Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family

oligosaccharides and seed vigour in Medicago truncatula

CÉLINE VANDECASTEELE¹, BÉATRICE TEULAT-MERAH², MARIE-CHRISTINE MORÈRE-LE PAVEN³,

OLIVIER LEPRINCE², BENOIT LY VU², LAURE VIAU³, LYDIE LEDROIT¹, SANDRA PELLETIER¹, NICOLE PAYET¹, PASCALE SATOUR³, CAMILLE LEBRAS², KARINE GALLARDO⁴, THIERRY HUGUET⁵, ANIS M. LIMAMI³,

JEAN-MARIE PROSPERI⁶& JULIA BUITINK¹

1Institut National de la Recherche Agronomique,²Agrocampus Ouest,³Université d’Angers, UMR 1191 Physiologie

Moléculaire des Semences, IFR 149 QUASAV, 49045 Angers, France,⁴Institut National de la Recherche Agronomique, UMR 102 Genetics and Ecophysiology of Grain Legumes, Domaine d’Epoisses, 21065 Dijon, France,⁵Laboratoire Symbioses et Pathologies des Plantes, INP-ENSAT, 31326 Castanet Tolosan, France and⁶Institut National de la Recherche Agronomique, UMR 1097 Diversité et Adaptation des Plantes Cultivées, 34130 Montpellier, France

ABSTRACT

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental con- ditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we inves- tigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determi- nants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chro- mosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds ofM. truncatulamight negatively affect seed vigour.

Key-words: drought; genetic variation; germination;

nutrients/nitrogen; seedling; storage carbohydrates.

Abbreviations: GolS, galactinol synthase; QTL, quantitative trait loci; RFO, raffinose family oligosaccharide; RFS, raffi- nose synthase; RIL, recombinant inbred line; STS, stachy- ose synthase; Suc, sucrose; TSW, thousand seed weight.

INTRODUCTION

Seed vigour is an estimate of how successfully a seed lot will establish under a range of conditions experienced in prac- tice and is determined by both seed germination and sub- sequent seedling growth until the autotrophic growth stage.

Environmental stress may cause significant reductions in rate and final percentage of germination, as well as reduc- tion in hypocotyl and radicle growth, leading to uneven stand establishment, poor crop performance and poor yield (reviewed by Bennett 2004; Finch-Savageet al. 2010). While a large amount of QTL analyses have been performed to determine the loci associated with morpho-agronomic traits relating to seed quality (composition, nutritional quality, size) (Black, Bewley & Halmer 2006) and those affecting dormancy and germination (Betteyet al. 2000; Clerkxet al.

2004; Bentsink, Soppe & Koornneef 2007; Finch-Savage et al. 2010; Dias et al. 2011), the genetic relationships between the seed composition and seed vigour is unclear.

Legume seeds are particularly well endowed with large amounts of soluble sugars, in particular the RFOs, repre- senting up to 8–10% of the dry weight (DW). RFO sugars accumulate at the final stages of maturation, concomitant with a decrease in Suc (Djemelet al. 2005; Rosnobletet al.

2007). During imbibition, even prior to germination, they are mobilized and suggested to support germination and further seedling growth (Blacket al. 2006; Rosnobletet al.

2007; Blöchlet al. 2008). Besides a carbon storage function, non-reducing sugars are also involved in protecting cellular integrity in systems that are exposed to drought and desic- cation (Hoekstra, Golovina & Buitink 2001; Peters et al.

2007; Nishizawa, Yabuta & Shigeoka 2008 and references therein). Next to these functions, sugars act as signalling molecules that modulate growth, development and stress responses with hormone-like properties (Weber, Borisjuk

& Wobus 2005; Rolland, Baena-Gonzalez & Sheen 2006).

A downside of RFOs is that they are difficult to digest and provide little metabolizable energy for humans and Correspondence: J. Buitink. Fax: +33 241 22 55 49; e-mail:

julia.buitink@angers.inra.fr

monogastric animals. However, whether the removal of RFOs via breeding has an impact on physiological seed quality is unclear. Whereas several studies demonstrated that a role for RFOs in seed storability is unlikely (Bentsink et al. 2000; Buitink, Hemminga & Hoekstra 2000; Clerkx et al. 2004), contradicting reports exist regarding their role in seed vigour. On the one hand, Neus, Fehr & Schnebly (2005) reported that low RFO lines from soybean were not affected in field emergence and other traits such as seed yield, maturity and fatty acid content compared to a high RFO line. On the other hand, inhibition ofa-galactosidase activity resulting in an inhibition of the breakdown of RFOs during imbibition of pea seeds delayed germination (Blöchl, Peterbauer & Richter 2007), but a similar study using soybean seeds did not confirm the link between RFO breakdown and germination rate (Dierking & Bilyeu 2009).

Seeds of soybean lines carrying a mutation in a myo-inositol phosphate synthase gene, conferring reduced myo-inositol, galactinol, raffinose, stachyose and phytin, were shown to be sensitive to imbibition (Obendorfet al. 2008).

