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Glauca Wasatch in F.×ananassa by repeated back-crosses (Hancock, 1999). Nevertheless, the considerable variation in flowering magnitude (inflorescence ...
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Hortic. Environ. Biotechnol. 58(6):585-590. 2017. DOI 10.1007/s13580-017-0373-0

Research Report

Segregation Ratio in Selfed and Crossed Progenies Demonstrates Single Dominant Gene Inheritance of Day-neutrality in Strawberry Jiwei Ruan1,2†, Chunmei Yang1,2†, Guoxian Wang1,2, Lifang Wu1,2, Shenchong Li1,2, Pang Tao3, Chengfei Liao2,4, Young Rog Yeoung5*, and Jihua Wang1,2* 1

Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kumming 650205, Yunnan, China National Engineering and Technology Research Center for Ornamental Horticulture, Kumming 650205, Yunnan, China 3 Horticulture Research Institute, Yunnan Academy of Agricultural Sciences, Kumming 650205, Yunnan, China 4 Research Institute of Tropical Eco-agricultural Sciences, YAAS, Yuanmou 651300, Yunnan, China 5 Department of Plant Sciences, Gangneung-Wonju National University, Kangnung 210-702, Republic of Korea

2

*Corresponding author: [email protected], [email protected] † These authors contributed equally to this work

Received December 18, 2016 / Revised April 27, 2017 / Accepted May 9, 2017 Ⓒ Korean Society for Horticultural Science and Springer 2017

Abstract. Studies on the inheritance of day-neutrality, a highly desirable trait in strawberry (Fragaria×ananassa), have not been conclusive because flowering phenotypes were evaluated based on different materials and experimental sites. In the present study, selfed and crossed progenies of Day-neutral (DN) ‘Albion’ and June-bearing (JB) ‘Akihime’ cultivars, namely ‘Albion’ × ‘Albion’, ‘Akihime’ × ‘Akihime’, and ‘Akihime’ × ‘Albion’, were established in 2013 and 2014. The flowering phenotype (DN or JB) of seedlings from each of the three progenies, as well as of parental plants, was evaluated for two successive years (2015 and 2016) in Central Yunnan, China, and their segregation ratios were used to elucidate day-neutrality inheritance. Single dominant gene inheritance was demonstrated for day-neutrality, although minor genes (from the JB parent ‘Akihime’) likely increase DN magnitude. During the juvenile stage (i.e., long before the first inflorescence), the major gene controlling day-neutrality may not be expressed. We provide basic information for DN gene mapping and cloning, as well as materials (progenies) for domestic DN strawberry breeding. Additional key words: day-neutral, Fragaria×ananassa, photoperiod-independent flowering, segregation ratio, seedlings, single dominant gene inheritance

Introduction Day-neutral (DN) strawberry (Fragaria×ananassa) is cultivated in annual, multiple cropping. Inflorescences are formed irrespective of the photoperiod (day-neutrality), and plants keep flowering and fruiting between 4°C and 29°C (Durner et al., 1984; Ahmadi et al., 1990). Therefore, DN cultivars are used in Summer and Autumn fruit production worldwide (Ballington et al., 2008; Ruan et al., 2011b, 2013a). In Central Yunnan (Latitude: 24°19′ to 27°03′ N; elevation: 1,700 to 2,300 m), about 700 ha of DN strawberry are cultivated annually, providing more than 50% of the fresh strawberries produced in China during Summer and Autumn. The day-neutrality character of DN strawberries was selected from its wild relative Fragaria virginiana ssp. glauca Staudt (Galletta et al., 1981; Durner et al., 1984). Inheritance of

day-neutrality has been intensively studied and several models have been suggested, ranging from single dominant gene (Ahmadi et al., 1990; Sugimoto et al., 2005) to polygenetic (Shaw, 2003; Serce and Hancock, 2005). Model of inheritance based on a major locus with large effects and minor quantitative trait loci (QTL) with additive effects (Shaw and Famula, 2005; Weebadde et al., 2008; Gaston et al., 2013) has also been proposed. The different models suggested may result from these studies being conducted at different sites and using different materials and crossing mates. Genotypic and environmental factors, mainly temperature, have interactive effects on the flowering of DN strawberry plants (Hancock et al., 2002; Hancock and Weebadde, 2008; Weebadde et al., 2008). Hancock et al. (2002) and Weebadde et al. (2008) found that differences in the percentages of DN seedlings obtained from identical progenies in different experimental sites may be due to temperature differences

