# Genetic variation for lignin content and cell wall digestibility in early maize lines derived from ancient landraces

Yves Y. Barrière, Alain A. Charcosset, Dominique Dénoue, Delphine D. Madur, Cyril Bauland, Jacques Laborde

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Yves Y. Barrière, Alain A. Charcosset, Dominique Dénoue, Delphine D. Madur, Cyril Bauland, et al. Genetic variation for lignin content and cell wall digestibility in early maize lines derived from ancient landraces. Maydica, 2010, 55 (1), pp.65-74. hal-02666723

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# GENETIC VARIATION FOR LIGNIN CONTENT AND CELL WALL DIGESTIBILITY IN EARLY MAIZE LINES DERIVED FROM ANCIENT LANDRACES

Y. Barrière¹*, A. Charcosset², D. Denoue¹, D. Madur², C. Bauland², J. Laborde³

¹ INRA, Unité de Génétique et d'Amélioration des Plantes Fourragères, BP6, FR-86600 Lusignan, France

² INRA, Unité de Génétique Végétale, Ferme du Moulon, FR-91170 Gif sur Yvette, France

³ INRA, Station Expérimentale du Maïs, FR-40590 St Martin de Hinx, France

Received February 3, 2010

**ABSTRACT** - Remarkable genetic improvement in maize yield, lodging resistance, biotic and abiotic stress tolerance were obtained during the 1955 - 2000 period in Europe. However, a decline in the average cell wall digestibility of maize hybrids has been observed during the 1980 - 2000 period. This trend has now ceased with the breeding of specialized silage varieties. The cell wall degradability of the best current hybrids does not yet equal that of better old types of the 1960s era such as INRA258, mostly because the germplasm currently used in maize breeding corresponds to progenies of resources initially chosen for grain maize breeding. Nearly 110 new lines were thus derived by self-pollination in old or unused landraces and accessions in order to test the interest of this kind of genetic resources in maize breeding for cell wall degradability traits. Based on *per se* and top-cross experiments, several lines representative of different and new germplasm displayed high or medium high cell wall digestibility. F7103 (Argentina-BBC143), F7104 (Argentina-BBC325), F7106 (Gelderland-VC150), F7114 (Zakarpatskaja-Zeltaja-Zubovidnaja), and F7121 (ComptonsEarly) are likely among the more promising new resource lines for improvement of cell wall digestibility in elite dent or flint germplasm. These lines covered a large genetic diversity as F7103 and F7104 are mostly lowland tropical, F7106 and F7121 mostly northern flint, F7114 mostly Oh43/Minnesota13 related. Two other lines of medium-high cell wall digestibility, F7101 (St Engrace) and F7112 (Mecklenburger), were shown to be significantly related to lowland tropical germplasm. Several landraces or old varieties also gave lines of low cell wall digestibility such as F7124 (Curaco-de-Velez) or F7126 (Baanbreker), both related to the northern flint group. Average yield of top-crossed lines was nearly 7 t/ha lower than the average value of the three control hybrids and only one hybrid (F7104 x UH002) had yield and earliness equal to those of control hybrids. These low values were related to the fact that investigated lines were derived from old resources that did not get the last 60 years genetic improvements, and also to the only use of a flint male for top-crosses that did not optimize the genetic distance with all top-crossed lines. Allele sequencing and association genetics should give markers for targeted introgression of new alleles of interest originating from this specific set of lines in elite lines.

**KEY WORDS:** Maize; Cell wall; Silage; Biofuel; Lignin; Digestibility; Degradability; Genetic resources.

## INTRODUCTION

Grasses are the major source of nutrients for wild and domesticated herbivores. Among grasses, maize is the most important annual forage crop in the European Union, in particular in northern Europe where nearly 3.5 million hectares are ensiled. In France, 90% of milk is produced by cows fed on silage maize. The great value of forage maize for livestock was quickly recognized since the periods of exploration of America and after its introduction in Europe. PARMENTIER (1791) wrote that “cows eat maize forage greedily, and it makes them yield a lot of milk”. Maize was, for long time, mostly used as fresh green fodder for animal feeding during summer and ensiling of maize was not common in France and in other European countries at the end of 1940s and early in the 1950s (CAUDERON et al., 1951). At that time, varieties were landraces and/or hybrids between landraces or synthetics. Silage development began with the first European-bred flint x dent hybrids which rapidly conquered European northern countries in the 1960s and 1970s after the introduction of US dent x dent hybrids in the early fifties.