So far, no QTL approach has been undertaken on legume seeds to investigate whether a genetic interaction exists between seed vigour characteristics and the amount and composition of soluble sugars, especially RFOs. A study addressing the link between RFOs and seed vigour charac- teristics on Arabidopsis indicated a co-location between Suc and germination under stress, but no co-location was found with RFOs. However, it should be noted that the main soluble non-reducing sugar inArabidopisis Suc with 2% of the DW of the seeds, whereas RFOs constitute only 0.6% of the DW. This is in contrast with seeds of legumes, such as those ofMedicago truncatulathat contain 7–9% of their DW in the form of RFOs, with the remaining 0.5–1%

in the form of the disaccharide Suc (Djemel et al. 2005;

Rosnobletet al. 2007).

The objective of this study was to investigate a possible co-location with genetic determinants involved in RFO metabolism and QTL controlling traits important for seed vigour, such as germination rate, radicle and hypocotyl growth under optimal and stress conditions, and seed weight. A negative correlation and several co-locations were observed between QTL for the ratio Suc/RFO and QTL regulating germination rate or radicle growth after germination, suggesting that the two characteristics are linked. The data argue that one should consider the conver- sion of Suc to RFO rather than the amount of RFOs in order to understand the role of RFOs in seed vigour of legumes.

MATERIALS AND METHODS Seed material and production

Two RIL populations were selected based on parental dif- ferences in either physiological or compositional traits for the characterization of genetic factors underlying these traits. The first one, named LR1, was derived from the cross between DZA315.26 and DZA45.6. The second one, named

LR4, was derived from the cross between Jemalong-6 and DZA315.16 (Pierre et al. 2008). Seeds from the LR1 and LR4 RILs were produced in greenhouses in Dijon and Angers, respectively, from November to May 2007.

Plants of the LR1 population were grown in Terra-Green (2/3) and clay pebbles (1/3) in duplicates on two separate trays. The location of the RIL on the trays was randomly determined. Plants of the LR4 population were grown in a mix of vermiculite and soil. Three plants of each RIL were randomly placed in the greenhouse. For both locations, growth conditions were 16 h photoperiod with minimum night and day temperatures of 16 and 19 °C, respectively.

The plants were automatically watered with a nutrient solu- tion of N–P–K (20–20–20). The plants were not inoculated with rhizobium, and the nitrogen supply was mainly mineral and non-limiting. The pods were harvested at maturity and threshed after 2 weeks of drying at 60% relative humidity (RH) and 20 °C.

Phenotyping

The traits were determined on 184 RILs and 171 RILs for LR1 and LR4, respectively. For each line, seeds of two (LR1) or three (LR4) plants were pooled together. Before measurements, the seeds were stored at 20 °C at 60% RH for 5 months to allow for dormancy release, after which they were stored an additional 7 months at 10 °C prior to the germination experiments. TSW was determined gravimetri- cally on 100 seeds from two repetitions of two plants for LR1 and on 1000 randomly chosen seeds pooled from three plants for LR4.

Sugar determination

Mature seeds of each line were ground and lyophilized, and DW was determined. One millilitre 80% methanol contain- ing melizitose as the internal sugar standard was added to 20–30 mg powder. After heating at 76 °C for 15 min, the liquid was evaporated under vacuum in a Speedvac AES1010 (Savant Instruments, Hyderabad, India). The residue was dissolved in 1 mL distilled water, and after appropriate dilution, sugars were analysed by high- performance liquid chromatography (HPLC) on a Carbo- pac PA-1 column (Dionex Corp., Sunnyvale, CA, USA) as described by Rosnobletet al. (2007). Three and two inde- pendent extractions and assays were performed from samples of 10 seeds and 100 seeds per line for LR1 and LR4, respectively.

Germination

After scarification with sandpaper, 80 seeds were imbibed on 14 cm plastic Petri dishes with Whatman no. 1 (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) that were placed randomly in a dark room. Seeds of the LR4 were imbibed in water at 20 °C (⫾0.5 °C). Seeds of LR1 were imbibed in water or at-0.5 MPa in a polyethyl- ene glycol solution (PEG) (8000; Sigma, St Louis, MO,

© 2011 Blackwell Publishing Ltd,Plant, Cell and Environment,34,1473–1487

USA) at 17 °C (⫾0.5 °C) instead of 20 °C to prevent temperature¥water potential¥genotype interaction (Brunelet al. 2009). The number of seeds that had germi- nated, scored by the protrusion of the radicle, was counted every 2 h (control) or at regular intervals (PEG) under green light. Germination data were fitted to a sigmoid curve using the equationy=a/{1+exp-[(x-x0)/b]}, from which T50was calculated. Germination rate is expressed as 1/T50.

Seedling growth

Seedling growth parameters were determined on the LR4 population under optimal conditions or during water deficit or nitrate excess, starting from a comparable developmental time point (protruded radicle length 0.5–1 cm) after germi- nation was completed.

For the water deficit experiment, germination was homogenized by imbibing seeds for 2 h at 20 °C, then 68 h at 4 °C, followed by 24 h at 20 °C in the dark. Ten germi- nated seeds with 5–10 mm emerged radicles were trans- ferred into 12 cm square plates with filter paper containing either water (control treatment in demineralized water) or a PEG solution at -0.50 MPa. The plates were wrapped with parafilm and inclined at 85° angle and placed at 20 °C in the dark. For dark-grown seedlings, final hypocotyl and radicle length in the dark were determined 120 h after seedling transfer. Elongation rates (mm h^-1) were calcu- lated between 48 and 72 h after transfer for dark-grown hypocotyls. Organ length was scored by marking the plate cover at regular intervals. To measure the length and cal- culate the growth rate, the covers were photographed and the digital pictures were analysed using the public domain image processing and analysis software ImageJ (http://

rsbweb.nih.gov/ij/).