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Jiwei Ruan, Chunmei Yang, Guoxian Wang, Lifang Wu, Shenchong Li, Pang Tao, Chengfei Liao, Young Rog Yeoung, and Jihua Wang

between sites. Hancock et al. (2002) and Honjo et al. (2011) showed that different DN materials and crossing mates resulted in different percentages of DN seedlings and different flowering magnitudes. Previous studies showed that the fruits produced by the ‘Albion’ cultivar have the highest sugar content among DN cultivars (Ruan et al., 2013b), and the greatest number of other desirable traits, including firm fruits (Ruan et al., 2013a). The typical June-bearing cultivar ‘Akihime’ also produces fruits with high sugar content but carries some weak traits, such as fruit softness and high susceptibility to powdery mildew. In the present study, selfed progenies of DN ‘Albion’ and JB ‘Akihime’ cultivars and the progeny of ‘Akihime’ × ‘Albion’ were used to elucidate the inheritance model of day-neutrality under the typical climate of Central Yunnan. Specific seedling traits such as inflorescence timing and leaf number between Spring and Summer inflorescences, have been used to identify DN seedlings at an early stage (Honjo et al., 2011). Recently, Gaston et al. (2013) showed that the dominant gene controlling day-neutrality also controls vegetative growth, including crown and runner forming. Thus, in the present study we surveyed inflorescence timing, crown number, runner number, and leaf petiole length of the seedlings resulting from ‘Akihime’ × ‘Albion’ progeny, to select morphologic traits that may be used to identify DN plants at an early stage. The results of this study, developed using specific crossing mates and under this specific climate, will facilitate the domestic breeding of DN cultivars and contribute fundamental information for future genetic studies aiming to map and clone genes controlling inflorescence timing and yield.

Materials and Methods Establishment of the Three Progenies Thirty JB ‘Akihime’ and 30 DN ‘Albion’ plants were placed in a table-top substrate culture system at the National Engineering and Technology Research Center for Ornamental Horticulture farm (Latitude: 24°18′ N, elevation: 1,760 m), Yuxi city, Yunnan province, China. These plants received complete nutrient solution through a drip irrigation system (emitters at 250 mm spacing, flow capacity of 2 L·min-1 per 30 m tube length; Ruan et al., 2013a). Controlled selfing and crossing of the two cultivars (using ‘Albion’ as the pollen donor) occurred when plants were 3-4 months old. Seeds were harvested when the fruits were fully ripe (40-50 days after pollination) and dried at 40°C for 72 h in a BGZ-240 oven (Boxun, Shanghai, China). After scarification with sulfuric acid for 10 min (Ito et al., 2011), seeds were sown in plug trays filled with peat moss (Jiffy, Moerdijk, Netherlands) and germinated for 20 days

