative and 22 reproductive morphological characteristics...70
Fig. 11. Polymorphism among Fragaria hayatae, white-fruited strawberry, F.
nilgerrensis, and other Fragaria species with primer UBC-4, UBC-16, and UBC-34 ...71 Fig. 12. Principal component analysis 2D scatter plot based on the correlation of 409
bands from 35 primers in Fragaria hayatae, white-fruited strawberry, F.
nilgerrensis, F. vesca, F. × ananassa ‘Taoyuan No. 3’, and Potentilla matsumurae ...72 Fig. 13. Dendrogram of Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F.
vesca, F. ×ananassa ‘Taoyuan No. 3’, and Potentilla matsumurae clustering by UPGMA method based on Jaccard coefficient derived from presence-absence data of 409 bands from 35 primers...73 Fig. 14. Relationship between terminal leaflet length, terminal leaflet width, and
terminal leaflet length × width with actual leaf area in Fragaria hayatae....74 Fig. 15. Effects of temperature and its duration on new leaf formation in Fragaria
hayatae ...75 Fig. 16. Effects of temperature and its duration on new leaf formation in Fragaria
hayatae ...76 Fig. 17. Effects of temperature and its duration on runner formation in Fragaria
hayatae ...77 Fig. 18. Effects of temperature and its duration on runner formation in Fragaria
hayatae ...78 Fig. 19. Effects of day/night temperature and day length on leaves and runners in
Fragaria hayatae...79 Fig. 20. Hybrid seedlings of Fragaria ×ananassa ‘Taoyuan No. 3’ × F. hayatae ....80 Fig. 21. Fruit of hybrid seedlings from Fragaria ×ananassa ‘Taoyuan No. 3’ × F.
hayatae ...81
List of Tables
Table 1. Fragaria ploidy level and geographic distributions ... 82 Table 2. Location of Fragaria hayatae and white-fruited strawberry used in this
study ... 83 Table 3. Descriptions of vegetative morphological characteristics used in this
study ... 84 Table 4. Descriptions of reproductive morphological characteristics used in this
study ... 85 Table 5. Leaf morphological characteristics of Fragaria hayatae, white-fruited
strawberry, F. nilgerrensis, F. vesca, and F. ×ananassa ‘Taoyuan No.
3’... 86 Table 6. Flower morphological characteristics of Fragaria hayatae,
white-fruited strawberry, F. nilgerrensis, F. vesca, and F. ×ananassa
‘Taoyuan No. 3’... 89 Table 7. Fruit morphological characteristics of Fragaria hayatae, white-fruited
strawberry, F. nilgerrensis, F. vesca, and F. ×ananassa ‘Taoyuan No. 3’... 91 Table 8. Results of principal component analysis performed on 19 vegetative
morphological characteristics among Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F. vesca, and F. ×ananassa ‘Taoyuan No. 3’ .... 92 Table 9. Results of principal component analysis performed on 19 vegetative
and 22 reproductive morphological characteristics among Fragaria hayatae, white-fruited strawberry, F. nilgerrensis, F. vesca, and F.
×ananassa ‘Taoyuan No. 3’ ... 93 Table 10. Primers and their polymorphism in the RAPD analysis... 95
Table 11. Effects of temperature and its duration on petiole length, leaf area, leaf formation rate, and runner formation rate in Fragaria hayatae ... 96 Table 12. Effects of temperature and its duration on flowering in Fragaria
hayatae ... 97 Table 13. Effects of day/night temperature and day length on number of leaves,
leaf area, petiole length, and number of runners in Fragaria hayatae... 97 Table 14. Effects of day/night temperature and day length on flowering in
Fragaria hayatae... 97 Table 15. Results of interspecific hybridization in Fragaria hayatae and F.
×ananassa ‘Taoyuan No. 3’ ... 98
Introduction
Fragaria hayatae Makino, also called as F. nilgerrensis Schlecht. ex J. Gay subsp.
hayatae in some studies, is the only endemic Fragaria species in Taiwan. Fragaria hayatae is characterized with anthocyanin in all parts of the plant (Hancock, 1999), which results in brownish red petiole, brownish red runner, red fruit, and purplish-red blush at the base of white petal. In a field trip collecting F. hayatae, one population of strawberry with green petiole, green runner, cream-white fruit and white petal was found by our lab in Shiaoshueshan (小雪山). We called the newly found strawberry
“white-fruited strawberry”.
Fragaria hayatae was recorded in “Flora of Taiwan” (Ohashi, 1993). However, it was classified as a subspecies of F. nilgerrensis by Staudt (1989; 1999), and in Flora of China (Li et al., 2003). According to the characteristics described by Staudt (1999), F.
nilgerrensis was characterized with yellowish, brownish or even reddish petiole, runner with or without anthocyanin, white to cream-colored fruit, and white petal, which is significantly different from F. hayatae. We considered F. hayatae to be distinct from F.
nilgerrensis. It may be that the white-fruited strawberries were brought by birds from China or they might be a mutant of F. hayatae or even a new species of Fragaria. In order to solve this problem, morphological characteristics of the white-fruited strawberry, F. hayatae, F. nilgerrensis and F. vesca were observed and surveyed, and the DNA marker of some diploid Fragaria species was analyzed to unravel the relationship and taxonomic position of F. hayatae and the white-fruited strawberry population.
Fragaria hayatae, being the only endemic Fragaria species of Taiwan, was never used in commerce despite its useful horticultural characteristics such as good taste
(Hayata, 1908) and significantly different odor from the cultivated strawberry. In order to understand the growth habit of F. hayatae, the effects of a series of temperature and photoperiod were studied.
Incorporation of traits from lower ploidy Fragaria species to the cultivated strawberry (octoploid) could be accomplished by artificially doubling chromosome numbers following interspecific hybridization (Hancock and Luby, 1993; Harbut and Sullivan, 2004). Hence reciprocal crosses were made between F. hayatae (diploid) and the cultivated strawberry ‘Taoyuan No. 3’ (octoploid) to study the interspecific hybridization compatibility.
