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# Genetic variation and QTL mapping of para-coumaric and ferulic acid contents in maize stover at silage harvest

A.S. Fontaine, M. Briand, Yves Y. Barrière

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A.S. Fontaine, M. Briand, Yves Y. Barrière. Genetic variation and QTL mapping of para-coumaric and ferulic acid contents in maize stover at silage harvest. Maydica, 2003, 48, pp.75-84. hal-02682277

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# GENETIC VARIATION AND QTL MAPPING OF PARA-COUMARIC AND FERULIC ACID CONTENTS IN MAIZE STOVER AT SILAGE HARVEST

A.S. Fontaine, M. Briand, Y. Barrière*

*Unité de Génétique et d'Amélioration des Plantes Fourragères, INRA, 86600 Lusignan, France*

Received October 15, 2002

**ABSTRACT** - Genetic variation for lignin content and phenolic acid [p-coumaric (pCA) and ferulic acid (FA)] content was investigated in a set of 25 maize lines (including 2 bm3 lines) whose DINAGZ cell wall digestibility ranged from 43.1 to 60.9%. Normal and bm3 lines belonged to the same continuum, and 6 normal lines had cell wall digestibility values non significantly different from values observed in bm3 lines. Significant variations were observed for etherified pCA, esterified and etherified FA, whether bm3 lines were included or not in the analysis. Esterified pCA was lower in bm3 lines, whereas esterified FA and etherified FA were not affected by the bm3 mutation. Two flint lines (F286 and F7012) had FA patterns very different from the one observed in others lines. Phenolic acids contents were also investigated in a RIL progeny between two of the 25 previously studied lines. QTL were found for ester pCA, ester FA, and total FA contents. No QTL were found in similar locations for pCA and FA contents, and this should be considered as a new evidence of the "independent" spatio-temporal pathway and regulation between these two hydroxycinnamic acids. No QTL colocalizations were observed between DINAGZ and ester pCA, and only one colocalization was observed between DINAGZ and FA QTL. Further improvement in silage maize breeding for higher digestibility value will arise from a dissection of wall digestibility under its underlying determinants.

**KEY WORDS:** Zea mays L.; Silage maize; QTL analysis; Yield; Digestibility; Lignin, p-Coumaric acid; Ferulic acid.

**Abbreviations:**

<table><tr><td>pCA</td><td>p-Coumaric acid</td></tr><tr><td>FA</td><td>Ferulic acid</td></tr><tr><td>NDF</td><td>Neutral Detergent Fiber</td></tr><tr><td>ADF</td><td>Acid Detergent Fiber</td></tr><tr><td>ADL</td><td>Acid Detergent Lignin</td></tr><tr><td>KL</td><td>Klason Lignin</td></tr><tr><td>IVDMD</td><td>In Vitro Dry Matter Digestibility</td></tr><tr><td>DINAGZ</td><td>In Vitro Digestibility of the "Non-(Starch, soluble Carbohydrates and crude Protein)" part.</td></tr></table>

## INTRODUCTION

Lignification is the basic cause of reduced digestibility in forage crops, but, according to CASLER and JUNG (1999), "while this point is scarcely debatable, the precise mechanism by which lignification reduces digestibility is unclear". Lignin itself is clearly the cell wall component whose content has the most detrimental effect on plant wall digestibility in ruminants. From investigations in different species of grass and dicotyledons forage, other factors also appeared involved (or were considered to be involved) in digestibility limitation, out of which i) the content in ferulic (FA) phenolic acid, and more especially the covalent cross-linkages between lignin and arabinose units of arabinoxylan chains by ferulic acid bridges, ii) the cross-linking between arabinoxylan chains by diferulates, iii) the ratio between guaiacyl (G) and syringyl (S) units in lignin, and iv) the content in p-coumaric (pCA). In grass cell walls, esterified pCA content increases with physiological maturation. A higher content in esterified pCA could be negatively correlated with wall digestibility because a higher pCA content could indicate a higher plant wall maturity, and/or a higher proportion of secondary wall in tissue. The pCA esters are mainly bound to the lignin polymers, via linkage involving the hydroxymethyl terminal group of S units, and are thereby deposited in the walls concomitantly to the secondary wall lignification (JUNG and DEETZ, 1993). Conversely, ferulic acid is the major hydroxycinnamic derivative in young cell walls. FA content first increases in young walls, and then plateaus, or sometimes declines, as the walls come to maturity. FA is primarily and intracellularly esterified to non cellulosic polysaccharides, such as grass arabinoxylans, and ferulic glycosides are then exported to the maturing wall (FRY et al., 2000). A part of FA esters are additionally linked to coniferyl alcohol by ether

<footnote>* For correspondence (fax 33 5 49 55 60 44; e-mail: barriere@lusignan.inra.fr).</footnote>
bonds (JACQUET *et al.*, 1995), but also to lignins by some other bonding patterns (RALPH *et al.*, 1992-1998; GRABBER *et al.*, 2000). FA esters act as lignin initiation sites and direct cell wall cross linking (RALPH *et al.*, 1995). FA cross-linking between guaiacyl residues and arabinoxylans could thus be detrimental to wall digestibility, acting as a reinforcement of the wall structure, and decreasing wall component accessibility to micro-organism enzymes (JUNG, 1996; GRABBER *et al.*, 1998).