A second experiment focused on early root growth on nitrate compared to water. Dry seeds were imbibed in 12 cm squared transparent plates on filter paper containing either 5 mmKNO3-for the nitrate treatment or water as a control. After incubating for 5 h at 20 °C, and then for 96 h at 4 °C in the dark, the plates were placed at a 45° angle at 20 °C with a 16 h photoperiod under a light intensity of 200mmol m^-2s^-1. Radicle length on nitrate was determined 168 h after imbibition, whereas elongation rate was deter- mined by measuring the growth increment between 96 and 168 h. Data analysis was performed as described earlier.

For both experiments, the plates were organized in a randomized complete-block design with two blocks. Each block contained one plate with 10 seedlings of one line for each treatment.

Statistical analyses

All statistical analyses were performed with SAS 8.1 (SAS Institute Inc., Cary, NC, USA). For parental lines, analyses of variance (ANOVAs) were carried out for all traits using the GLM procedure. Means of the lines were then com- pared with Student–Newman–Keuls test. Regarding the RIL populations, seedling growth and sugar data were

submitted to ANOVA using the GLM procedure. As a sig- nificant block effect was observed for seedling growth, we generated adjusted means by regression on the block effect by using the least square method (lsmeans command of SAS GLM procedure). Phenotypic correlations between traits were investigated using the CORR procedure.

QTL analysis

Genetic maps used for LR1 and LR4 were based on 60 and 126 markers, respectively. MTE and Mtic are microsatellite markers. MTE markers are anchored on the physical map.

The LR4 genetic map comprises 105 MTE and Mtic markers, as well as 21 EST markers named MANG and MDIJ that were added to improve QTL positions. Genetic maps were made using MAPMAKER/EXP version 3.1b software (Landeret al. 1987). The LR1 and LR4 maps cover 562 and 620.6 cM, respectively.

QTL mapping was carried out using MCQTL (Jourjon et al. 2005) with the iterative QTL mapping method (iQTL) that aims to automatically find a multiple QTL model (Charcossetet al. 2001). Cofactors were selected by forward regression usingF-test for a global genome-wide type I risk of 10%. TheFthreshold for QTL detection was automati- cally determined for all traits by 1000 iterations of permu- tation tests at a global genome-wide type I risk of 5%. For each QTL identified, additive genetic effect, percentage of phenotypic variance explained (R²),F-test value as well as confidence intervals were determined by the software.

Equivalent LODs were then calculated. A globalR², repre- senting the percentage of phenotypic variance explained by all detected QTL, was calculated by the multiple QTL model.

Epistatic interactions were detected using Epistat soft- ware package (Chase, Adler & Lark 1997). An automated search for all pairwise interactions was performed, using an initial likelihood ratio cut-off of 8. The associated Monte Carlo simulation program was used to analyse 1 000 000 resampled subgroups as a means of estimating P values.

Interactions for which the two markers were within 50 cm of each other were removed to avoid linkage effects. Inter- actions were declared significant whenPvalues were below 0.0001 and whenPvalues with markers in the neighbour- hood were significant.

The software Biomercator (Arcadeet al. 2004) was used to draw the QTL on a single map by iterative projection of loci from the LR1 map onto the LR4 map. The imple- mented method was used to deduce confidence intervals for the additive QTL based on population size and R² explained by the QTL as described by Darvasi & Soller (1997). Maps were redrawn by MapChart (Voorrips 2002).

In silicomapping and expression analysis A blast search using the nucleotide sequence of sucrose synthase (SUS) (Medtr3g086770), RFS (Medtr3g114540) and GolS (Medtr1g102760) was performed on the Medi- cago genome (http://www.medicago.org) to obtain the

physical position on the chromosomes for the different iso- forms of the three enzymes (E-value>2e-19). Because the markers are physically anchored to the genetic map, the closest genetic marker to the different genes was used to place the SUS, RFS and GolS genes in silico. To detect sequence polymorphisms in the coding sequence, RT- PCR fragments were cloned and sequenced three times.

Detected amino acid differences between the parents were analysed for their impact on protein function using the SIFT program (http://sift.jcvi.org/) (Ng & Henikoff 2003).

Total RNA was isolated according to Verwoerd, Dekker

& Hoekema (1989) from dry mature seeds, and quality was verified by a bioanalyser (Experion; Bio-Rad, Hercules, CA, USA). Reverse transcription reactions were performed on 1mg of total RNA using the QuantiTect Reverse Tran- scription Kit (Qiagen, Valencia, CA, USA). Quantification of transcript levels of STS was performed by RT-qPCR using an ABI PRISM 7100 Sequence Detection System and SYBR Green (Applied Biosystems, Foster City, CA, USA) as a double-stranded DNA-specific fluorescent dye, using 5′-CGTTTGATGACCCCAAAATC-3′ and 5′-ATC ACCCAGCCACTCAAAAG-3′ as amplification primers.