Literature Review
1. Taxonomy and Distribution of the Strawberry Species
Strawberry belongs to the family Rosaceae, subfamily Rosoideae, tribe Potentilleae and genus Fragaria L. Among tribe Potentilleae, Duchsnea Smith and Potentilla L. are the closest to the genus Fragaria. Over 20 species (Table 1) are recognized in the Fragaria genus, which is divided into four fertility groups mainly associated with ploidy levels including diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), hexaploid (2n
= 6x = 42), and octoploid (2n = 8x = 56) species (Darrow, 1966; Hancock, 1999; Folta and Davis, 2006). The basic chromosome number in Fragaria is x = 7 (Staudt, 1989).
Fragaria species are distributed throughout the holarctic zone with a few species spread into the tropics (Fig. 1). With the exception of the diploid F. vesca that is distributed in both Eurasia and America, all species are confined to a single continent.
Central Asia and the Far East are the two centers of diversity for the diploid species.
The tetraploid species are limited to east and southeastern Asia. The only hexaploid species is originated in Europe. The octoploid species are mainly distributed in North and South America, but one species is endemic to the Far East (Southern Kuriles) (Staudt, 1989).
Six diploid Fragaria species were used in this study. Their characteristics are described as follow.
1.) Fragaria vesca L.
There are four subspecies in this group: subsp. vesca is distributed in woods of Europe and Asia; subsp. americana is distributed in woods of eastern North America to British Columbia; subsp. bracteata is distributed in woods of western North America;
and subsp. californica is distributed in California (Hancock, 1999).
Fragaria vesca is a perennial herbaceous plant. The plant height is 5-30 cm.
Leaves are green and 3-foliolate, rarely pinnately 5-foliolate. Shape of leaflets is obovate, elliptic or broadly ovate and 1-5 cm long. Inflorescences are corymbiform, with 2-4 (or 5) flowers. Inflorescences are about the same or taller than leaf petioles.
Flowers are bisexual and hermaphroditic, approximately 1.3 cm wide, white, and with 5 petals. Ovoid fruits are highly aromatic and flesh is soft. Although some white fruits form, most of the plants have red fruits when mature. Achenes are raised or superficial.
Runnerless forms exist (Hancock, 1999; Li et al., 2003).
2.) Fragaria viridis Duch.
This strawberry is distributed in Europe, eastern and central China and Canary islands.
Fragaria viridis is a perennial herb. Plants are 15-25 cm tall. Leaves are ternately compound, dark green, with ovate to elliptic leaflets. Inflorescences are erect with 4-10 flowers, and are often exserted above than leaves. Flowers are hermaphroditic and petals overlap. They are yellowish green when blooming then turn white. Fruits are obloid or globose, and the color is light green when mature. Fruits have firm texture and are fragrant. Achenes are yellowish green, in shallow pits or level with surface or superficial (Deng and Lei, 2005; Lei et al., 2006).
3.) Fragaria mandschurica Staudt
This species of strawberry is distributed in the Russian Far East, extending westward to Lake Bajkal, Mongolia, Manchuria, and North Korea.
Fragaria mandschurica is a perennial herb. Plants are 5.0-26.0 cm (x = 14.9) tall.
Leaves are ternately compound, bright green, and with rhombic-ovate to obovate cuneate leaflets. Terminal leaflets are 32.0-68.0 mm (x = 49.1) long, and length-width
index is 1.24-2.14 (x = 1.54). Inflorescences mostly surpass leaves, with 2.0-12.0 (x
= 4.9) flowers. Flowers are bisexual, hermaphroditic, and 13.0-29.0 mm (x = 20.5) in diameter with 5-7 petals (x = 5.2) which are white with yellow claw. Fruits are ovoid to broadly ovoid, 9.0-23.0 mm (x = 12.6) long, 8.0-26.0 mm (x = 11.2) wide, and color only in skin (Royal Horticultural Society (R. H. S.) Color Chart Red 45B-A to 46A when ripe). This strawberry has juicy flesh, melty texture, slightly acidulous with pleasing, and sometimes strong fruity flavour and taste. Achenes are yellow to light brown when mature, and they are in shallow pits or superficial. Fragaria mandschurica closely resembles the tetraploid F. orientalis (Staudt, 1989), but it can be morphologically distinguished by that the former is hermaphroditic and the latter is dioecious and trioecious (Staudt, 2003).
4.) Fragaria pentaphylla Lozinsk
This species is distributed in the Sino-Himalayan region.
Fragaria pentaphylla is a perennial herb. Plants are 2.3-29.0 cm (x = 12.3) tall.
Leaves are ternately compound, and dark green. The proximal part of petiole is mostly with two or rarely up to four accessory leaflets, which are smaller and less toothed than ternate ones. Shape of leaflets is elliptic to narrowly elliptic. Terminal leaflets are 1.0-5.0 cm (x = 2.9) long and the length-width index is 1.10-2.09 ( x = 1.59).
Inflorescences are mostly shorter than leaves, with 1-5 flowers (x = 2.1). Flowers are bisexual, hermaphroditic, and 13.0-27.0 mm (x = 21.4) in diameter, with 5 -7 white petals. Fruits are ovate to widely ovate, 7-24 mm long, 8-18 mm wide, and color only in skin (R. H. S. color chart: Red 44A and B and Red 45 B when ripe). Fruits are slightly juicy, with spongy texture, but nearly without prominent smell and taste, somewhat acidulous. Achenes are reddish brown when mature, deeply imbedded or in shallow pits.
There is a forma alba distributed on Mt. Gyala Peri and North of Tsangpo Gorge,
southeast Tibet. The fruits of this form have widely depressed ovoid shape, and the color is white to cream colored (R. H. S. Color Chart: Yellow-white 158 B-C when ripe). The taste of fruits is quite pleasant, sweetish caramel-like. Other characteristics vary in the range of the typical forma (Staudt and Dickoré, 2001).