Different studies were devoted to phenolic acid contents in maize. GALETTI *et al.* (1996) and MORRISON *et al.* (1998) confirmed an increase of pCA in maize stover or internodes of increasing maturity. GALETTI *et al.* (1996) showed an increase of esterified FA in maize stover of increasing maturity, but, conversely, MORRISON *et al.* (1998) observed that FA contents rose and peaked in younger internodes, and declined in older internodes. According to JUNG (1996), this decline during maturation was related to continued deposition of lignin and polysaccharides, while no further FA was deposited in the wall during the latest steps of secondary wall formation. A significant genetic variation was first proved by JUNG et BUXTON (1994) for esterified and etherified pCA and FA content in the two lowest elongated internodes of 45 lines harvested at silking time. ARGILLIER *et al.* (1996) showed significant variation for esterified and etherified phenolic acid contents in basal stalks of 6 maize hybrids at silage harvest. Genetic variation in pCA and FA contents was more recently investigated at silage harvest by MÉCHIN *et al.* (2000) in basal stalks of a set of maize lines, highlighting that significant differences existed between lines, and that these traits were involved in variation of wall digestibility. In this paper, the genetic variation in pCA and FA contents was investigated on a broader genetic basis in whole maize plants of which ears were removed (namely stover) at silage harvest. Moreover, phenolic acid contents were also estimated in a RIL progeny, and QTL mapping was then investigated for pCA and FA content in maize stover, and compared to QTL simultaneously found for lignin content and wall digestibility.

# MATERIALS AND METHODS

## Germplasm used and field experiments

A set of 25 maize inbred lines, including two brown-midrib-3 (bm3) genotypes, were chosen from preliminary experiments as representative of a great genetic variation in wall digestibility, and representative of a broad base germplasm (F2, F2bm3, F7, F4,

F10, F66, F251, F286, F324, F7012, F7021, F7023, early flint), (F113, F252, F271, F288, F317, F7019, F7025, F7026bm3, W117, W401, early dent), (F838, Lam496, W95115, medium late dent), F2 and F2bm3, and F7025 and F7026bm3, are near-isogenic lines, respectively. The bm3 gene originates from a natural mutation and segregates as a simple mendelian recessive character (EMERSON, 1935). The bm3 plants exhibit a reddish brown pigmentation of the leaf midrib, have a reduced lignin content, and have greatly improved digestibility and ingestibility (review in CHERNEY *et al.*, 1991, and BARRIÈRE and ARGILLIER, 1993). Lines were cropped at Lusignan (France), for two successive years (2000 and 2001), in randomized block designs with three replicates of two 5.2 m long rows.

Investigated RILs have been previously developed at INRA Lusignan (France) by single seed descent from the cross between the elite inbreed early dent line F271, of low cell wall digestibility, and the early dent line F288, of medium digestibility (BARRIÈRE *et al.*, 2001). RILs *per se* (135 RILs), and both parents, were evaluated for one year (2001) in Lusignan, in a generalized alpha-lattice design with two replicates of one 5.2 m long row for the tested RILs, and six replicates for the parents.

In both experiments, row spacing was 0.75 m and the density was 95,000 plants per hectare. Irrigation was applied in Lusignan during summer to prevent water stress (90 to 120 mm per year). At the silage harvest stage (about 28 to 32% of dry matter (DM)), ears of all plants were hand removed, and the resulting stover were then machine-harvested with a forage chopper. A representative sample of 1 kg chopped material per plot was collected. The samples were oven-dried (65°C) and ground with a hammer mill, to pass through a 1 mm screen.

## Chemical analyses and digestibility estimate

In the 25 lines experiment, the neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) of dried stem samples were measured according to GOERING and VAN SOEST (1970). The lignin content was also determined by the Klason procedure (DENCE and LIN, 1992). Lignin contents were expressed as the weight percentage in NDF. The ratio ADL/KL was considered as an estimate of the most resistant part of lignin. The *in vitro* dry matter digestibility (IVDMD) was estimated according to AUFRÈRE and MICHALET-DOREAU (1983). Cell wall digestibility was investigated using the DINAGZ estimate [*in vitro* digestibility of the "non starch, non soluble carbohydrates and non crude protein" part] (ARGILLIER *et al.*, 1995; BARRIÈRE and EMILE, 2001), according to the formula DINAGZ = 100 x (IVDMD - starch - soluble carbohydrates - crude protein) / (100 - starch - soluble carbohydrates - crude protein).

Hydroxycinnamic acid contents were measured after treating NDF fractions with NaOH according to the procedure previously described by MORRISON III *et al.* (1993) and used by MÉCHIN *et al.* (2000), which involves two alkaline treatments differing in their severity. Esterified hydroxycinnamic acids were released by subjecting NDF samples (100 mg) to a mild alkaline hydrolysis (5 ml of 2 M NaOH, room temperature, 20 h, under N₂ and with mechanical shaking). Other NDF samples (100 mg) were placed in a Teflon screw-cap vial containing 5 mL of 4 M NaOH and purged with nitrogen. This vial was then sealed in a stainless steel reaction vessel, which was placed in an oven at 170°C for 2 hours. This severe alkaline treatment allows the release of both the esterified and the etherified hydroxycinnamic acids. The samples recovered from mild and severe alkaline hydrolyses were then subjected to the following extraction procedure. An internal standard (*p*-anisic acid) was added to the reaction medium prior to
centrifugation (380 × g for 10 min). The supernatants were acidified to pH 2 with concentrated HCl and kept at 4°C overnight before the next centrifugation step. Hydroxycinnamic acids were extracted from the aqueous supernatant with ethyl acetate and analyzed by high-performance liquid chromatography (HPLC), according to CHABBERT *et al.* (1994). The concentration of etherified FA was calculated as the difference between FA amounts released by the severe and mild alkaline treatments. In the present study and taking into account the literature data (RALPH *et al.*, 1994; HATFIELD *et al.*, 1999), only the contents in esterified pCA, esterified FA, total FA and etherified FA were reported and discussed in relation to cell wall digestibility.