from March to April, 2014. Emerging ‘Albion’ × ‘Albion’ (n = 109), ‘Akihime’ × ‘Akihime’ (n = 132), and ‘Akihime’ × ‘Albion’ (n = 828) seedlings were planted in gutters in May, 2014 and maintained in a table-top substrate culture system (Ruan et al., 2013a) until the evaluation of their morphologic traits and flowering phenotypes. Runner plantlets of seedlings belonging to the three progenies were rooted in pots as described by Ruan et al. (2011a) in September and early October, 2015. Potted runners were planted in gutters and maintained in the table-top substrate culture system described above until the evaluation of their flowering phenotypes. The flowering phenotypes of seedlings that failed to generate potted runners (102 out of the 818 surviving ‘Akihime’ × ‘Albion’ seedlings, and 14 out of the 102 surviving ‘Albion’ בAlbion’ seedlings) were inferred from corresponding older plants sown. Potted ‘Albion’ and ‘Akihime’ individuals were planted in gutters (100 individuals of each cultivar) and maintained in the table-top culture system described above until their flowering phenotypes were evaluated. Two replicates were performed (2014 and 2015). As the temperature at the experimental site can affect flowering phenotypes, temperatures were recorded at 30-min intervals during the growing seasons using a temperature data logger sensor TP-2200 (a-volt, Beijing, China). Morphologic Traits of the ‘Akihime’ × ‘Albion’ Progeny Lateral crowns and runners were removed from each of the 483 out of the 824 surviving ‘Akihime’ × ‘Albion’ seedlings planted and their numbers were recorded. The number of days from planting to inflorescence emergence was also recorded for each of the 483 seedlings, and the petiole length of their youngest fully-expanded leaf was measured. Flowering Phenotyping Seedlings of the three progenies were maintained for five months for flowering phenotyping and cloned plug plant generation. Flowering phenotypes were evaluated by assigning a subjective flowering score and by counting the number of recently-emerged inflorescences per seedling during late Summer (July to August) 2015 and 2016. Newly-emerged inflorescences were removed and their numbers were recorded at seven-day intervals. A 1-5 flowering score was assigned using the modified method of Shaw and Famula (2005): 1 = more than six recently-emerged inflorescences; 2 = four or five recently-emerged inflorescences; 3 = two or three recently-emerged inflorescences; 4 = one recently-emerged inflorescence; and 5 = no recently-emerged inflorescences.

Hortic. Environ. Biotechnol. 58(6):585-590. 2017.

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Segregation of Day-neutrality Plants in the three progenies presenting only one or no recently-emerged inflorescences (flowering score = 4 or 5) during late Summer (July-August, 2015 and 2016) were considered JB plants, whereas those presenting other flowering scores were considered DN plants. Under the null hypothesis of genetic control by a single dominant locus, the allele for day-neutrality is dominant over the allele for short-day flowering. Since ‘Albion’ is considered heterozygous, the selfed progeny of DN parents, the selfed progeny of JB parents, and the crossed progeny were expected to fit 3:1, 0:1, and 1:1 DN:JB genotype ratios, respectively. Chi-square statistics were calculated to test whether the segregation ratio of day-neutrality fitted these expectations. Morphologic Identification of DN Seedlings at Early Stages Morphological indicators of DN at early stages were screened in seedlings from ‘Akihime’ × ‘Albion’ progeny. Pairwise correlations between the number of lateral crowns, the number of runners, the number of days from planting to inflorescence emergence, and the petiole length of the youngest fully-expanded leaf, and the trait representing DN (i.e. number of inflorescences) were obtained using SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, USA).

Fig. 1. Frequency distribution of the 483 ‘Akihime’ × ‘Albion’ seedlings, according to the time of emergence of their first inflorescence in late 2014 and early 2015.

Results and Discussion

positively affected the day-neutrality trait, while negatively affecting some vegetative traits such as runnering. Earlier studies (Nicoll and Galletta, 1987; Bradford et al., 2010) also showed that DN genotypes produced fewer runners and more crowns than JB genotypes. Furthermore, differences in leaf number between Spring and Summer inflorescences have been used to identify DN seedlings at an early stage (Honjo et al., 2011). However, in our study the number of runners and crowns did not differ between DN and JB seedlings. This may be due to the four morphological traits being evaluated long before the first flowering, i.e., at the juvenile stage (Jahn and Dana, 1970). The lack of differences in crowning and runnering between DN and JB seedlings may result from lack of expression of the major gene controlling the DN genotype at this early stage.