5.) Fragaria nilgerrensis Schlecht.
This strawberry is mainly distributed in the temperate zone of mountainous regions of the Nilgiri Hills, southwestern India, the Khasi Hills, northeastern India, East Himalaya, northeastern Burma, northern Vietnam and Southwest and Central China.
Fragaria nilgerrensis is a perennial herb with 2.2-23.5 cm in height. Leaves are ternately compound, dark green. Terminal leaflets are subcircular or broadly obovate, 17.0-54.0 mm long, length-width index 0.61-1.74 (x = 1.17). Inflorescences are below, equaling or above the leaves, with 1-13 flowers (x = 8.5). Flowers are hermaphroditic, 13-20 mm (x = 16.4) in diameter, with 5 (occasional 6-7 in primary flowers) white petals. Fruits are subglobose to depressed subglobose or slightly conoidal, white to cream colored skin and flesh (R. H. S. Color Chart: Yellow 4D, Yellow 8D, Yellow 11D), slightly hairy, distinctly fruity, of a somewhat sweetish taste and strong fruity flavor. Achenes are yellowish brown, usually deeply imbedded. Runners are with or without anthocyanin (Staudt, 1999).
6.) Fragaria hayatae Makino
This species is not recorded from outside Taiwan.
Fragaria hayatae is a perennial herb with 2.5-15.7 cm in height. Leaves are ternately compound, terminal leaflets are 10.5-30.5 mm long, 10.0-23.5 mm wide, length-width indices are 1.05-1.40 (x = 1.18). Inflorescences are with 2-5 flowers, which are bisexual, 7-16 mm (x = 14.2) in diameter with 5 petals. Petals are white and clawed with anthocyanin. Fruits have pinkish to red skin (R. H. S. Chart: Orange Red
31 D, Orange Red 32 D, Orange Red 33 C and D), and are slightly hairy. The cortex and pith are white to cream colored. Fruits have no distinct taste and smell. Achenes are yellowish brown to reddish brown, and imbedded. Runners are usually with anthocyanin (Staudt, 1999).
2. Brief History of Strawberry Domestication
Fragaria vesca, the alpine or wood strawberry, was the first strawberry domesticated in the Old World. It was originally cultivated in gardens by Romans and Greeks. This strawberry was cultivated all across Europe by the 1300s and had reached its widest popularity in 1500s and 1600s before the introduction of strawberry species from the New World. It is now generally restricted to home gardens and most of the varieties grown are everbearers (Darrow, 1966; Hancock, 1999; Hancock et al., 2008).
Fragaria moschata, the musky-flavored strawberry was planted in European gardens by the late 15th century. Its fruit was used by English, Germans and Russians.
Fragaria viridis, the green strawberry, was also cultivated for ornamental use across Europe at that time. Neither F. moschata nor F. viridis is of current commercial importance (Darrow, 1966, Hancock, 1999, Hancock et al., 2008).
Fragaria virginiana, the Virginian or scarlet strawberry, was introduced from Canada and Virginia to Europe, possibly by Jacques Cartier, and it had thus replaced the dominant role of F. vesca in the 1600s. All clones imported to Europe were of wild origin as the North American aboriginals did not cultivate strawberries. The first F.
virginiana imported from Canada often bear small fruit and were green where the fruit was under shading. Those imported from Virginia made an impact in the horticulture industry. Because of their large fruit size, high yields and deep red color, F. virginiana was known as the scarlet strawberry (Hancock, 1999).
Fragaria chiloensis was domesticated 1000 years ago by indigenous Chilean
Mapuches and was spread widely by the Spanish during the colonization period (Hancock et al., 2008). One Chilean clone of F. chiloensis was brought to Europe in the early 1700s by Captain Amédée Frézier, a French spy, but the strawberries he introduced were all female and had insufficient hardiness to be widely grown. Fragaria chiloensis became more import after F. ×ananassa was accidentally born. It is currently grown in a small extent in Chile, although it was once grown widely, and it was replaced by F. ×ananassa since the late 1800s (Hancock, 1999; Hancock et al., 2008).
Unusual seedlings appeared in Brittany (a former province of France) and other gardens in Europe with unique combinations on morphological characteristics after introduction of F. chiloensis. The French botanist Antoine Nicholas Duchesne (1766) determined that they were hybrids of F. chiloensis × F. virginiana and named them as F.
×ananassa due to the fragrant of fruits similar to pineapple (Ananas). The dessert strawberry, F. ×ananassa, now dominates cultivation of the strawberries (Hancock, 1999).
3. Strawberry Species in Taiwan
Fragaria hayatae, the Hayata’s strawberry or Taiwan strawberry, is the only species of Fragaria native in Taiwan (Ohashi, 1993). It is endemic and does not belong to any threatened category of the IUCN list (Naruhashi et al., 1999). Taiwan strawberry is distributed at 2,000 to 3,700 m altitude in the Central Mountain Region, and it is usually found in somewhat moist, open places such as exposed slopes along road cut, hiking trails or mountain meadow (Naruhashi et al., 1999).
Fragaria hayatae was first discovered at Mountain Morrison (Yushan) by T.
Kawakami and U. Mori during their collection journey in October 1906. Kawakami noted “the fruit of this Fragaria is very delicious” (Hayata, 1908). The morphology was described, but the species name was not given due to incompleteness of specimen (lack
of flower) at this collection (Chen, 1995). It was later named as variety minor of F.
vesca by Hayata (F. vesca L. var. minor Hayata) (Kawakami, 1910) and its descriptions were recorded in “Materials for a Flora of Formosa” at 1911. The complete Latin description was given by Makino, who published it as F. hayatai Makino (Makino, 1912; Chen, 1995) and this was the scientific name recorded in “Flora of Taiwan”
(Ohashi, 1993).
The taxonomy and scientific name of the Taiwan strawberry was controversial.