In RIL experiments, because of the great number of samples, NDF, ADF, ADL, KL, and IVDMD were estimated using near infrared reflectance spectroscopy (NIRS). Calibration equations, previously described in ROUSSEL *et al.* (2002), were provided by SHB Libramont (Belgium), and calibration regressions were validated by a laboratory analysis of 40 samples. Cell wall digestibility was investigated using the DINAGZ estimate. pCA and FA contents were predicted with newly developed NIRS calibrations based on all samples from the line experiments and 30 samples of the RIL experiments (NIRS system 6500 spectrophotometer, with wavelengths spaced every 4 nm from 1100 to 2500 nm). Coefficients of determination between laboratory analysis and NIRS prediction, and standard errors of prediction, were 0.90 and 1.07 for esterified pCA, 0.66 and 0.42 for esterified FA, 0.61 and 0.47 for total FA, respectively. Among RIL progenies, values of esterified and total pCA were very close, with equal average values (13.0 and 13.1 mg/g NDF, respectively) and equal ranges, indicating that most of the pCA was only esterified, and consequently the total pCA was not further considered.

## Statistical analysis

For the set of 25 maize inbred lines, analyses of variance were carried out following the standard procedure of a fixed model with year, block, genotype, and genotype x year interaction effects. In the RIL experiment, analyses of variance were carried out following the standard procedure of a fixed model with block, sub-block and genotype effects. The Modli software (KOBILINSKY *et al.*, 1983) was used for computation of variance analyses in both experiments. RIL mean values were used in QTL analysis. Line mean values over replicates (and years in the 25 lines experiment) were used for correlation estimates and multi

ple regression analysis. Multiple regression analyses were computed with the Splus software (Venable and Ripley, 1994) with the “leaps” and “lfsist” procedures. These procedures give the best fitting regression with a chosen number of variables using the full subset of explanatory variables. The “lfsist” procedure also allows the computing of regressions with chosen variables.

## Linkage map and QTL identification in the RIL progeny

The linkage map of the F288xF271 RIL progeny was available from a previous work (BARRIÈRE *et al.*, 2001) with 108 SSR markers on 131 RILs. Some chromosomal areas remained unmarked, and were very probably monomorphic, because F271 and F288 both had line Co125 in their pedigree. As previously described in BARRIÈRE *et al.* (2001), QTL mapping was based on the mean values of 131 RILs *per se*, using the method of Composite Interval Mapping [CIM, ZENG (1994)] implemented in the PLABQTL computer package (UTZ and MELCHINGER, 1995 and 1996). A LOD threshold of 2.0 was used, yielding an individual type I error rate close to 0.25% and an experimentwise error rate of 30% (result obtained by the permutation test method of CHURCHILL and DOERGE (1994)) considered as suitable for the biological interpretation of QTLs and linkage patterns. LOD thresholds equal to respectively 2.7, 3.0 and 4.0 allowed an experimentwise error rate close to respectively 10, 5 and 1%. QTL positions were estimated where the LOD score reached its maximum in the region under consideration.

## RESULTS AND DISCUSSION

### Variance analysis and mean values in the 25 lines set

Average stover dry-matter yield of lines was 3.3 t/ha, and average stover dry-matter content at harvest was 29.7%, in agreement with expected values for harvest at the silage maturity stage in early and medium early lines. Whether the bm3 genotypes were included or not, year effects were highly significant for all investigated traits ($P < 0.01$), except etherified FA (Table 1). Genotype effects were also