Values were based on three repetitions. The constitutively expressed mRNAMsc27was used as a housekeeping gene for standardizing data and following RT-qPCR efficiency according to Bolingueet al. (2010). Changes in transcript abundance were estimated as fold change relative to the expression in the genotype J6.

RESULTS

Variations in seed and seedling traits, and seed sugar composition in two RIL populations of M. truncatula

Based on parental differences, traits associated with seed vigour were determined on two different RIL populations, LR1 and LR4. The major characteristics investigated in this study were germination rate (1/T50) and post-germinative growth (rate of radicle growth and radicle length with and without excess nitrate and final radicle and hypocotyl length, as well as hypocotyl growth with and without water deficit) (Table 1). Most traits showed normal distributions for both populations, except for germination percentage under water deficit conditions (Supporting Information Figs S1 & S2). Germination rate under optimal conditions was measured in both RIL populations (Table 1; Support- ing Information Figs S1 & S2). Because a recent study of Brunel et al. (2009) showed that the parents of the LR1 population differed considerably in germination rate at -0.5 MPa, the germination rate under water deficit was also determined on seeds from this population. Indeed, water deficit decreased strongly the rate of germination based on the means of the population (Table 1; Supporting Informa- tion Fig. S1).

Post-germinative growth was characterized only in the LR4 population, with measurements of hypocotyl growth and final radicle and hypocotyl length under optimal or

water deficit conditions in the dark to simulate hetero- trophic growth. Under these conditions, the final lengths of all hypocotyls and radicles were reached after 5 d. Water deficit resulted in an increase of radicle length and a decrease of hypocotyl length. Parents of the LR4 population strongly differed in the size of the final hypocotyl length upon water deficit, with a much stronger reduction in final length for DZA315.16 (Table 1). In addition, radicle growth and length after the first 7 d of imbibition were studied in the presence or absence of nitrate in the light to investigate the effect of nutrition status on radicle growth. Nitrate excess resulted in a reduction in radicle length and growth rate (Table 1;

Supporting Information Fig. S2).

Additional traits related to seed weight and sugar com- position were measured on both RIL populations (Table 1).

The phenotyping data followed a normal distribution for TSW, Suc and total non-reducing sugar amounts (Support- ing Information Figs S1 & S2). The frequency distributions for the amount of stachyose and verbascose, as well as for the ratio between these two sugars, were clearly bimodal, suggesting that only a small number of gene factors are involved in the conversion of stachyose into verbascose in the studied genetic backgrounds. The parental lines of LR1 (DZA315.26 and DZA45.6) differed strongly in the amount of stachyose and verbascose, with a 42-fold difference in the verbascose/stachyose ratio (0.073 versus 3.03, see Table 1).

The parents of LR4 showed a 4.4-fold difference in this ratio (0.018 versus 0.080).

The total amount of RFOs only slightly differed between the parents of the LR1 (71.60 versus 75.16mg mg^-1) or LR4 (67.06 versus 59.98mg mg^-1). In contrast, the Suc amount was different between the parents of both the LR1 (5.54 versus 3.42) and LR4 (4.52 versus 6.8mg mg^-1) population.

This difference explained most of the variation in Suc/RFO ratio between the parents (Table 1).

Correlation analysis of seed traits

Correlation analysis was performed to identify putative links between seed traits (Tables 2 & 3). Several physiologi- cal traits were found to be correlated with each other for LR4 (Table 3), such as hypocotyl growth with the final hypocotyl length in the dark, both in control and during water deficit. Similarly, radicle growth and length were posi- tively correlated under control and nitrate conditions.

Positive correlations were also found for all radicle length values, irrespective of the environmental conditions (control, water deficit, nitrate excess, as well as darkness or 16 h photoperiod). A significant, but weak, positive correla- tion was found between radicle growth or radicle length and germination rate, and this relationship remained when seedlings were grown under stress conditions. A weak cor- relation was detected between final hypocotyl and final radicle length under unstressed conditions (r=0.23**), whereas in water deficit conditions, the correlation became stronger (r=0.40***). For the LR1 population (Table 2), a strong correlation was found between germination rate

© 2011 Blackwell Publishing Ltd,Plant, Cell and Environment,34,1473–1487

and final germination percentage during water deficit (r=0.54***).

For both populations, certain physiological traits correlated with sugar amounts and composition. All traits pertaining to radicle growth were weakly, but significantly, correlated with the amount of Suc and RFOs (Table 3). In addition, mainly Suc and the ratio Suc/RFO correlated negatively with germination rate under water deficit (LR1) or on water (LR4). Intriguingly, when seedlings were exposed to a 16 h photoperiod, a positive correlation was found between verbascose/stachyose ratio and radicle growth parameters, irrespective of the presence of nitrate (Tables 2 & 3). No correlation was found between sugars and hypocotyl traits.

The correlation analysis of the sugar composition showed that in both populations, the amounts of all RFOs were

linked (Tables 2 & 3), which can be expected considering the sequential addition of a galactose moiety to Suc to form raffinose (trisaccharide), stachyose (tetrasaccharide) and verbascose (pentasaccharide). Stachyose levels correlated negatively with raffinose and verbascose levels. This is dif- ferent from soybean, where stachyose and raffinose are positively correlated (Neuset al. 2005). Suc levels were cor- related with the RFO levels in LR1, but not in LR4. In addition, Suc and the Suc/RFO ratio were correlated with TSW, but in the opposite direction for LR1 or LR4.