Maize hybrids of the late fifties to the late seventies involved flint lines which mostly belonged to Lacaune and Yellow Bade groups and dent lines which were mostly related to Wisconsin and Min-

<footnote>* For correspondence (fax +33 549 55 60 44; e.mail: yves.barriere@lusignan.inra.fr).</footnote>
Minnesota bred germplasms. LG11, INRA258, Blizzard G188, Brillant DK202 and Capella were thus among the most popular maize early hybrids used for silage in Europe during the period of 1970-1980. The registration of Dea (1980) in France and Golda (1984) in Germany illustrated major changes in dent, and to a lesser extent flint, maize germplasm. Golda had an early BSSS-related line as female and a Co255-type flint x dent line as male, while Dea had an Iodent-related line as female and the INRA F2 line as male. Since Dea registration, the Iodent germplasm has become the foundation of nearly all early hybrids and the basis of further early maize improvements in Europe. Early and medium early dent parental lines of current hybrids are related to Iodent and BSSS families, sometimes introgressed of later dent germplasms. Early European flint parental lines of current hybrids are often related to INRA F2 and introgressed by dent germplasm, with no further use of lines related to INRA F7 or Galicia Ep1. Moreover, a few early European hybrids have even two dent parents. These germplasm changes together with considerable breeding efforts mostly focused on grain maize have allowed remarkable genetic improvement in grain yield, close to 0.1 t/ha.year, but also in plant standability, and biotic and abiotic stress tolerance. However, unfavorable correlative consequences for silage cell wall degradability, and consequently on silage energy value, have been observed. Two thirds of hybrids registered in France before 1980 had cell wall degradability higher or equal to that of LG11, while this was true for only one eighth of hybrids registered between 1994 and 2002 (BARRIÈRE et al., 2004). The decline in the average cell wall digestibility value observed since 1980 has now ceased with the breeding of specialized silage maize and the use of a digestibility criterion in forage maize registration since 1998 in France. The cell wall degradability of the best modern hybrids does not yet equal that of the best older types such as INRA258, but it is currently close to the one of Dea-type hybrids. In middle-long to long cycle maize genotypes (FAO 400 to 800), mostly bred for grain production but of interest for high biomass production, the average cell wall digestibility and energy value were shown to be nearly 15% lower than in early genotypes (LUNDVALL et al., 1994; BERARDO et al., 1999; BARRIÈRE et al., 2004, 2009).

Further improvements in maize cell wall digestibility towards silage cell wall degradability and energy value, but also straw-based biofuel produc-

tion, in the USA or in Europe, might require targeted (re)-introduction and/or use of specific germplasm in current elite germplasm. Flint genetic resources with high cell wall degradability are mostly related to a few European-bred genotypes. Among INRA lines created so far, F4 related to Northern flint germplasm, F7 originating from the Lacaune landrace, F66 related to the Sost landrace, F286 and F324 related to both F7 and Argentina origins are likely resources for most of the favorable alleles involved in high cell wall degradability (BARRIÈRE et al., 2009). In dent material, genetic resources with high cell wall degradability are also limited to a few origins, despite the fact that dent groups represent the largest share of available genetic resources in temperate maize. Out of publicly available early dent lines, W117 and F113 were shown several times to have high cell wall degradability and other old lines such as F107, F115 and W33 are also suspected to be of interest for this trait. These lines often have a common ancestry in the old open-pollinated variety Minnesota13. However, not all Minnesota13 derived lines have high cell wall degradability and favorable alleles for cell wall degradability also exits in other dent groups including Lancaster origins. The Minnesota13 variety was bred for grain yield and earliness at the University of Minnesota after the purchase of a common yellow dent corn cultivar in 1893 in St. Paul (TROYER, 1999), and alleles of interest for cell wall related traits in this landrace does not thus result of specific breeding. Several progenies of F113 and W117 have been bred for higher cell wall degradability at INRA Lusignan, such as F7019 and F7082 reaching 107 and 113% of F113 cell wall degradability value, respectively. In later germplasm, dent lines have been bred in the Wisconsin Quality Synthetic (BUENDGEN et al., 1990; FREY et al., 2004), based on top-cross with Stiff Stalk related lines. Released lines such as W94129 and W95115 have lignin contents significantly lower than in lines of similar earliness.