<table><caption>TABLE 1 - Variance analysis of cell wall digestibility and lignification traits in the 25 lines set (mean square (MS) of effects were significant at $P < 0.01$ ** and $P < 0.05$ *).</caption><thead><tr><th></th><th>DINAGZ</th><th>NDF</th><th>KL/NDF</th><th>ADL/NDF</th><th>ADL/KL</th><th>Est pCA</th><th>Est FA</th><th>total FA</th><th>Ether FA</th></tr></thead><tbody><tr><td colspan="10">with bm3 lines</td></tr><tr><td>MS Year</td><td>52.5**</td><td>408.1**</td><td>65.4**</td><td>1.10**</td><td>511.6**</td><td>40.1**</td><td>2.47**</td><td>0.99*</td><td>0.34</td></tr><tr><td>MS Genotype</td><td>105.6**</td><td>61.1**</td><td>12.5**</td><td>3.47**</td><td>32.7**</td><td>34.7**</td><td>1.72**</td><td>1.73**</td><td>0.31**</td></tr><tr><td>MS GxY</td><td>6.2*</td><td>16.9**</td><td>1.1*</td><td>0.33**</td><td>6.6</td><td>0.5</td><td>0.28**</td><td>0.27*</td><td>0.06</td></tr><tr><td>Residual MS</td><td>3.3</td><td>1.8</td><td>0.6</td><td>0.10</td><td>5.0</td><td>0.3</td><td>0.06</td><td>0.10</td><td>0.09</td></tr><tr><td colspan="10">without bm3 lines</td></tr><tr><td>MS Year</td><td>62.4**</td><td>381.0**</td><td>65.2**</td><td>1.03**</td><td>506.7**</td><td>34.4**</td><td>2.34**</td><td>1.08*</td><td>0.24</td></tr><tr><td>MS Genotype</td><td>87.6**</td><td>65.9**</td><td>9.8**</td><td>2.52**</td><td>24.3**</td><td>24.1**</td><td>1.85**</td><td>1.86**</td><td>0.31**</td></tr><tr><td>MS GxY</td><td>6.7*</td><td>18.2**</td><td>1.1*</td><td>0.35**</td><td>6.4</td><td>0.5</td><td>0.28**</td><td>0.26*</td><td>0.06</td></tr><tr><td>Residual MS</td><td>3.5</td><td>1.8</td><td>0.6</td><td>0.10</td><td>5.3</td><td>0.3</td><td>0.06</td><td>0.10</td><td>0.09</td></tr></tbody></table>
<table><caption>TABLE 2 - Means and confidence limits of cell wall digestibility and lignification traits in the 25 lines set (hydroxycinnamic acid contents were given as mg/g NDF).</caption><thead><tr><th></th><th>DINAGZ %</th><th>NDF %</th><th>KL/NDF</th><th>ADL/NDF</th><th>ADL/KL</th><th>Est pCA</th><th>Est FA</th><th>total FA</th><th>Ether FA</th></tr></thead><tbody><tr><td>F271</td><td>43.1</td><td>60.8</td><td>16.6</td><td>6.31</td><td>37.8</td><td>11.3</td><td>4.84</td><td>6.40</td><td>1.56</td></tr><tr><td>W401</td><td>43.2</td><td>58.5</td><td>15.5</td><td>4.83</td><td>31.4</td><td>14.5</td><td>6.41</td><td>7.75</td><td>1.33</td></tr><tr><td>F252</td><td>45.2</td><td>56.8</td><td>16.0</td><td>5.25</td><td>33.0</td><td>11.5</td><td>5.63</td><td>7.19</td><td>1.56</td></tr><tr><td>F838</td><td>46.7</td><td>49.7</td><td>18.5</td><td>6.83</td><td>37.0</td><td>14.9</td><td>5.36</td><td>6.71</td><td>1.35</td></tr><tr><td>F288</td><td>48.1</td><td>53.1</td><td>16.2</td><td>5.52</td><td>34.0</td><td>11.1</td><td>5.39</td><td>6.94</td><td>1.54</td></tr><tr><td>F2</td><td>48.6</td><td>53.9</td><td>16.6</td><td>5.73</td><td>34.3</td><td>10.0</td><td>5.30</td><td>6.57</td><td>1.27</td></tr><tr><td>F317</td><td>48.7</td><td>53.1</td><td>17.2</td><td>5.77</td><td>33.4</td><td>11.5</td><td>5.86</td><td>7.20</td><td>1.35</td></tr><tr><td>F7021</td><td>48.7</td><td>56.7</td><td>17.8</td><td>6.46</td><td>36.1</td><td>10.2</td><td>5.29</td><td>6.85</td><td>1.56</td></tr><tr><td>Lan496</td><td>50.1</td><td>50.1</td><td>17.1</td><td>5.94</td><td>34.5</td><td>9.9</td><td>5.10</td><td>6.59</td><td>1.49</td></tr><tr><td>F251</td><td>50.5</td><td>59.5</td><td>15.8</td><td>5.61</td><td>35.5</td><td>9.8</td><td>5.05</td><td>6.38</td><td>1.34</td></tr><tr><td>F7023</td><td>50.9</td><td>54.9</td><td>18.1</td><td>6.71</td><td>37.1</td><td>10.9</td><td>5.09</td><td>6.36</td><td>1.28</td></tr><tr><td>F7</td><td>51.8</td><td>62.1</td><td>14.1</td><td>4.88</td><td>34.3</td><td>7.9</td><td>4.65</td><td>5.79</td><td>1.13</td></tr><tr><td>F286</td><td>51.8</td><td>54.0</td><td>15.3</td><td>4.93</td><td>32.5</td><td>10.5</td><td>6.03</td><td>6.57</td><td>0.54</td></tr><tr><td>F7019</td><td>51.9</td><td>51.4</td><td>16.1</td><td>5.60</td><td>35.3</td><td>9.0</td><td>5.80</td><td>7.19</td><td>1.39</td></tr><tr><td>W117</td><td>52.5</td><td>50.7</td><td>16.5</td><td>5.40</td><td>33.1</td><td>12.8</td><td>5.39</td><td>6.67</td><td>1.28</td></tr><tr><td>F7025</td><td>53.0</td><td>49.1</td><td>15.4</td><td>5.27</td><td>34.4</td><td>11.0</td><td>6.25</td><td>7.40</td><td>1.15</td></tr><tr><td>F113</td><td>53.0</td><td>51.3</td><td>16.1</td><td>5.59</td><td>34.7</td><td>8.9</td><td>5.72</td><td>7.17</td><td>1.44</td></tr><tr><td>F324</td><td>54.1</td><td>52.4</td><td>17.2</td><td>5.20</td><td>30.8</td><td>8.2</td><td>5.30</td><td>6.30</td><td>1.00</td></tr><tr><td>F66</td><td>54.4</td><td>51.3</td><td>15.6</td><td>4.92</td><td>31.4</td><td>7.6</td><td>4.85</td><td>6.41</td><td>1.56</td></tr><tr><td>F7012</td><td>55.1</td><td>60.2</td><td>17.9</td><td>6.74</td><td>37.8</td><td>7.3</td><td>3.77</td><td>5.04</td><td>1.27</td></tr><tr><td>W95115</td><td>55.3</td><td>50.9</td><td>14.8</td><td>4.91</td><td>33.2</td><td>7.3</td><td>4.82</td><td>6.30</td><td>1.48</td></tr><tr><td>F2bm3</td><td>55.4</td><td>53.7</td><td>12.8</td><td>3.95</td><td>31.2</td><td>6.0</td><td>5.44</td><td>7.00</td><td>1.55</td></tr><tr><td>F10</td><td>55.5</td><td>59.6</td><td>13.5</td><td>4.42</td><td>32.7</td><td>9.1</td><td>5.46</td><td>7.00</td><td>1.54</td></tr><tr><td>F7026bm3</td><td>59.9</td><td>52.2</td><td>14.3</td><td>4.04</td><td>28.6</td><td>4.2</td><td>5.64</td><td>6.73</td><td>1.09</td></tr><tr><td>F4</td><td>60.9</td><td>57.9</td><td>13.1</td><td>5.04</td><td>38.9</td><td>8.1</td><td>4.62</td><td>5.88</td><td>1.26</td></tr><tr><td>mean</td><td>51.5</td><td>54.6</td><td>15.9</td><td>5.43</td><td>34.1</td><td>9.7</td><td>5.32</td><td>6.66</td><td>1.33</td></tr><tr><td>c. limit</td><td>1.6</td><td>1.9</td><td>0.7</td><td>0.31</td><td>1.9</td><td>0.5</td><td>0.26</td><td>0.30</td><td>0.23</td></tr></tbody></table>