QTL associated with seed and seedling characteristics

To visualize co-locations of QTL, all the additive QTL iden- tified were summarized on a consensus genetic map of the Table 1. Phenotypic analysis for seed- and seedling-related traits of the LR1 and LR4 populations and the parental lines

Traits Abbreviations Pop Means Min Max

Parental lines

J6

DZA 315–16/26

DZA 45–6 Germination under optimal and water deficit conditions

Germination rate (h^-1) 1/T50 LR1 0.038 0.026 0.047 0.038^a 0.039^a

LR4 0.050 0.037 0.061 0.052^a 0.049^a

Germination rate at-0.50 MPa (h^-1) 1/T50wd LR1 0.025 0.016 0.044 0.015^b 0.028^a

Final germination % at-0.5 MPa %Gwd LR1 97.2 75.8 100.0 89^b 99^a

Post-germinative growth under optimal and stress conditions (water deficit and nitrate excess)

Final hypocotyl length (mm) FHL LR4 32.4*** 24.9 42.7 30.7^a 30.7^a

Final hypocotyl length at-0.50 MPa (mm) FHLwd LR4 23.3*** 13.5 34.6 26^a 17^b Hypocotyl growth rate (mm h^-1) HG LR4 0.086*** 0.026 0.151 0.068^b 0.115^a Hypocotyl growth rate at-0.50 MPa (mm h^-1) HGwd LR4 0.136*** 0.057 0.232 0.152^a 0.110^b

Final radicle length (mm) FRL LR4 45*** 28.4 69.8 45.8^a 43.2^a

Final radicle length at-0.50 MPa (mm) FRLwd LR4 75*** 40.7 105.6 84.5^a 77.7^a Radicle length 16 h photoperiod (mm) RLL LR4 59.9*** 41.8 81.3 52.6^b 61.9^a Radicle length 16 h photoperiod KNO3(mm) RLLNO3 LR4 50.5*** 32.4 81.2 49.8^a 47.3^a Radicle growth rate 16 h photoperiod (mm h^-1) RGL LR4 0.306*** 0.2 0.4 0.301^a 0.313^a Radicle growth rate 16 h photoperiod KNO3(mm h^-1) RGLNO3 LR4 0.242*** 0.1 0.4 0.276^a 0.194^b Seed weight

TSW (g) TSW LR1 4.16 3.19 5.17 4.13^b 4.79^a

LR4 3.90 2.99 4.83 3.71^b 3.97^a Soluble, non-reducing sugar content

Suc (mg mg^-1DW) Suc LR1 6.97*** 2.11 21.4 5.54^a 3.42^b

LR4 7.05*** 3.10 11.67 4.52^b 6.80^a

Raffinose (mg mg^-1DW) Raff LR1 4.66*** 1.90 12.9 2.22^b 3.96^a

LR4 2.41*** 1.38 3.86 1.57^a 2.05^a

Stachyose (mg mg^-1DW) Stach LR1 40.7**** 8.56 93.3 66.64^a 17.88^b

LR4 60.12*** 46.2 77.3 64.08^a 53.62^b

Verbascose (mg mg^-1DW) Verbas LR1 27.1*** 3.35 89.7 4.89^b 54.25^a

LR4 3.73*** 0.95 8.51 1.41^b 4.31^a

Verbascose/stachyose Verbas/Stach LR1 4.0*** 0.31 11.9 0.073^b 3.03^a

LR4 0.066*** 0.016 0.158 0.018^b 0.080^a

Total RFO sugars RFO LR1 72.85*** 32.7 127.9 71.63^a 75.16^a

LR4 66.24* 56.8 81.1 67.06^a 59.98^b

Suc/RFO Suc/RFO LR1 0.100*** 0.024 0.264 0.075^a 0.044^b

LR4 0.107*** 0.045 0.193 0.067^b 0.113^a

Total sugars (mg mg^-1DW) Sstot LR1 79.6*** 38.0 149.3 79.29^a 79.51^a

LR4 75.41*** 65.4 88.1 71.28^a 66.78^b

a,bStudent–Newman–Keuls method (between the two parental lines for each trait).

***Significantly different between lines atP<0.001. Unmarked values were not statistically analysed.

two RIL populations (Fig. 1). Detailed information is avail- able in Tables 4 and 5 for LR1 and LR4, respectively. In total, 59 QTL were detected for 20 traits related to seed and seedling vigour, seed weight and sugar composition. They were distributed into 15 chromosomal regions of the eight chromosomes (Fig. 1). Some of these regions contained iso- lated QTL (LG6), but most of them shared co-located QTL (e.g. long arms of LG1, LG3 and LG8, and short arm of LG5).

A total of 23 QTL were detected that are related to seed or seedling vigour, explaining between 6.8 and 22% of the variation. Most of the QTL were organ specific, such as the ones on LG1 and on LG6 for hypocotyl and the one on LG5 for radicle growth. Common QTL were identified for seed- ling growth under darkness and in 16 h photoperiod (LG3 for both organs and LG8 for radicle).