Staudt (1999) examined herbarium specimens and live materials of F. nilgerrensis Schltdl. ex J. Gay and F. hayatae cultivated in Berlin, Cologne and Merzhausen, Germany. He addressed that F. hayatae should be a subspecies of F. nilgerrensis and made a combination of the Taiwan strawberry as F. nilgerrensis Schltdl. ex J. Gay subsp. hayatae (Makino) G. Staudt. The subspecies hayatae was distinguished by having anthocyanin in all parts of plants, even the berries (Staudt, 1989). The Taiwan strawberry was categorized as F. nilgerrensis var. nilgerrensis in Flora of China, although Ikeda and Ohba (two of the three editors of the Fragaria section of Flora of China) believed that the plants from Taiwan should be separated as F. hayatae, because they differd from F. nilgerrensis in having 1(-3)-flowered inflorescences and the shapes of petals are obovate to broadly obovate, white with a reddish purple base. Whereas F.
nilgerrensis had (1 or)2-5(or 6)-flowered inflorescences and the shape of petals were orbicular, white throughout (Li et al., 2003 ).
4. Breeding Potential of the Wild Strawberries
Since 1960, the majority of the genetic makeup of strawberry cultivars in North America came from only seven nuclear and ten cytoplasmic sources (Sjulin and Dale, 1987; Dale and Sjulin, 1990). This germplasm base also predominated in the international breeding programs (Hancock et al., 1993). Narrow genetic base may lead
to lethal inbreeding effects and lack of diversity to adapt to new environment (Hancock and Luby, 1993). The cultivated strawberry came from an accidental cross of F.
chiloensis and F. virginiana, but little use of the native germplasm has been made by breeders until recently (Hancock et al., 1993). Disease resistance, stress adaptability, and characteristics in wild strawberries can be used to expand the germplasm of the cultivated strawberry and improve its pleasant characteristics (Hancock and Luby, 1993). The key to use native germplasm in plant breeding is to catalog their horticultural useful traits (Hancock et al., 2003). Wild clones of Fragaria species were collected and evaluated for germplasm conservation and for utility in strawberry breeding.
Species with the same ploidy level can often be successfully crossed and all octoploid Fragaria species were completely interfertile (Hancock and Luby, 1993).
Fragaria chiloensis and F. virginiana, the octoploid species, which were the progenitors of cultivated strawberry, are the suitable materials for improving its genetic base. Although the lower ploidy strawberries were more difficult to be crossed with the octoploid F. ×ananassa (Hancock and Luby, 1993), they were valuable for holding potential to improve modern strawberry cultivars by introducing traits such as unique flavors and disease resistance while increasing genetic diversity (Harbut and Sullivan, 2004).
4.1. The octoploid species
The first step of utilizing wild strawberries should be collection of germplasm.
Possible useful traits could be speculated by the original habitat of the collection site.
Staudt (1999) and Hancock et al. (2001a) assumed that there were horticulturally useful genes in the native Fragaria since they distributed in a broad geographical range covering various biotic and abiotic stress environment. Native F. virginiana was
collected for representatives of North America from Pacific Northwest in 1985 and Northern Rocky Mountains in 1989 (Luby et al., 1992). The diverse habitat of which F.
virginiana was collected, ranged from dry pine forests to wet meadows. It suggested that certain accessions could possess resistance to drought or water-saturated soil. Some F. virginiana accessions were found near timberline where growing seasons were 6-8 weeks, with frost or snow occurring any time of the year indicating possible cold hardiness or blossom frost tolerant resource. Collections from sites with alkaline soils may be sources of higher pH (Luby et al., 1992). Similarly, F. chiloensis clones were collected with representatives of its wide geographical range and important horticultural traits like very large fruit, resistance to powdery mildew, red stele caused by Phytophthora fragariae, leaf spot, aphids, and two-spotted spider mites were observed (Hancock et al., 2001a). A bulk collection representing all octoploid strawberry species in North and South America was evaluated by Hancock et al. (2003). In this study, F.
chiloensis was generally superior in crown number, fruit weight, soluble solids and seed set while F. virginiana was superior for runner production, peduncle length, fruit number, fruit color, and winter hardiness.
After selecting elite accessions of the wild strawberries, the performance test should be conducted at various sites to affirm if the characteristics were truly genotypic or resulted from interaction between genotype and environment. The work of Hancock et al. (2001b) demonstrated that fruit characteristics could be assessed in one single site since the fruit weight, skin color, flesh color and firmness coincided throughout the five sites (Maryland, Oregon, Minnesota, Michigan, and Pennsylvania). However, multiple sites were necessary to predict physiological adaptations and disease resistance since the percentage bed fill, foliar disease incidence, 50% bloom date and number of flowering cycles showed interactions between genotype and location (Hancock et al., 2001b).
A subsequent step was to cross the elite accessions of wild strawberries with the cultivated strawberry or another elite wild strawberry accession for evaluation of the hybrid progeny and the transferability of traits from the parents. It was reported that fruits of hybrid from the cross of wild F. virginiana with F. ×ananassa were generally too soft and often irregular in appearance, but the fruits displayed high level of fertility, high flavor and highly productive behavior (Hancock et al., 2001a). Hancock et al.
(1993) and Luby et al. (2008) considered that reconstruction of F. ×ananassa using superior clones of F. chiloensis and F. virginiana might be more efficient than simply backcrossing to F. ×ananassa because the hybrid of wild strawberries possessed much higher proportion of unique genes that will be available for recombination in later generations.
4.2. The lower ploidy species
Fragaria species occupied various environments and they should carry some horticultural useful traits (Hancock et al., 2008). Fragaria vesca (2x) was often found in the same habitats as F. virginiana but frequently occupied drier and coarser sites where F. virginiana was absent. It suggests that F. vesca might be a source of extreme drought tolerance (Luby et al., 1992). F. moschata (6x) was found under heavy shade (Hancock and Luby, 1993). Harbut and Sullivan (2004) indicated that a species adapted to shade might maintain higher CO2 assimilation rate, and it could be beneficial for production in greenhouse, low light areas and high plant populated areas.