highly significant for all traits, and appeared lower than year effects only for NDF content, KL/NDF and ADL/KL. Genotype x year interaction for NDF content was, as expected, highly significant and rather high. However, genotype x year interactions were non significant for ADL/KL, esterified pCA and etherified FA, and clearly lower than genotype effects for DINAGZ, KL/NDF, ADL/NDF and esterified FA. A large variation was observed for DINAGZ wall digestibility that ranged from 43.1 to 60.9% (Table 2). Six normal lines had DINAGZ values not significantly different from values observed in the bm3 lines. Moreover, DINAGZ wall digestibility was significantly higher in F7026bm3 than in F2bm3, highlighting the effect of the genetic background on the improvement brought by the bm3 gene. ADL/NDF was the lowest in bm3 lines, whereas bm3 lines were only among the lowest in KL/NDF. Lines with a high lignin content were found both in flint and dent, and both in early and later germplasm. Similarly, lines with low lignin content were found in the diverse genetic origins. Line W95115, bred by J. G. Coors in

the Wisconsin Quality Synthetic (WQS) for low lignin content, appeared here effectively with a good DINAGZ and a relatively low lignin content. These results indeed corroborated works of MÉCHIN et al. (1998, 2000), that first gave evidence of the fact that normal and bm3 lines belonged to the same continuum for both lignin content and wall digestibility. Esterified pCA content ranged from 7.3 to 11.3 mg/g NDF, and was lower in bm3 as previously observed by KUC et al. (1968), LAPIERRE et al. (1988), CHABBERT et al. (1994) and BARRIÈRE and GUILLET (2001). Esterified FA and etherified FA contents were not affected by the bm3 mutation, as previously reported by BARRIÈRE and GUILLET (2001). Average contents in pCA or FA were lower in this experiment than previously reported, but investigations of JUNG and BUXTON (1994), ARGILLIER et al. (1996), or MÉCHIN et al. (2000) were done on only basal stems or internodes. Relatively to hydroxycinnamic acid contents, some lines differed from others. The line W401, which had both a fairly low lignin content and a low DINAGZ, had also one of the highest content in hydroxycinnamic
<table><caption>TABLE 3 - Correlations in the 25 lines set between cell wall digestibility (DINAGZ), lignin contents and phenolic contents in the cell wall, with and without bm3 lines above and below the diagonal, respectively. Correlations higher than 0.40 and 0.51 were significant at P = 0.05 and 0.01, respectively.</caption><thead><tr><th></th><th>DINAGZ</th><th>NDF</th><th>KL/NDF</th><th>ADL/NDF</th><th>ADL/KL</th><th>Est pCA</th><th>Est FA</th><th>total FA</th><th>Ether FA</th></tr></thead><tbody><tr><td>DINAGZ</td><td>-</td><td>-0.16</td><td>-0.53</td><td>-0.47</td><td>-0.18</td><td>-0.78</td><td>-0.29</td><td>-0.39</td><td>-0.25</td></tr><tr><td>NDF</td><td>-0.11</td><td>-</td><td>-0.20</td><td>0.03</td><td>0.30</td><td>-0.05</td><td>-0.40</td><td>-0.37</td><td>0.05</td></tr><tr><td>KL/NDF</td><td>-0.46</td><td>-0.28</td><td>-</td><td>0.88</td><td>0.37</td><td>0.51</td><td>-0.10</td><td>-0.08</td><td>0.04</td></tr><tr><td>ADL/NDF</td><td>-0.33</td><td>0.05</td><td>0.86</td><td>-</td><td>0.76</td><td>0.49</td><td>-0.32</td><td>-0.26</td><td>0.15</td></tr><tr><td>ADL/KL</td><td>-0.07</td><td>0.28</td><td>0.21</td><td>-0.68</td><td>-</td><td>0.28</td><td>-0.48</td><td>-0.41</td><td>0.18</td></tr><tr><td>Ester pCA</td><td>-0.72</td><td>-0.16</td><td>0.37</td><td>0.26</td><td>-0.03</td><td>-</td><td>0.40</td><td>0.42</td><td>0.06</td></tr><tr><td>Ester FA</td><td>-0.39</td><td>-0.39</td><td>-0.06</td><td>-0.31</td><td>-0.50</td><td>0.58</td><td>-</td><td>0.92</td><td>-0.20</td></tr><tr><td>Total FA</td><td>-0.48</td><td>-0.37</td><td>-0.03</td><td>-0.24</td><td>-0.44</td><td>0.59</td><td>0.92</td><td>-</td><td>0.21</td></tr><tr><td>Ether FA</td><td>-0.23</td><td>0.04</td><td>0.08</td><td>0.18</td><td>0.16</td><td>0.02</td><td>-0.19</td><td>0.20</td><td>-</td></tr></tbody></table>