For LR1, although no QTL were identified for germi- nation in optimal conditions, three QTL were detected for germination rate under water deficit, two on LG1 and the other on LG3. The QTL found for final percentage of ger- mination (%Gwd) was positioned in the same region on LG3. For LR4, two QTL for rate of germination were found that together explained 34% of the variation. Under the dark/light regime in the presence of nitrate, one of the QTL for post-germinative radicle growth co-located with a QTL for germination rate (LG7). One QTL controlling hypocotyl growth and final hypocotyl length under water deficit treatment in LR4 co-located with a QTL for ger- mination rate under water deficit in LR1 (LG1). A similar investigation on the effect of low temperature on final hypocotyl length using the LR5 population ofM. trunca- tulaalso identified a QTL in this region (Diaset al. 2011), indicating that this chromosomal region is involved in heterotrophic growth variation under different abiotic stresses. Significant epistasis was found for seedling growth under stress conditions (Fig. 2; Supporting Information Tables S1 & S2). These interactions between loci were related to hypocotyl elongation under water deficit

conditions (red lines, Fig. 2) and radicle length on nitrate (grey line, RLLNO3, Fig. 2).

A total of 28 QTL were detected for soluble sugar com- position and amount. One major QTL for amounts of raffi- nose, stachyose and verbascose was found on the long arm of LG4 for both RIL populations (Tables 4 & 5). In the same chromosomal region, a QTL for the ratio verbascose versus stachyose explained 80% of the variation, and the direction of the additive effect of raffinose and verbascose was opposite of that of stachyose, suggesting that an under- lying mechanism is related to the conversion of these sugars. This was the only QTL detected for RFO levels for LR1, whereas for LR4, additional minor QTL (7.6–11%

variation) were found for RFO levels (LG5 for verbascose, LG8 for raffinose, and both LG3 and 7 for stachyose) (Fig. 1; Table 4). QTL related to the Suc amount and to the Suc/RFO ratio were located on LG2 and LG3 for LR1, and on LG1, LG3 and LG7 and LG8 for LR4. For LR1, QTL controlling Suc amount and the Suc/RFO ratio were also found in the major region involved in RFO amount and composition on LG4.

Several epistasic interactions were found for the amount and composition of RFOs in both populations (Supporting Information Tables S1 & S2; Fig. 2), in par- ticular for stachyose and verbascose (and their ratio). All interactions were with markers positioned on the long arm of LG4, the chromosomal region containing the major additive QTL associated with RFO amount and composi- tion (Tables 4 & 5). The other interactive markers were on the short and long arms of LG1 (MTE2 region and MTE6 region, respectively), on LG2 (MTE14 region), on LG3 (MTE17 and MTE117 regions) and on the short arm of LG4. Most of the chromosomal regions that interacted were the same for both populations (Fig. 2). When the DZA315.16/26 alleles at loci on the long arm of LG4 (major QTL) are combined with other alleles at the other loci, verbascose amount increased in LR4 whereas verbas- cose amount decreased in LR1.

Table 2. Correlations between seed vigour traits during water deficit, sugar composition and seed weight of the LR1 population

Trait 1/T50 1/T50wd %Gwd TSW Suc Raff Stach Verbas Sstot Suc/RFO Verbas/stach

1/T50 1 0.17*

1/T50wd 1 0.54*** -0.18* -0.26**

%Gwd 1 0.16* -0.21* 0.20* 0.17* -0.37*** -0.18*

TSW 1 -0.33*** 0.17* -0.36*** -

Suc 1 0.58*** -0.32*** 0.53*** 0.37*** 0.78*** 0.47***

Raff 1 -0.25* 0.73*** 0.56*** 0.28*** 0.55***

Stach 1 -0.72*** 0.49*** -0.61*** -0.88***

Verbas 1 0.22* 0.44*** 0.85***

Sstot 1 -0.22* -

Suc/RFO 1 0.59***

Verbas/Stach 1

Significance is indicated by asterisks. *P<0.05, **P<0.01, ***P<0.001.

1/T50, rate of germination; 1/T50, germination rate at-0.5 MPa; %Gwd, final germination % at-0.5 MPa; TSW, thousand seed weight; Suc, sucrose; Raff, raffinose; Stach, stachyose; Verbas, verbascose; Sstot, total sugars; Suc/RFO, ratio Suc over oligosaccharides; Verbas/Stach, ratio verbascose over stachyose.