Morphological Traits and Identification of DN Seedlings In the 483 ‘Akihime’ × ‘Albion’ seedlings the number of runners varied from 1 to 17, and the number of crowns from 0 to 7; inflorescences took 112-248 days to emerge, and petiole length ranged from 80 to 280 mm (data not shown). The first inflorescences emerged between September 2014 and January 2015, being most common in September and November (Fig. 1). No significant correlations were found between each morphological trait and the number of inflorescences (Fig. 2). Thus, none of the four morphological traits screened could be used to identify DN seedlings at an early stage in the ‘Akihime’ × ‘Albion’ progeny. However, the number of days to first inflorescence emergence decreased with the number of inflorescences, a trend that was consistent with the results of Honjo et al. (2011), where early flowering seedlings usually showed high DN magnitude. Gaston et al. (2013) showed that a major dominant gene

Evaluation of Flowering Phenotype Day-neutrality has been introgressed in cultivated strawberry because it is a highly desirable trait that extends the fruit production period and contributes to higher fruit yield (Durner et al., 1984). Classification of DN strawberry plants is usually based on qualitative observations that include assessing growth and flowering (Nicoll and Galletta, 1987; Serçe and Hancock, 2005), inflorescence emergence scoring (Shaw and Famula, 2005), or more complex criteria, such as flowering on mother and clone plants and the evaluation of DN during the second year of culture (Ahmadi et al., 1990). In the present study, late Summer (July and August) flowering was used to evaluate the segregation of the DN trait in a large population based on previously described methods (Serçe and Hancock, 2005; Shaw and Famula, 2005). The number of recently-emerged inflorescences per seedling in the ‘Akihime’ × ‘Albion’ progeny varied from 0 to 22 in 2015, and from 0 to 31 in 2016 (Table 1). The other two progenies

Data Processing Data were analyzed using category and Chi square tests in Excel 2010 (Microsoft Corporation, Redmond, WA, USA). Figures were plotted in SigmaPlot 10.0.

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Jiwei Ruan, Chunmei Yang, Guoxian Wang, Lifang Wu, Shenchong Li, Pang Tao, Chengfei Liao, Young Rog Yeoung, and Jihua Wang

A

B

C

D

Fig. 2. Relationships between flowering magnitude (registered in 2015) and four morphological traits (measured in 2014) in the 483 ‘Akihime’ × ‘Albion’ seedlings. A: number of runners, B: number of crowns, C: days to first inflorescence emergence, and D: petiole length. Table 1. Segregation ratios of day-neutral (DN) and June-bearing (JB) seedlings among the three progenies and their parents in late Summer (July and August) 2015 and 2016, and their fit to the dominant locus hypothesis Progenies and parents ‘Albion’ ‘Akihime’ ‘Albion’× ‘Albion’ ‘Akihime’ בAkihime’ ‘Akihime’ בAlbion’

X2

x

Year

N

Inflorescence number

DN: JB

DN (%)

2015

100

2-7

100:0

100

-

2016

100

2-8

100:0

100

-

2015

100

0-1y

0:100

0

-

2016

100

z

0-1

0:100

0

-

2015

102

0-12

78:24

76.47

0.85

2016

101

0-16

77:24

76.24

0.77

2015

124

0-1

0:124

0

0

2016

119

0-1

0:119

0

0

2015

818

0-22

427:391

52.20

3.59

2016

790

0-31

394:396

49.87

0.94

x

Data shown in this column do not deviate significantly from the expected 3:1 (‘Albion’× ‘Albion’), 0:1 (‘Akihime’ בAkihime’), and 1:1 (‘Akihime’ בAlbion’) ratios at p < 0.05 (X2 0.05,1 = 3.84). Nine out of 100 plants produced one inflorescence in late Summer 2015. z Ten out of 100 plants produced one inflorescence in late Summer 2016. y

and the parents presented much lower numbers (Table 1). The frequency distribution of late Summer inflorescence numbers and flowering scores in the ‘Akihime’ × ‘Albion’ progeny in 2015 and 2016 revealed a bimodal pattern (Fig. 3).