acids. Line F7012 had an esterified and total FA content lower than all the other lines, but its etherified FA value was in the variation range of other genotypes. This could be related to different defects in enzyme activities, including enzymes involved in glucoside transport. Line F286 had an etherified FA content much lower than all other lines, and line F324, which a progeny of F286, had also a low content in etherified FA. In the cell wall, polysaccharide ferulate esters undergo radical coupling reactions involving wall-associated oxydases, such as peroxydases and laccases. RANOCHA et al. (2002) indeed showed that laccase down-regulation caused accumulation of total soluble phenolics in poplar, and alteration of cell wall structure, without lignin content modification. F286 appeared altered in a trait involved in binding with lignin and had a high cell wall digestibility. F286 could thus be hypothesized as altered in the functioning of one specific oxydative mechanism. However, since only β-ether are quantifiable by alkaline hydrolysis (RALPH, 1996), a greater propensity of coupling with more resistant linkage could not be completely excluded in F286. This last hypothesis did not appear very likely, especially as this line was highly digestible.

## Relationships between cell wall digestibility and cell wall component traits in the 25 lines set

Very low correlations were observed between cell wall content (NDF) and DINAGZ cell wall digestibility (Table 3), corroborating data obtained by INRA Lusignan from in vivo experiments (BARRIÈRE et al., 2003). These results proved the feasibility of breeding highly digestible forage maize with given starch and soluble carbohydrates contents. Correlations between DINAGZ and any of wall component traits were low, except with esterified pCA, and to a much

lower extent, with KL/NDF and ADL/NDF which were highly correlated (Table 3). These correlations did not differ greatly when bm3 hybrids were included or not. Lines having DINAGZ equal to 53-55% had also an ADL/NDF, KL/NDF or ADL/KL distributed among the minimum - maximum range (Table 2). Five lines had an ADL/NDF close to 6.5%, differing by less than 0.5%, but their DINAGZ ranged from 43.1 to 55.1%. Wall lignin content only explained 25% of the observed variation of wall digestibility in this set of genotypes, whether lignin content was estimated with KL/NDF or ADL/NDF. Higher values observed for KL/NDF than for ADL/NDF were due to the solubilization of lignin components by the ADL treatment (HATFIELD et al., 1994). A genetic variation existed between lines for the soluble part of lignin, because the ADL/KL ratio was highly significant. However, this ADL/KL ratio, which was more related to ADL/NDF than to KL/NDF, was not related to DINAGZ, and did not appear of direct interest in plant breeding. Breeders must use a digestibility trait in forage maize digestibility improvement, as lignin content can be a poor predictor of digestibility in sets of plants of similar maturity.

As previously observed (BUXTON and RUSSELL, 1988; JUNG, 1989), pCA content was related to lignin content, but esterified pCA unexpectedly explained 61% of the observed variation in DINAGZ, a much greater part than the one explained by lignin content. Conversely, correlations between esterified or etherified FA and ADL/NDF or KL/NDF contents were very low, and correlations between esterified or etherified FA contents and DINAGZ were low. These last results corroborated the results of CASLER and JUNG (1999) on smooth bromegrass showing that the amount of FA etherified to lignin and the lignin content were independent features. Esterified FA and
etherified FA were also not correlated. This could be
expected since FA esters are deposited primarily dur-
ing primary cell wall deposition, and FA ethers dur-
ing secondary cell wall deposition. But this also indi-
cated that the availability of FA esters was not the
major determinant of coupling with lignin. The best
fitting regression between DINAGZ and the studied
lignification traits, with two variables, explained 65%
(57% without bm3 lines) of the observed variation,
and was based on esterified pCA and etherified FA
contents. When esterified pCA content was replaced
in the regression by KL/NDF, the explained variation
was only 34%. However, without esterified pCA con-
tent, the regression with KL/NDF fitted better with
total FA than with etherified FA content, and ex-
plained 47.0% of the observed DINAGZ variation
(45% without bm3 lines). When considering only the
eleven lines with the highest KL/NDF, ranging from
16.2 to 18.5%, and whose range in DINAGZ was
about 10 percent points, the best fitting regression
with two variables was again based on esterified
pCA and etherified FA contents, and explained 63%
of the observed DINAGZ variation. Reciprocally,
when considering the twelve lines with the lowest
KL/NDF (without bm3 lines), ranging from 13.1 to
16.1%, and whose range in DINAGZ was about 18
percent points (equal to the entire range of variation
in DINAGZ), the best fitting regression with two vari-
ables was then based on esterified pCA content and
KL/NDF, and explained 77% of the observed DI-
NAGZ variation. Very similar results were obtained
when splitting the set of lines round the median val-
ue of KL/NDF (over and under 15.8%, giving two
groups of 15 and 8 lines, respectively). As a tentative
conclusion from this set of lines, when the wall
lignin content ranged from low to medium values,
the lignin content could be a major factor of wall di-
gestibility. Reciprocally, when the wall lignin content
ranged from medium to high values, an increase in
lignin content could not be the most detrimental fac-
tor to wall digestibility, but esterified pCA and etheri-
fied FA could explain the remaining variation in DI-
NAGZ. However, the S/G ratio, not investigated
here, should be taken into account for a better un-
derstanding of lignin characteristics involved in
maize wall digestibility (MÉCHIN *et al.*, 2000).