© 2011 Blackwell Publishing Ltd,Plant, Cell and Environment,34,1473–1487

Table3.Correlationsbetweenseedandseedlingvigourtraits,sugarcompositionandseedweightoftheLR4population Trait1/T50FRLFRLwdRLLRLLNO3RGLRGLNO3FHLFHLwdHGHGwdTSWSucRaffStachVerbasSstotSuc/RFOVerbas/Stach 1/T5010.18*0.28***0.30***0.25**0.24**-0.26**-0.30***-0.38***-0.25**-0.36*** FRL10.34***0.46***0.50***0.31***0.43***0.23**0.17*0.17*-0.21** FRLwd10.46***0.36***0.34***0.31***0.40***0.42*** RLL10.74***0.84***0.64***0.22**0.20**-0.26***-0.20**0.16*-0.24**-0.27***-0.24** RLLNO310.57***0.90***0.17*-0.22**-0.20*-0.19*-0.25**-0.25**-0.26** RGL10.56***0.22**0.22**-0.19*-0.28***-0.29**-0.19*-0.28*** RGLNO31-0.22**-0.21**-0.17*-0.21**-0.24**-0.23** FHL10.32***0.65***0.28*** FHLwd10.20*0.82*** HG10.18* HGwd1 TSW10.30***0.16*0.29***-0.16* Suc10.30***0.97*** Raff1-0.60***0.85***0.83*** Stach1-0.78***0.78***-0.33***-0.83*** Verbas1-0.33***0.18*0.99*** Sstot1-0.40*** Suc/RFO10.21** Verbas/Stach1 Significanceisindicatedbyasterisks.*P<0.05,**P<0.01,***P<0.001. 1/T50,germinationrate;FRL,finalradiclelength(dark);FRLwd,finalradiclelengthat-0.5MPa(dark);RLL,radiclelength(16hphotoperiod);RLLNO3,radiclelengthin5mmKNO3(16hlight);RGL,radiclegrowth rate(16hlight);RGLNO3,radiclegrowthratein5mmKNO3(16hlight);FHL,finalhypocotyllength(dark);FHLwd,finalhypocotyllengthat-0.5MPa(dark);HG,hypocotylgrowthrate(dark);HGwd,hypocotylgrowth at-0.5MPa(dark);TSW,thousandseedweight;Suc,sucrose;Raff,raffinose;Stach,stachyose;Verbas,verbascose;Sstot,totalsugars;Suc/RFO,ratioSucoveroligosaccharides;Verbas/Stach,ratioverbascoseoverstachyose.

MANG10.0 MTE751.3 MTE23.2 MANG23.7 MDIJ115.4 MTE6120.4 MTE9721.7 MTE7730.2 MTE6251.9 MTE557.4 MTE7859.0 MTE664.3 MDIJ368.6 MTE8875.0 MTE780.7 MTE883.4 MTE11486.3 RLLNO3-LR4

FHLwd -LR4 HGwd -LR4

TSW-LR4 Suc-LR4 SucRFO-LR4 1/T50wd -LR1 1/T50wd -LR1

TSW-LR1

LG 1

TSW-LR4 TSW-LR4

Suc-LR1 SucRFO-LR1

LG 2

FRLwd -LR4 RLL -LR4

Stach -LR4 Suc-LR4 SucRFO-LR4 SucRFO-LR1

1/T50wd -LR1

%Gwd -LR1

LG 3

RGL-LR4 Raff -LR4 Stach -LR4 Verbas -LR4 StachVerbas -LR4

SStot -LR4 Suc-LR1 SucRFO-LR1

Raff -LR1 Stach -LR1 Verbas -LR1 StachVerbas -LR1

LG 4

TSW-LR4 Verb -LR4

SStot -LR4 TSW-LR1

1/T50-LR4

RLL -LR4 RGL-LR4

RLLNO3-LR4 RGLNO3-LR4

LG 5

FHL-LR4

LG 6

Suc-LR4 SucRFO-LR4 Stach -LR4

1/T50-LR4 RLLNO3-LR4

LG 7

Suc-LR4 SucRFO-LR4 Raff -LR4 Raff -LR4

FRL-LR4 RLL -LR4 RGL-LR4

LG 8

RFS-> RFS->

STS-> RFS-> RFS->

RFS-> RFS-> RFS->

GolS-> GolS->

GolS-> GolS->

SS-> SS->

SS->

SS->

SS-> SS-> SS-> Figure1.QTLontheLR4–LR1consensusmapobtainedfromtheprojectionoftheLR1geneticmapontoLR4geneticmapwithBiomercator.SupportintervalsandpeaksofQTLhave beenrecalculatedbythesoftware,LR4line,LR1dashedlines.TheapproximatepositionofgenesinvolvedinRFOorSucmetabolismwasobtainedbyinsilicomappingusingtheanchored MTEmarkers.1/T50,germinationrate;1/T50wd,germinationrateat-0.5MPa;%Gwd,finalgermination%at-0.5MPa;FRL,finalradiclelength(dark);FRLwd,finalradiclelengthin -0.5MPa(dark);RLL,radiclelength(16hlight);RLLNO3,radiclelengthin5mmKNO3(16hlight);RGL,radiclegrowthrate(16hphotoperiod);RGLNO3,radiclegrowthratein5mm KNO3(16hphotoperiod);FHL,finalhypocotyllength(dark);FHLwd,finalhypocotyllengthin-0.5MPa(dark);HG,hypocotylgrowthrate(dark);HGwd,hypocotylgrowthratein -0.5MPa(dark);TSW,thousandseedweight;Suc,sucrose;Raff,raffinose;Stach,stachyose;Verbas,verbascose;Sstot,totalsugars;Suc/RFO,ratioSucoveroligosaccharides;Verbas/Stach, ratioverbascoseoverstachyose;GolS,galactinolsynthase;SS,sucrosesynthase;RFS,raffinosesynthase;STS,stachyosesynthase.Colourcodesforthedifferenttraitsarerepresentedas follows:sugar,green;germination,lightblue;germinationunderwaterdeficit,purple;TSW,brown;radiclegrowth,darkblue;radiclegrowthunderwaterdeficit,black;hypocotylgrowth, orange;hypocotylgrowthunderwaterdeficit,red.

