Abstract
BACKGROUND:
Understanding the genetics of flowering in the strawberry (Fragaria×ananassa) will aid in the development of breeding strategies.
OBJECTIVE:
To search for quantitative trait loci (QTL) associated with remontancy and weeks of flowering in the strawberry.
METHODS:
Previously collected phenotypic data from two non-remontant ‘Honeoye’×remontant ‘Tribute’ strawberry populations and simple sequence repeats (SSR) markers were used to search for QTL associated with repeat flowering, weeks of flowering and runner production, as well as the ability to produce flowers and runners at 17, 20 and 23°C.
RESULTS:
As was discovered in other studies, we found a major QTL that regulated remontancy and weeks of flowering on homeologous linkage group IV of ‘Tribute’. This QTL also had a negative effect on runner production and a positive influence on flower production under high temperatures. A number of additional QTL were discovered that significantly (LOD >3.0) influenced flower and runner production.
CONCLUSIONS:
Remontancy/non-remontancy is controlled by a major gene/locus and several minor modifying ones.
Introduction
The seasonal flowering response of strawberry cultivars (Fragaria×ananassa Duchesne ex Rozier), has classically been described as short day (June-bearers), day-neutral and long-day (everbearing) [1, 2]. However, evidence is accumulating that the classification of strawberry genotypes based on their photoperiodic response is complicated by the effect of temperature [3–7]. Genotypes previously classified as short-day, will flower under long days when temperatures are cool, and genotypes that have been called day-neutral do not flower at all under warm temperatures above a threshold [1, 8]. For this reason, day-neutrals are more accurately described as remontant and short-day types as non-remontant.
Genetic control of day-neutrality or remontancy in the octoploid strawberry has long been debated and several hypotheses have been proposed including a single dominant gene [9, 10], a single major gene with modifier genes [11], dominant complementary genes [12], and polygenic control [5, 14].
In the last decade, molecular marker and QTL approaches have been used to identify the genes associated with remontancy in F.×ananassa and a major gene and several minor genes have been identified. Sugimoto et al. [10] studied a population derived from ‘Ever berry’ (remontant) and ‘Toyonoka’ (non-remontant) and identified a RAPD (Randomly Amplified Polymorphic DNA) marker associated with remontancy. Weebadde et al. [5] used amplified fragment length polymorphisms (AFLPs) to identify QTL regulating remontancy in a population of non-remontant ‘Honeoye’×remontant ‘Tribute’ (H×T) phenotyped at five locations across the USA. They identified one QTL associated with remontancy that was common to the eastern states Minnesota (MN), Michigan (MI), and Maryland (MD), in addition to three QTL specific to MN, and one QTL specific to MI and California (CA).
Castro et al. [15] used SSR markers to identify QTL for remontancy and runner production using the phenotypic data collected in the ‘Honeoye’בTribute’ population of Weebadde et al. [5]. They scored day-neutrality qualitatively as repeat flowering after July 17 and quantitatively as number of weeks of flowering. When qualitatively scored, they found a QTL that mapped on LG IV of the ‘Tribute’ map, regardless of planting location. QTL for weeks of flowering were also found on LG IV in MD and CA, and for stolon production in MN, MI and OR.
In another population segregating for remontancy (non-remontant ‘CF1116’×remontant ‘Capitola’), Gaston et al. [16] identified a single major QTL on LG IV that controlled the length of flowering and runner production. The QTL had opposite effects on flowering (positive effect) and runnering (negative effect), suggesting that both traits share a common physiological control. Gaston et al. [16] also found several minor QTL regulating what they called “perpetual flowering” on other linkage groups.
All of the above studies were done in ambient field conditions, so they provide little information on the influence of temperature on the expression of remontancy. To directly test the influence of temperature in the regulation of flowering in F.×ananassa, Mookerjee et al. [17] made the same cross as Weebadde et al. [5] and evaluated the flowering patterns of a different set of progeny under three temperature regimes (17, 20, and 23°C) in a greenhouse under long days. In addition, replicates of the same genotypes were grown under field conditions in MI and OR and were evaluated for their flowering patterns. Their data indicated that high temperature tolerance plays a role in the expression of remontancy, as most of the progeny that produced more flowers at 23°C than at 17 °C in the greenhouse were remontant in the field in MI and OR.
Herein, we describe a QTL analysis for remontancy and runner production on the two H×T populations generated by Weebadde et al. [5] and Mookerjee et al. [17] using many of the same SSR markers used by Lewers et al. [18] and Gaston et al. [16]. The goals of this study were to: 1) further validate the existence of a major QTL for remontancy and weeks of flowering in F.×ananassa on LG IV, 2) determine if the level of floral heat tolerance in Mookerjee et al.’s [17] H×T population co-segregates with a QTL determining remontancy and weeks of flowering, and 3) determine whether the QTL for remontancy in the H×T population also regulates runner production as in the ‘CF1116’בCapitola’ of Gaston et al. [16].