Variance analysis and mean values
in the F288 x F271 RIL progeny

Average RIL stover dry-matter yield at harvest
was 3.2 t/ha, and average dry-matter content was
30.2%, in agreement with the usual value observed

at silage maize maturity. Genotype effects were
highly significant for all investigated traits, including
phenolic acid contents (Table 4). Ranges of varia-
tion for DINAGZ and lignin contents were higher in
this experiment with stover parts of plants than the
values previously observed over 7 locations with
the whole plants of the same RIL progenies (ROUSSEL
et al., 2002). The parental lines F288 and F271 had
very close pCA and FA contents (Table 4), as it was
also observed through the line comparison experi-
ments (Table 2), but largely transgressive lines were
observed in the RIL progeny. The best fitting regres-
sion between DINAGZ and the studied lignification
traits, with two variables, explained 77% of the ob-
served variation, and was based on ADL/NDF and
etherified FA, both with negative coefficients. Un-
like to the above study in 25 lines, ester pCA con-
tent did not appeared as greatly related to DINAGZ
in the F288 x F271 progeny (r = -0.31), whereas
ether FA content was strongly related (r = -0.78).
Similarly, in a representative subset of 40 RIL of the
Co125 x W401 progeny, the best fitting regression
explaining cell wall digestibility was based on lignin
content and total phenolic content, and explained
80% of the observed variation (MARVIN et al., 1995).
In these F288 x F271 and Co125 x W401 progenies,
lignin content explained about 50% of the variation
in cell wall digestibility. 25 to 30% additional varia-
tion was explained by phenolic acid contents.

QTL mapping for phenolic acids
in the F288 x F271 RIL progeny

QTL for DINAGZ, Hemicellulose/NDF, Cellu-
lose/NDF and lignin content observed in this one
location experiment with stover parts of plants
(Table 5) were in agreement with multilocal previ-
ous observations in whole plants of the same RIL
progeny (ROUSSEL et al., 2002), except one QTL ex-
tra localization for DINAGZ and ADL/NDF in posi-
tion 4-110 (bin 4.08). However, MÉCHIN et al. (2001)
had also observed a QTL for DINAG in the bin 4.08
in the Io x F2 progeny. QTL were found for ester
pCA, ester FA, and total FA contents, but although
genotype variation was highly significant for etheri-
fied FA contents, no significant QTL for this trait
were observed in this progeny. The part of genetic
variation explained by each QTL ranged from 7.5 to
10.3% for ester pCA, from 7.6 to 8.7% for ester FA,
and from 6.8 to 12.1% for total FA. Similar values
were observed for KL/NDF (6.7 to 9.9%), but a
higher part of the genetic variation was explained
by each QTL for ADL/NDF (10.3 to 22.1%) and for
<table><caption>TABLE 4 - Mean and range values, and variance analysis of cell wall digestibility and lignification traits in the RIL experiment (genotype mean square (gen MS) of traits were all significant at P < 0.01, res MS = residual MS).</caption><thead><tr><th></th><th>DINAGZ</th><th>Hemicellulose /NDF</th><th>Cellulose /NDF</th><th>KL/NDF</th><th>ADL/NDF</th><th>ester pCA</th><th>ester FA</th><th>total FA</th><th>ether FA</th></tr></thead><tbody><tr><td>F271</td><td>39.3</td><td>38.8</td><td>52.5</td><td>18.9</td><td>8.7</td><td>12.3</td><td>5.11</td><td>6.40</td><td>1.29</td></tr><tr><td>F288</td><td>50.3</td><td>40.2</td><td>52.9</td><td>16.3</td><td>6.9</td><td>12.9</td><td>5.91</td><td>7.23</td><td>1.31</td></tr><tr><td>mini</td><td>29.1</td><td>35.2</td><td>50.0</td><td>12.9</td><td>6.1</td><td>9.8</td><td>4.06</td><td>5.60</td><td>0.93</td></tr><tr><td>maxi</td><td>52.7</td><td>42.5</td><td>56.2</td><td>21.7</td><td>9.9</td><td>16.5</td><td>6.25</td><td>7.67</td><td>1.89</td></tr><tr><td>mean</td><td>43.7</td><td>39.5</td><td>52.7</td><td>17.2</td><td>7.9</td><td>13.0</td><td>5.46</td><td>6.83</td><td>1.37</td></tr><tr><td>gen MS</td><td>45.12</td><td>4.35</td><td>2.60</td><td>4.27</td><td>1.10</td><td>2.51</td><td>0.35</td><td>0.27</td><td>0.053</td></tr><tr><td>res MS</td><td>3.42</td><td>0.57</td><td>0.43</td><td>0.57</td><td>0.09</td><td>0.52</td><td>0.07</td><td>0.08</td><td>0.014</td></tr></tbody></table>

DINAGZ (10.1 to 13.8%). No QTL were found in similar location for pCA and FA contents, confirming possibly the “independent” spatio-temporal pathway and regulation between this two hydroxycinnamic acids, in agreement with the available biochemical data in growing cell walls (RALPH et al., 1995; JUNG, 1996; FRY et al., 2000). Conversely, QTL for ester FA and total FA contents were all found exactly in the same locations, with higher LOD for total FA content than for ester FA content in positions 2-72 and 3-76. The QTL found for ester and total FA contents in position 3-76 could be directly involved in the FA biosynthesis, transportation or linkage to wall components, because it did not colocalize with any QTL of wall component contents. Similarly, all four QTL found for ester pCA contents did not colocalize with any other investigated traits, and they

could then be specific to pCA transportation or linkage to lignin units. The lack of QTL colocalization between DINAGZ and ester pCA corroborated the weak relationship between these two traits in this RIL progeny, whereas a strong relationship between these traits observed in the set of 25 lines. From a study in a 48 RIL progeny descended in the late Cm37 x T232 hybrid, NI et al. (1998) reported that markers, located in the vicinity of bm2, bm3 and bm4 genes, were associated with FA ether content. They also reported that the marker npi568, located in bin 7.02, was associated with pCA and FA esters, and also S/G ratio. No colocalization of QTL for phenolics was found in the F288 x F271 and Cm37 x T232 progenies, but the QTL found in one RIL progeny were likely a sub-set out of all QTL involved in pCA or FA biosynthesis, transportation or