Disease resistance can be found in some lower ploidy Fragaria species. Resistance to Phytophthora cactorum (crown rot or leather rot on fruit) was found in several F.
vesca clones and its hexaploid and decaploid derivatives indicated that the wood strawberry might be a source of crown rot resistance and this ability was inheritable (Gooding et al., 1981). Xue et al. (2005) screened eleven non-octoploid Fragaria
species for resistance to Xanthomonas fragaria Kennedy and King, the bacterial angular leaf spot which may reduce yield for up to 75%. Their results indicated that some accessions of F. pentaphylla (2x) and F. moschata (6x) either showed no symptoms (highly resistant), hypersensitive reactions (resistant), or restricted water-soaked lesions (moderately resistant) and the two species harbored diversified resistant source. Bors and Sullivan (1997) observed immunity to aphids and leaf diseases in F. nilgerrensis, and winter hardiness and excellent leaf disease resistance in F. moschata.
Some useful fruit characteristics can be found in the lower ploidy strawberries.
Fruit of F. viridis (2x) had a spicy, cinnamon-like flavor (Bors and Sullivan, 1997); the flavour of mature fruit of F. nilgerrensis (2x) was described as similar to melons or peaches (Oda et al., 1990 in Noguchi et al., 2002), apricots and/or bananas (Staudt et al., 1975); F. moshchata tasted like ‘Concord’ grape when grown in the greenhouse (Bors and Sullivan, 1997). These strawberries could add new elements to typical strawberries and have the potential in aroma breeding. Fragaria pentaphylla had very bright red and firm fruits (Bors and Sullivan, 1997) which would be ideal for strawberry shipping (Harbut and Sullivan, 2004). Fragaria nilgerrensis subsp. hayatae (F. hayatae) was found to have anthocyanins in all parts of the plant (Hancock, 1999), and this unique characteristic may lead to high antioxidant capacity (Harbut and Sullivan, 2004).
4.3. Crossbility
1.) Crosses between diploids
Four subspecies of F. vesca were used as female parent to cross with four diploid Fragaria species (F. nilgerrensis, F. nubicola, F. pentaphylla and F. viridis) in the study of Bors and Sullivan (2005a). Although the rate of fruit set varied from 39% (F.
vesca × F. nilgerrensis) to 89-100% (other combinations), hybrids were obtained from all combination (Bors and Sullivan, 2005a), indicating that crosses between these
diploid species were generally successful.
2.) Crosses between diploids and hexaploids
Interspecific hybridization was administrated by Evans (1974) using F. vesca, F.
viridis, F. nubicola, F. nilgerrensis, and F. (vesca × viridis) as female plants to cross with F. moschata (6x) and only two seedlings were obtained from the 43 pollinated flowers and the two seedlings died before true leaves formed. This result indicated crossing barriers between these species. However, the diallel crosses of F. moschata with F. nubicola and F. viridis in the study of Bors and Sullivan (2005b) were more successful. They got 1.4 healthy plants/ pollination in the combination of F. moschata × F. viridis, 3.3 healthy plants/ pollination in F. nubicola × F. moschata and 0.1 healthy plants/ pollination in F. viridis × F. moschata. The success rate was raised probably because the germination technique was improved using in vitro culture (Bors and Sullivan, 2005b).
3.) Crosses between diploids and octoploids
Interspecific hybridization was conducted in numerous studies and clear crossing barriers were observed (Evans, 1974; Li et al., 2000; Marta et al., 2004). In F. × ananassa ‘Honeoye’ × F. vesca ‘Changsen’, a relative low number of hybrid seedlings (84 hybrid seedlings from 303 seeds) was obtained. The germination rate of F. vesca pollen on F. ×ananassa stigmas was low, some germinated pollens did not penetrate the stigma. Elongation of pollen tubes in the style was irregular and the growth of embryos and endosperms was aberrant (Li et al., 2000). In the study of Marta et al.
(2004), pollen tube growth was observed to arrest in the first-third of the style and it produced only two aborted seeds in the cross of F. ×ananassa and F. vesca. In the reciprocal cross, 35 seeds were obtained, but the germination rate was only 14% (5 seeds) and seedlings died shortly after germination (Marta et al., 2004). The work of Li
et al. (2000) and Marta et al. (2004) revealed pre-zygotic and post-zygotic barriers between the interspecific hybridization of octoploids and diploids.
The above studies suggest that crosses in the same ploidy level are much easier than interploidy hybridizations. Although species at lower ploidy levels were more difficult to cross with F. ×ananassa, they had not been ignored by plant breeders (Hancock and Luby, 1993). Incorporation of traits from lower ploidy Fragaria species into the cultivated strawberry had been accomplished by artificially doubling chromosome numbers and making numerous crosses (Hancock and Luby, 1993).
Evans (1977) came up with the system called synthetic octoploid (SO system) in which germplasms of 2x, 4x, and 6x Fragaria species were incorporated into octoploid hybrids. In this system, Fragaria species of the lower ploidy were crossed to obtain tetraploid hybrids and the hybrids were further treated with colchicine resulting in octoploid hybrids that contained various germplasms (Evans, 1977; Harbut and Sullivan, 2004). It bypassed ploidy level differences and facilitated introgression of 2x, 4x and 6x species into the cultivated strawberries (Evans, 1977; Bors and Sullivan, 2005).
The use of this method has led to two SO clones, Guelph SO1 (Evans, 1982a) and Guelph SO2 (Evans, 1982b). Guelph SO1 originated from colchicine treated tetraploid hybrids between F. moschata (6x) and F. nubicola (2x). It was a staminate clone possessing late flowering, upright flower stalks, high number of flower stalks and its flavor, aroma and flesh color of fruit were distinctive to F. moschata (Evans, 1982a).