Material and methods
Mapping population
DNA from a Pseudo testcross population of 174 progeny of ‘Honeoye’ (non-remontant)בTribute’ (remontant) was used to build the linkage map [5]. Out of this mapping population, 112 genotypes had been phenotyped in the field by Weebadde et al. [5] in 2005 and 2006. We also extracted DNA from the 54 progeny of the same cross made in 2009 by Mookerjee et al. [17], which had also been phenotyped in the field and greenhouse in 2011.
Phenotypic evaluations
The phenotypic data collected previously by Weebadde et al. [5] were used to determine QTL, along with the more recent data collected in the field and greenhouse in 2011 [17]. The presence and absence of flowers was recorded every week from the beginning of flowering until at least early August. Progeny that flowered in the spring (May-June) and in the long days of summer after 23 July were categorized as remontant. Progeny that flowered only in the spring (May-July 22) before the longest day of the year were categorized as non-remontant (Table 1).
In the greenhouse studies by Mookerjee et al. [17], three replicates of each genotype were grown in a completely randomized design in temperature controlled greenhouses at 17°C, 20 °C, and 23°C, and under 16 hr photoperiod using supplemental lights. The number of flowers and runners were counted every week from Dec 2010 to Mar 2011. All open flowers and runners were removed after counting every week (Table 1).
Genotyping
Selection of SSRs
SSR loci developed from F.×ananassa, F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, and F. viridis Weston, [18–26] were screened for production of polymorphic bands in a subset of 54 progeny and the parents. Of the 157 SSR markers that were screened, 118 were selected based on the presence of polymorphism and distinct scorable bands on 6% polyacrylamide gels.
DNA amplification
Young leaf samples from the parents and progeny were collected from greenhouse grown plants. DNA was extracted using the DNeasy Plant mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. DNA amplification was performed in 20μL reactions containing 1×GoTaq® Green Master Mix (Promega Corporation, Madison, WI), 0.5 mM of forward and reverse primer, and 1μL of 60 ng/μl DNA template. Amplifications were performed in a C1000TM Thermal Cycler (Biorad, Hercules, CA) using the PCR protocol: Initial denaturation: 94°C for 2 min; 34 cycles of 1 min at 94°C, 1 min at annealing temperature (= Tm+2°C), 1.5 min at 72°C; and a final extension step of 10 min at 72 °C, hold at 15°C.
Polyacrylamide gel electrophoresis
PCR amplified products were size separated using a 6% denaturing polyacrylamide gel (15 mL of 40% Acrylamide/Bis Solution; BioRad, Hercules, CA), 10 mL 10×TBE buffer, 42 g Ultra Pure Urea (Invitrogen, Carlsbad, CA), 500μL 10% APS, 100 mL TEMED (BioRad, Hercules, CA). The PCR amplicons were denatured (95°C for 5 min, hold at 4°C) and loaded on to 38 cm×50 cm Sequi-Gen GT system (BioRad, Hercules, CA) that was preheated for 20–30 min. The gels were run at 80 W for 3.5 hrs, and visualized with silver staining [27]. The fragment sizes were estimated by comparing with 10 and 50 bp ladders (Invitrogen, Carlsbad, CA).
Linkage map
The Single Dose Restriction Fragment (SDRF) [28] approach was used for scoring the markers. Each segregating fragment was treated as an individual allele and the genotypes were scored for presence/absence of the allele. Markers present in both the parents that segregated in a 3:1 ratio were coded as dominant markers and those that segregated in a 1:1 ratio were coded as codominant markers. The dominant and codominant markers were used to develop the linkage map using JoinMap v 3.0 [29], with a minimum threshold LOD score value of 2.8, maximum recombination frequency of 0.3, and the Kosambi mapping function. The linkage groups were visualized using MapChart 2.2 [30].
QTL identification
MapQTL v 5.0 [31] was utilized for QTL identification using the Multiple QTL Mapping (MQM) or Composite Interval Mapping (CIM) approach. The population was derived from two heterozygous parents and was coded as CP to include the three types of marker data: 1: codominant markers segregating in ‘Honeoye’, 2: codominant markers segregating in ‘Tribute’, 3: Dominant markers present in both parents. Markers identified as significant by the Kruskal-Wallis test were used as cofactors. The significant LOD score at p≤0.05 was determined from 1000 permutations with the dataset. Significant QTL regions along with the linkage groups were visualized using MapChart 2.2 [30].