<table><caption>TABLE 5 - Putative QTL identified for wall constituents and phenolics in the wall from 131 RIL in the F288 x F271 progeny (genomic location of the QTL, and, in brackets, LOD value).</caption><thead><tr><th>bin</th><th>DINAGZ</th><th>Hemicellulose /NDF</th><th>Cellulose /NDF</th><th>KL/NDF</th><th>ADL/NDF</th><th>ester pCA</th><th>ester FA</th><th>total FA</th></tr></thead><tbody><tr><td>1.02</td><td>-</td><td>1-112 (2.1)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>2.04</td><td>-</td><td>2-74 (3.7)</td><td>2-70 (3.7)</td><td>-</td><td>-</td><td>-</td><td>2-72 (2.4)</td><td>2-72 (3.5)</td></tr><tr><td>3.03/04</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3-74 (2.3)</td><td>3-76 (3.7)</td></tr><tr><td>3.05</td><td>3-136 (3.0)</td><td>-</td><td>-</td><td>-</td><td>3-140 (4.1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>3.08</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3-238 (2.2)</td><td>-</td><td>-</td></tr><tr><td>4.08</td><td>4-110 (4.2)</td><td>-</td><td>-</td><td>-</td><td>4-110 (2.0)</td><td>-</td><td>4-110 (2.6)</td><td>4-110 (2.0)</td></tr><tr><td>4.09/10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>4-220 (2.8)</td><td>-</td><td>-</td></tr><tr><td>6.01</td><td>6-8 (3.5)</td><td>-</td><td>-</td><td>6-0 (2.8)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>6.04</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6-78 (3.1)</td><td>-</td><td>-</td></tr><tr><td>6.06</td><td>6-186 (3.3)</td><td>6-162 (8.1)</td><td>6-160 (5.5)</td><td>6-162 (2.8)</td><td>6-182 (7.1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>7.01</td><td>-</td><td>-</td><td>-</td><td>7-104 (2.0)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>8.05</td><td>8-40 (3.1)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>9.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>9-14 (2.6)</td><td>-</td><td>-</td></tr><tr><td>9.02</td><td>-</td><td>-</td><td>-</td><td>9-80 (2.2)</td><td>9-100 (3.1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>9.05</td><td>-</td><td>9-142 (2.6)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
linkage to lignin units. Moreover, no colocalization
were seemingly found between any QTL of pheno-
lic acid found in the F288 x F271 progeny and any
public data of mapping of lignin pathway enzymes.

CONCLUSION

After the work of MÉCHIN *et al.* (2000) in maize
basal stalks, a great range of variation in hydroxy-
cinnamic acid contents was proved in the stover of
a set of lines and a RIL progeny that both had a
great range in lignin content and wall digestibility.
Lignin content was the primary determinant of wall
digestibility, but hydroxycinnamic acids were also
proven having a significant effect on wall digestibili-
ty. A higher content in esterified pCA could be neg-
atively correlated with wall digestibility because a
higher pCA content indicated higher plant wall ma-
turity, and/or a higher proportion of secondary wall
in tissue. FA cross-linking between guaiacyl residues
and arabinoxylans was clearly negatively related to
wall digestibility. This is one of the factors detri-
mental to wall digestibility that silage maize breed-
ers have to take into account. In most cases, QTL
related to pCA or FA contents did not colocalize
with QTL lignin content, and thus appeared to be
very likely related to the distinct physiological
mechanisms of the cell wall building. This was in
agreement with the reasonable genetic indepen-
dence that CASLER and JUNG (1999) achieved by
breeding divergent clones of smooth bromegrass for
their lignin or etherified FA contents. Efficient
breeding of forage maize for a higher energy value
can be based first on a cell wall digestibility criteri-
on such as DINAGZ. But further improvements will
arise from a dissection of wall digestibility in its un-
derlying determinants, lignin content, S/G ratio, and
hydroxycinnamic acid contents. This dissection
should be favored as it allows the gathering of com-
plementary traits favorable to digestibility in elite
line through marker assisted selection. It is also all
the more relevant as lignin content is also involved
in plant standability, and water and sap conduction.
The silage maize ideotype should combine a favor-
able combination of lignin content and structure,
phenolic acids content, linkages between FA and
lignin, and diferulate cross-linking between arabi-
noxylans, in order to allow the best compromise
between agronomic and feeding value traits.

ACKNOWLEDGMENTS - We thank the maize breeding compa-
nies involved in the ProMaïs - INRA network "DINAG" that con-

tribute to the funding of this forage maize research. We thank V.
Méchin for her helpful criticisms during the writing of this paper.
We also thank all contributors to experiments and analysis, espe-
cially D. Denoue, V. Menanteau and A. Beauchaud.

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## ERRATUM

**Maydica 46: 69-74 (2001)**

Title "Stability of corn hybrids (Zea Mays L.) for grain yield in Argentina".

In the authors a mistake was done during proofs correction by deleting the name Weilenmann de Tau.

The authors of the article regret for this mistake.

The authorship of the paper is: J. Luquez, L. Jura, E. Weilenmann de Tau