The origin of Guelph SO2 came from crossing the amphidiploid (4x) hybrid of F. vesca (2x) and F. viridis (2x) with F. moupinensis (4x) and the chromosome of the interspecific hybrid was doubled again to form the synthetic octoploid strawberry. SO2 was a staminate clone and had a reasonable resistance to powedery mildew, leaf scorch and leaf blight (Evans, 1982b). SO1 and SO2 were evaluated by means of outcrossing
(recurrent selection) for their potential to contribute horticultural useful traits in strawberry improvement. The yield and berry weight of some hybrid progenies were improved to be as good as or greater than the average of the check cultivars within 3-5 generations (Sangiacomo and Sullivan, 1994).
Fragaria ×ananassa ‘Toyonoka’ (female parent) and F. nilgerrensis ‘Yunnan’
(male parent) were crossed, their hybrid chromosome number was doubled and then backcrossed to F. ×ananassa ‘Pajaro’ in the study of Noguchi et al. (2002) in order to breed a new aromatic strawberry. This hybrid strawberry performed aroma of peach, light pink skin and soft flesh and was registered as Kurume IH No.1 in Ministry of Agriculture, Forestry and Fisheries of Japan at 2005. Some decaploid strawberries had been produced and released from crosses of F. ×ananassa and F. vesca, namely
‘Spadeka’, ‘Annelie’ and ‘Sara’ (Bauer, 1979 and Trajkovski, 1997).
The works of Bauer (1979), Evans (1982a, 1982b), Sangiacomo and Sullivan (1994), Trajkovski (1997) and Noguchi et al. (2002) verified possibility to incorporate germplasms of lower ploidy Fragaria into the cultivated strawberries via using the species of higher ploidy level as female parent when possible, or try to increase the chromosome complement of the species at lower level, or to use means such as embryo culture mentioned by Evans (1974).
5. Application of Morphological Traits in Fragaria Species
It was reported that morphological characteristics can be used for determining interspecific relationship (Harrison et al., 1997; Sargent et al., 2004), intraspecific relationship (Catling and Porebski, 1998) and cultivar identity (Dale, 1996; Nielsen and Lovell, 2000). The guidelines of International Union for the Protection of New Varieties of Plants (UPOV) provide detailed descriptions covering general habit, leaf, flower and fruit characteristics of strawberries (Annonymous, 1995; Annonymous, 2008) which all
are useful for plant breeders.
Forty-four morphological traits were evaluated on three subspecies of F. virginiana (subsp. glauca, subsp. platypetala and subsp. virginiana) and F. chiloensis subsp. lucida collected in North America. The results of principal component analysis based on genetic distances successfully separated the four subspecies into four distinct groups (Harrison et al., 1997). Sargent et al. (2004) examined quantitative and qualitative morphological characteristics of eight diploid Fragaria species (F. daltoniana, F.
iinumae, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla, F. viridis and F.
vesca). After summarizing the 14 quantitative characteristics by the principal component analysis, the diploid Fragaria species were separated into three distinctive groups, F. vesca, F. nilgerrensis and the rest of Fragaria species. Although the quantitative morphological characteristics did not give clear resolution to some species, they could be distinguished by qualitative characteristics such as bright pink fruit of F.
daltonia and tertiary leaflets of F. pentaphylla (Sargent et al., 2004).
Fourteen morphological characteristics were used to measure 95 plants representing the four subspecies of F. chiloensis (subsp. lucida and subsp. pacifica from North America, subsp. chiloensis from South America and subsp. sandwicensis from Hawaii) in order to provide a better resolution to the intraspecific relationship. The Hawaiian subspecies, F. chiloensis subsp. sandwicensis was entirely distinct from the other subspecies in having longer leaflets and longer hairs on the undersurface of the leaflets and more numerous leaflet veins. The South American subsp. chiloensis differed from the North American subspecies in having mostly 6-10 petals whereas the latter have 5-6 (rarely 7) petals (Catling and Porebski, 1998).
To distinguish 32 common strawberry cultivars grown in North America, a key of mostly vegetative characteristics was developed by Dale (1996) based on observation
and the UPOV guideline. The key was aimed for initial identification of varieties when fruits and flowers are not available. Nielsen and Lovell (2000) indicated that vegetative characteristics (leaf blistering, length: breadth ratio, base shape and teeth shape of the terminal leaflet) and reproductive characteristics (petal spacing, length : breadth ratio of petal, calyx : corolla ratio, fruit size, fruit length : breadth ratio, fruit shape, width of band without achenes on fruit, insertion of achenes on fruit and insertion of calyx on fruit) could be used for identifying strawberry cultivars cultivated in Auckland, but it is insufficient to use the vegetative or reproductive characteristics alone for identifying cultivars since they may share either floral or leaf characteristics.
6. Application of RAPD Markers in Fragaria Species
Expression of morphological traits could be affected by environmental factors such as climate conditions or cultivation procedures (Hu, 2001; Kuras, et al., 2004). While traditional identifications of cultivars were based on morphological traits, Nielsen and Lovell (2000) had pointed out the necessity of using alternative method for definitive identification such as molecular markers because it was difficult to put together a key based only on morphological markers. The dendrogram generated from morphological traits and molecular traits of strawberry cultivated in Argentina were not correlated (García et al., 2002) also demonstrated morphological traits alone was not sufficient in differentiating cultivars.
Analysis of molecular markers could be assessed at any developmental time and any organ, only few samples are needed for multiple analysis, DNA samples could be conveniently kept for long term use and procedures are standardized (Lin, 2001).
The technique of random amplified polymorphic DNA (RAPD) was first reported by Williams et al. in 1990. The DNA fragments were amplified by polymerase chain reaction (PCR) using short synthetic primers (generally 10 bp) of random sequence. The
amplified products were separated by gel electrophoresis and polymorphisms were detected as the presence or absence of particular band size. RAPD is quick and easy to assay. Only low quantities of template DNA is required since PCR is involved. No sequence data is needed because the random primers are commercially synthesized and available. The primer length is short and the annealing temperature is relative low which might result in mismatch of primer and sequence when complementary sequence does not exist, therefore the experiment should be repeated to check consistency.