Both variables presented a sharp peak corresponding to a general absence of late Summer flowering (inflorescence number = 0 and flowering score = 5); the other peak indicated that some seedlings presented a large number of inflorescences,

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Fig. 3. Frequency distribution of the number of late Summer inflorescences and flower scores obtained for ‘Akihime’ × ‘Albion’ seedlings in 2015 (n = 818) and 2016 (n = 790).

most recently emerged (inflorescence number > 6 and flowering score = 1). Thus, the highest number of seedlings observed in November 2014 (Fig. 1) might be attributed to late germination and to the delayed growth of some DN seedlings, which postponed their inflorescence emerging time. Segregation of DN in the Three Progenies Conventionally, plants within the same progeny not showing inflorescences in late Summer are classified as JB plants and the others as DN plants. However, in this study, 9% and 10% of the JB ‘Akihime’ parent plants produced one inflorescence in 2015 and 2016, respectively (Table 1). This may be due to cool temperatures and to the medium daylength during late Summer (July and August) in Central Yunnan (Fig. 4). Therefore, plants in the three progenies producing only one inflorescence (10.13 to 12.11% for crossed progenies in 2015 and 2016, Table 1) were classified as JB plants, and those with two or more recently-emerged inflorescences were classified as DN plants. The number of surviving seedlings in ‘Albion’ × ‘Albion’, ‘Akihime’ × ‘Akihime’, and ‘Akihime’ × ‘Albion’ progenies were, respectively: 102, 124, and 818 in 2015 and 101, 119, and 790 in 2016. The percentages of DN seedlings in selfed ‘Albion’, selfed ‘Akihime’, and ‘Akihime’ × ‘Albion’ progenies were, respectively: 76.47%, 0%, and 52.22% in 2015, and 76.24%, 0%, and 49.88% in 2016 (Table 1). Our data fit the hypothesis of genetic control by a single locus where the allele for day-neutrality is dominant over the allele for JB flowering, as the DN:JB ratios in the selfed progeny of DN parents, selfed progeny of JB parents, and crossed progeny fit the expected 3:1, 0:1, and 1:1 ratios, respectively. Cultivated strawberry is an allo-octoploid (2n=8x=56) with a AAA`A`BBB`B` genome, and disomic behavior is predominant for many traits (Brighurst, 1990). A single dominant

Fig. 4. Daily maximum and minimum temperatures registered from May to August 2015 and 2016 at the National Engineering and Technology Research Center for Ornamental Horticulture farm. Data were measured 300 m away from the greenhouse, in open field.

locus control of DN, as found in this study, is consistent with the results reported by Gaston et al. (2013). The DN trait was introgressed into cultivated strawberry from F. viginiana ssp. Glauca Wasatch (Powers, 1954; Galletta et al., 1981). The control of DN by a major locus in cultivated strawberry is consistent with the introgression of a single gene controlling this trait originating from wild octoploid F. viginiana ssp. Glauca Wasatch in F.×ananassa by repeated back-crosses (Hancock, 1999). Nevertheless, the considerable variation in flowering magnitude (inflorescence number and flowering score) among DN ‘Akihime’ × ‘Albion’ seedlings suggests that additional minor genes may also affect dayneutrality. Some of these minor genes may have been inherited from the JB parent ‘Akihime’, as many ‘Akihime’ × ‘Albion’ progenies showed higher flowering magnitude

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Jiwei Ruan, Chunmei Yang, Guoxian Wang, Lifang Wu, Shenchong Li, Pang Tao, Chengfei Liao, Young Rog Yeoung, and Jihua Wang

than the DN parent ‘Albion’ (Table 1). In conclusion, the present study demonstrates single, dominant gene inheritance of day-neutrality in three strawberry progenies in Central Yunnan, China. Moreover, it is possible that minor genes (from the JB parent ‘Akihime’) also increase DN magnitude, and the major gene controlling DN may not be expressed during the juvenile stages (i.e., long before the first inflorescence). This study provides fundamental information for DN gene mapping and cloning, as well as materials (progenies) for breeding of domestic DN strawberry varieties. Acknowledgments: This research was supported by the Establishment of National Engineering and Technology Research Center for Ornamental Horticulture (2012FU125X10), and by the Yunnan Science and Technology Plan Project (2016IA001).

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