Results and discussion
The 118 SSR markers resulted in the amplification of 704 segregating SDRF. Out of the 704 segregating SDRF, 396 were present only in ‘Honeoye’ and 308 were present only in ‘Tribute’. The 396 SDRF in ‘Honeoye’ cover 962.8 cM in 19 linkage groups with the average distance between makers of 9.9 cM (Table 2). The 308 markers in ‘Tribute’ cover 922.3 cM in 23 linkage groups with the average distance between markers of 11.8 cM (Table 2). The linkage groups of ‘Honeoye’ and ‘Tribute’ were named from I to VII using the markers previously placed on the diploid Fragaria vesca map by Sargent et al. [23] (Table 2).
As in the work of Castro et al. [15], we found a major QTL for repeat flowering and weeks of flowering on LG IV in the families of ‘Honeoye’בTribute’ generated by Weebadde et al. [5] and Mookerjee et al. [17]. Gaston et al. [16] also found a major QTL on LG IV for the number of inflorescences emerging from the end of May to the beginning of August. The marker locus ChFam011_129 on LG IV was associated with remontancy in the open fields of CA-05, MD-05, MN-05, MI-05, MI-06, MI-11, OR-05, and OR-11 (Table 3). The r2 varied from 12.6 to 34.0, and the effect ranged from 0.3 to 0.6. The same marker locus was also associated with weeks of flowering in the open fields of MI-05 and OR-05, with r2 varying from 11.1 to 16.7 and effects ranging from 1.4 to 4.4. In addition, ChFam011_129 was associated with the number of flowers produced in the greenhouse trials at all temperatures, with r2 ranging from 24.1 to 30.1, and effects varying from 74.5 to 113. Castro et al. (2014) also found a QTL on LG VI associated with remontancy and weeks of flowering in ‘Tribute’ with the marker ChFam011_129.
It appears that the QTL represented by ChFam011_129 or closely associated genes also regulate runner production antagonistically, as the same marker locus was associated negatively with runner production in the open field trials of MN-05 and OR-11, with r2 varying from 14.5 to 14.9 and effects ranging from –8.3 to –15.4. In the greenhouse temperature trials, ChFam011_129 was also found to be negatively associated with runner production at 23°C (r 2 = 34.4 effect = –25.4) (Table 3).
Besides the major QTL regulating remontancy and weeks of flowering, we identified a number of other QTL regulating these traits in both ‘Tribute’ and ‘Honeoye’, suggesting that the control of remontancy is by one major gene and a number of modifiers as proposed by Shaw and Famula, [11]. In OR-05, a QTL was identified on LG V of ‘Tribute’ with an r2 of 13.4% and an effect of +0.4. Several QTL for weeks of flowering were identified in MI-05, MI-11 and OR-05 for weeks of flowering on LG III of ‘Tribute’. Their r2 varied from 10.1 to 15% , and their effects from 0.1 to 2.9 weeks of flowering. A negative QTL for weeks of flowering was identified on LG I of ‘Honeoye’ with an r2 of 21.7 and effect of –3.1 (Table 4).
Like remontancy, runner production appears to be regulated as a polygenic trait, as several QTL were identified that regulated this trait. QTL for number of runners were identified on LG I and II of ‘Tribute’ in the open field trials in CA-05 and OR-05, with r2 ranging from 16.3 to 18.4 and effects from –3.1 to +1.1. Two QTL were also identified on LG II of ‘Honeoye’ at 17 C in the greenhouse trails. The r2 values ranged from 29.6 to 30.5 % and their effects ranged from –1.0 to –1.1 (Table 4).
It appears that heat tolerance plays a role in the expression of remontancy and duration of flowering. In the greenhouse temperature trials, the presence of the allele represented by ChFam011_129 had a dramatic effect of +113 on total numbers of flowers at 23°C, and a negative effect of –25.4 on runner production. An antagonistic QTL (marker locus band ARSFL19_295) was found in ‘Honeoye’ that had an effect of +1.8 on the ratio of flowers at 17 °C vs. 23°C, meaning that fewer flowers were produced at 23°C vs 17 °C in the presence of this allele (Table 4).
In summary, as in the work of Castro et al. [15] and Gaston et al. [16], we identified a major QTL regulating remontancy and weeks of flowering. This QTL or genes closely linked to it also regulated runner production in an antagonistic fashion and influenced the ability to produce flowers under high temperatures. There were also several additional QTL identified regulating flower and runner production, suggesting that these traits are controlled polygenically, with a major gene and several minor modifying genes.
Footnotes
Acknowledgments
The authors thank Dechun Wang for help with the linkage map and QTL analysis, and Pete Callow who helped collect the field data in the previous studies of ‘Tribute’בHoneoye’. “RosBREED: Enabling marker-assisted breeding in Rosaceae” is supported by the USDA-NIFA-Specialty Crop Research Initiative by a combination of federal and matching funds (grant number 2009-51181-05808).