© 2011 Blackwell Publishing Ltd,Plant, Cell and Environment,34,1473–1487

Co-location of QTL for seed vigour and seed weight with QTL for sugar composition

Several QTL controlling soluble sugar composition and amount co-located with both of the QTL for germination rate of LR4 (on LG5 and LG7), as well as for one of the QTL controlling germination under water deficit for LR1 (on LG3). Furthermore, sugar QTL co-located also with QTL detected for post-germinative radicle growth on several chromosomal regions (on LG4, LG5, LG7 and LG8). Consistently, additive effects at QTL for Suc, Suc/

RFO, raffinose and verbascose were in the opposite direc- tion of co-segregated QTL for germination and radicle growth/length under optimal or stress conditions. In con- trast, additive effects of stachyose QTL were in the same direction. This corresponds to the phenotypic correlation of these sugars with each other, with stachyose levels nega- tively correlated with raffinose and verbascose levels. Most of the soluble sugar QTL that co-located with seed and seedling vigour traits were related to Suc amount or the ratio of Suc versus RFOs, rather than the composition of the oligosaccharides (Fig. 1). The analysis of the epistatic interactions shows interaction of the locus on LG4 with several other markers co-locating with Suc/RFO QTL at LG1 and LG3, but also with markers that are co-locating with germination rate under water stress and radicle length on LG1 and LG5 (Fig. 2).

A total of six QTL were detected for seed weight in LR4, out of which two were also found in LR1 (LG1 and LG5).

Four QTL for seed weight co-located with QTL for Suc amount (on LG1, LG2 and LG8) or total amount of sugars (LG5), as well as QTL for germination rate (LG5 and LG7) and for radicle length (LG8).

Considering the correlation between seed weight and germination (Table 3), and co-location of the respec- tive QTL in LR4 (Fig. 1), we verified whether the seed weight did not introduce a bias in the correlation between

germination rate and Suc amount or Suc/RFO ratio. For this purpose, the RIL population was separated into three seed size classes (i.e. small, medium and large seed weight). Sub- sequently, a correlation analysis between germination and Suc amounts was carried out within each class (data not shown). For all three classes, a significant correlation remained between the two traits, with a respectivervalue of 0.38 (P<0.001), 0.19 (P<0.01) and 0.41 (P<0.001) for small, medium and large seed size. A second analysis was performed by comparing the ratio of Suc/RFO of the 20 lines of the LR4 population with the fastest germination with the ratio of the 20 lines that germinate the slowest (data not shown). The Suc/RFO ratio was significantly dif- ferent (P=9.8e-07) between the fast- and slow-germinating seeds. Faster-germinating seeds had a lower Suc/RFO ratio than slower-germinating seeds.

In silicomapping of RFO metabolism genes and characterization of a major gene involved in stachyose/verbascose conversion inM.

truncatulaseeds

To investigate whether genes involved in sugar metabolism can explain the QTL for sugar composition and ratio, anin silicomapping was performed on genes encoding enzymes involved in RFO or Suc metabolism, and placed on the genetic map using the anchored MTE markers (Table 6;

Fig. 1). Several soluble sugar QTL co-located with these genes, indicating that they might be the regulatory loci underlying the sugar variation. The Suc and Suc/RFO QTL on LG1 (explaining 10% of the variation) co-located with two genes encoding GolS (GolS) (Medtr1g102760 and Medtr1g102770), whereas the Suc/RFO QTL on LG3, detected for both RIL populations (explaining 18 and 22%

for LR1 and LR4, respectively) co-located with two genes encoding RFS (RFS) (Medtr3g114540 and Medtr3g119630) Table 4. Summary of detected QTL for seed-related traits ofMedicago truncatulaof the LR1 population

Trait LG

Peak position

Support

interval LOD R²(%)

Additive effect

‘DZA315.26’ GlobalR² Threshold^a

Rate of germination at-0.5 MPa (1/T50) 1 1.6 0–12.9 2.9 7.0 -1.29 h 26.6 2.47

1 30.1 19.8–36 7.7 16.6 +2.39 h

3 53.8 46.1–56.2 5.4 12.3 -1.79 h

Final germination % at-0.5 MPa 3 51.8 41.8–56.2 4.3 9.9 -1.31 h 2.40

TSW 1 1.6 0–11.8 3.2 7.6 -0.106 g 17.4 2.49

5 0 0–8.2 5.5 11.4 -0.143 g

Suc 2 6.0 0–19.3 5.9 16.2 +1.49mg mg^-1DW 30.7 2.52

4 65.3 62.9–65.3 8.2 21.0 -1.43mg mg^-1DW

Raffinose 4 65.3 62.6–65.3 11.1 25.9 -0.92mg mg^-1DW 2.50

Stachyose 4 65.3 65.2–65.3 106.4 77.4 +22.3mg mg^-1DW 2.56

Verbascose 4 65.3 65.0–65.3 89.6 74.4 -17.3mg mg^-1DW 2.31

Verbascose/stachyose 4 65.3 65.2–65.3 151.4 83.6 -3.79 2.61

Suc/RFO 2 0 0–7.5 11.1 27.7 +0.0181 61.0 2.47

3 44.9 37.5–54.0 6.5 18.4 +0.0142

4 65.3 64.2–65.3 21.0 42.1 -0.0246

aThreshold value for QTL detection (5%).