Standardized experimental procedures are required to prevent the low reproducibility nature of RAPD. The markers of RAPD are dominant and non locus-specific, the bands can not be interpreted in terms of loci and alleles, and fragments of similar size may not be homologous (Lin, 2001; Hu, 2001; Spooner et al., 2005).
RAPD markers were reported to be useful for determination of genetic relationship in the genera level or the species level. In a study analyzing relationship between Fragaria, Potentilla and Duchesnea of northwest Argentina, the phenogram obtained from RAPD characteristics revelaed that F. vesca and F. ×ananassa cluster together, whereas Potentilla tucumanensis and Duchesnea indica form a separate cluster. These results were identical to the phenogram generated from morphological and anatomical characteristics (Ontivero et al., 2000). Three subspecies of F. chiloensis were evaluated for their varation and genetic relationship by RAPD in the study of Porebski and Catling (1998). The results indicated a clear division between the North American subspecies and the South American subspecies by the similarity index of 0.16, on the other hand, the two North American subsp. lucida and subsp. pacifica were less separated from the similarity index of 0.64-0.88.
There were reports about application of RAPD markers used for cultivar identification in Fragaria species. Thirteen RAPD primers generating 37
genotype-specific bands could be used for cultivar identity for six main varieties of F.
×ananassa (‘Camarosa’, ‘Sweet Charlie’, Selva’, ‘Milsei Tudla’, ‘Chandler’, and
‘Pájaro’) cultivated in Argentina (García et al., 2002). When RAPD and ISSR (Inter Simple Sequence Repeat) were assessed for suitability of determining strawberry relationship, cultivars sharing the same pedigree were usually clustered together in the dendrogram generated from both of the molecular markers (Kuras et al., 2004). In the study of Milella et al. (2006), 65 genotypes of strawberry collected from Etna mountain of Italy and one cultivar ‘Madane Moutot’ which was possibly the ancestral genotype of the Etna strawberries were evaluated and their close genetic relationship was assured via RAPD markers.
RAPD markers were found to be linked to specific genes and these markers could facilitate the breeding process (marker-assisted breeding). Two Japanese culativars
‘Ever Berry’ (everbearing type) and ‘Toyonoka’ (Junebearing type) and their F1 cross hybridization progeny were analyzed for RAPD markers linked to the everbearing gene (controlled by a single dominant gene), and five markers were found to construct a likage map, with OPE07-1 and OPB05-1 being closest to the everbearing gene (Sugimoto et al., 2005). Seven RAPD markers were found to be linked to the Phytophthora fragariae resistant gene (Rpf1) in the cultivated strawberry by bulk segregant analysis, with OPO-08A and OPO-16A being closest to the Rpf1 gene (Haymes et al., 1997).
7. Growth and Development of Strawberry
Strawberry is a perennial plant usually described as herbaceous but was actually a true woody plant by the evidence of secondary xylem in roots and crowns (Darnell et al., 2003). The plant body is comprised of a rosette central stem or crown from which leaves, roots, runners (stolons) and inflorescences emerge (Hancock, 1999). The crown
is terminated by a bud generally containing 5 to 7 developing leaves enclosed within the stipules of the last emerged leaf (Guttridge, 1985). At the top of each leaf along the crown is an axillary bud (Hancock, 1999) which produce runners under long day conditions and develop into branched crowns or remain dormant under short day conditions (Konsin et al., 2001). Inflorescences emerge from the apical meristem of the crown while the uppermost axillary bud continues the vegetative extension of the crown (branched crowns). Flower initiation may occur in the branched crowns when there are more than two leaf primordia and the environment is flower-inductive (Guttridge, 1985;
Hytönen et al., 2004).
The cultivated strawberry was traditionally categorized by fruiting behavior as Junebearers and everbearers. Junebearers produce a single flush of flowers every year (single cropping). Everbearers produce several flushes of flowers in the growing season (multiple cropping). The flowering of strawberry is greatly influenced by temperature and photoperiod. Photoperiod requirement for flowering in strawberry can be divided into three groups which were long day plants, short day plants and day-neutral plants.
Junebearers are considered as facultative short day plants, with flower initiation under short day if the temperature is above about 15℃ or irrespective of the day length if the temperature is below 15 ℃ (Guttridge, 1985; Hancock, 1999; Taylor, 2002).
Everbearers are considered as long day plants because their flowers initiate under long day conditions when temperature is moderate (Hancock, 1999; Darnel et al., 2003).
Day-neutral plants were introduced by Bringhurst and Voth (1980) (in Taylor, 2002) to describe multiple cropping cultivars derived from crosses between Junebearing types and F. virginiana subsp. glauca (everbearing type). Day-neutrals are relatively insensitive to day length for flower initiation (Darnel et al., 2003). Strict categorization is difficult due to the continuum of photoperiodic responses observed in different
genotypes (Darnell et al., 2003).
The physiology of the perennial cycle in June-bearing strawberry was described by Battey et al., (1998). The temperature was cool and the day length was short after August, hence vegetative growth gradually slowed down and flower initiation was triggered. While the temperature got colder in November, strawberry plants underwent a dormant period until next spring, when the temperature was warm and the day length was long, the vegetative growth was recovered and the flower buds which were already differentiated bloomed and set fruit through March to August.
Wen (1984) had reviewed that strawberry seedlings are able to accept the stimulus of short day and low temperature for flower differentiation when there are more than four true leaves and for runner plants, there should be more than four to five expanded leaves. In the study of Verheul et al. (2006) comparing the performance of F. × ananassa ‘Korona’ at 4, 8 or 12 weeks of plant age after flower inductive treatment, flowers were induced in 4-week-old runner plants with only three to four leaves. The number of inflorescence and total number of flowers increased as the plant age was increased.
Sønsteby and Nes (1998) investigated the critical number of short day cycles necessary to induce flower in four cultivars of strawberry (‘Korona’, ‘Elsenta’, ‘Bounty’
and ‘Senga Sengana’) under three temperature region (9℃, 15℃ and 21℃). For
‘Korona’ and ‘Elsanta’, 16 short day cycles were required for flower initiation when the temperature was 15℃, while longer short day cycles were required for lower and higher temperatures. In ‘Bounty’ and ‘Senga Sengana’, flowers were inducted almost irrespective of short day treatment. Twelve days of inductive cycle was required for
‘Chandler’ under 9 hours of short day and 16℃, while a longer inductive cycle of 19 days was required under night interruption (Zhang et al., 2000). Verheul et al. (2006)
investigated the optimum short day cycles (14, 21 and 28 days) for flower induction in
‘Korona’ and found that 28 short day treatment resulted in highest number of inflorescences and flowers per plant.
Materials and Methods
1. Morphological Variations among Fragaria hayatae, White-fruited Strawberry and other Fragaria Species
1.1. Plant materials and management
Ten accessions of F. hayatae (H1, H2, H4-8, H10, H12 and H13), one accession of white-fruited strawberry, F. nilgerrensis, F. vesca, F. ×ananassa were used in this experiment. F. hayatae and white-fruited strawberry were collected from central mountain regions of Taiwan during 2006-2008 (Table 2). All strawberries were obtained as runner plants except F. vesca, which was grown from seeds purchased from Known-You Seed Company (Kaohsiung, Taiwan). The plants were cultivated in the outdoor bench of Department of Horticulture, National Taiwan University. All strawberries were planted in 3 in. red plastic pots with King Root Plant Medium #1 : peat moss =1:1 as medium. Bagasse compost (Taiwan Sugar Co., Tainan, Taiwan) was mixed in the medium in 1:10 by volume. Slow release fertilizer Hi-Control No. 1 (N:P:K=14:12:14, Taiwan Horticultural Co., Ltd, Taipei, Taiwan) was used one month after the runner plants rooted. The plants were fertilized with 1000× soluble fertilizer Wonder Grow (N:P:K=20:20:20, Taiwan Horticultural Co., Ltd, Taipei, Taiwan) every week and irrigated with tap water when needed. 500× potassium bicarbonate (KHCO3, AG168 Co., Ltd., Taipei, Taiwan) and 500× narrow range oil (paraffin oil, AG168 Co., Ltd., Taipei, Taiwan) were sprayed when needed to prevent powdery mildew, spider mites and aphids.
1.2. Characteristic measurement
Morphological characteristics and conduction method were referred from UPOV document TG/22/9 (1995), TG/22/10 (2008) and Sargent et al. (2004). The characteristics and standards of measurement were listed in Table 3 and 4. More than ten plants from each collection were used for observation.
1.3. Data analysis
The means of the characteristics were compared to test for significant difference using General linear model (GLM program) and Duncan’s multiple range test (SAS version 8.01, SAS Institute Inc., Cary, N.C., USA). For clustering analysis, these data were first standardized by subtracting the accession mean from the grand mean for each characteristic then divided by the characteristic standard deviation. Euclidean distant coefficient matrix was derived from the standardized morphological characteristic means. The matrix was analyzed using the unweighted pair-group method with arithmetic mean (UPGMA) (SAHN clustering program, NTSYS-pc version 2.11L, Applied Biostatistics Inc., NY, USA). For principle coordinate analysis, the standardized morphological characteristic matrix were used to derive Pearson product-moment correlation coefficient matrix from which the principle components were extracted and projected in two or three dimensions (EIGEN, PROJ, MXPLOT and MOD3D programs, NTSYS-pc version 2.11L, Applied Biostatistics Inc., NY, USA).
Euclidean distant coefficient:
Eij =
∑
k(
Xki −Xkj)
2Eij : the taxonomic distance between individual i and j Xki: the value of the kth variable of individual i Xkj: the value of the kth variable of individual j
2. Molecular Variations among Fragaria hayatae, White-fruited
Strawberry, and Other Fragaria Species 2.1. DNA samples
Fourteen accessions of F. hayatae Makino and one accession of white-fruited strawberry collected from central mountain regions of Taiwan during 2006-2008 were listed in Table 2. F. vesca L. seedlings were sown from the seeds, which were purchased from Known-You Seed Co. (Kaohsiung, Taiwan). F. ×ananassa ‘Taoyuan No. 3’ was purchased from Shen-Neng Chang’s farm at Hsinchu, Taiwan. DNA samples of F. nilgerrensis Schlecht., F. mandschurica Staudt, F. pentaphylla Lozinsk.
and F. viridis Duch. were provided by Institute of Horticulture, Jiangsu Academy of Agricultural Science, Nanjing, China. Potentilla matsurae was gathered from Hohuanshan and included in this study as outgroup.
2.2. DNA Extraction
DNA samples were extracted by a modified CTAB method (Torres et al., 1993;
Lin, 2004). One or two young leaves (ca. 0.1 g fresh weight) were cleansed with RO water and pat dried by paper towel and fixed with liquid nitrogen. Liquid nitrogen fixed leaves were grounded into fine powder with ceramic mortar and pestle. 1 ml of grinding buffer (2% (w/v) CTAB, 0.1 M Tris-HCL pH 8.0, 20 mM Na-EDTA pH 8.0, 1.4 M NaCl, 0.4% (w/v) β-mercaptoethanol) was added into the mortar before the sample thawed and continued grinding until material became slurry. The slurry was transferred into 1.5 ml eppendorf, inserted into Styrofoam float and incubated in 60℃ water bath for one hour, then removed from water bath and let cool to room temperature. Sufficient (ca. 800 µL) chloroform: isoamyl alcohol solution (24:1) was added into the eppendorf.
The eppendorf was vortexed until color of mixture appeared uniform, and was centrifuged at 10,000 x g for 5 minutes to separate phases. The upper (aqueous) phase