The germplasm currently used in maize breeding mostly corresponds to resources chosen for grain maize breeding, or their progenies, even if several breeding companies also have programs devoted to silage and now biofuel purposes. Consequently, it is questionable whether it is of interest to carry out investigations of cell wall traits in old accessions and exotic resources unused so far, in order to discover new mechanisms or alleles allowing significant wall degradability improvement. Nearly 110 lines were
thus derived by self-pollination in old and/or un-used landraces in order to test the interest of these genetic resources in maize breeding for cell wall degradability traits.

## MATERIALS AND METHODS

Investigated genetic resources were chosen from lists of plant genetic resource centers as landraces or old hybrids having no progenies in early line collection of INRA and public institutes (Table 1). A specific focus was made on European resources cropped at the beginning of maize breeding including those known to be used in cattle feeding. The well-known landrace Gelber-Badisher-Landmais was added as control.

Germplasm has been obtained from the Center of Genetic Resources of the Netherlands in Wageningen for Dutch, German, and Russian resources, Spanish and Portuguese landraces, and a few US origins. Most of Canadian and US landraces were obtained from the Plant Gene Resources of Canada in Saskatoon. French landraces were obtained from the Centre de Ressources Génétiques Maïs in INRA Montpellier. Argentina and Chili landraces corresponded to accessions obtained many years ago by INRA breeder M. POLLACSEK during a stay in South America. Among these resources, VC150 is the only Dutch variety available in collections. It most likely originates from an old landrace of the province Gelderland (www.cgn.wur.nl). Goudster was the first early maize hybrid developed in Europe (1952). It has been used as forage in the Netherlands and Northern Europe (BUNTING, 1978), followed in 1953 by Foliant (CIV6). Baanbreker (1941) and Noordlander (1938) are two older Dutch cultivars.

In order to investigate the genetic variation within each landrace, it was planned to obtain three lines in each of the origins. These lines were developed through independent self-pollinations tracing back to different individual initial plants, and with the objective to reach different genotypes and phenotypes (plant height, leaf more or less erected, silking date, ...) within each landrace or cultivar. Lines per se value experiments were carried out at INRA Lusignan in 2003, 2004, and 2005, corresponding to S4, S5, and S6 generations of inbreeding. F7025 and F7026bm3 (brown-midrib3) INRA dent lines, which are two sister lines of medium and high cell wall digestibility obtained from the Iodent

x F113bm3 hybrid, were added as control lines. The well-known INRA F2 line was also added as flint control line. Topcross experiment was carried out at INRA Lusignan in 2007 and 2008, with UH002 flint line as pollinator. Line seed quantities were limited and did not allow top-cross production with two males of contrasted heterotic patterns. Anjou304, Nexxos and PR38V12 which have a medium-high, medium-low, and medium-low cell wall digestibility, respectively, were added as control registered hybrids. Lines and top-crosses were evaluated in lattice designs with 2 replicates. All experimental plots were 5.2 m long single rows of 37 plants. Row spacing was 0.75 m, and the density was 90,000 plants ha⁻¹. Irrigation was applied according needs during summer to prevent water stress. Plots were machine-harvested in a single day with a forage chopper at silage maturity (nearly 35% DM on average). A representative sample of 1 kg chopped material per plot was collected for dry-matter (DM) content estimates and biochemical analysis. Samples were dried in a ventilated oven (65°C), and dry samples were ground with a hammer mill to pass through a 1-mm screen for further analyses.

Neutral Detergent Fiber (NDF) and Acid Detergent Lignin (ADL) were investigated according GOERING and VAN SOEST (1970). Hemicelluloses (Hcell) and cellulose (Cell) were estimated as usual as NDF - ADF and ADF - ADL, respectively. Because these compounds are constituent of the cell wall, Hcell, Cell, and ADL were expressed as percentage of NDF (Hcell/NDF, Cell/NDF, ADL/NDF, respectively). The *in vitro* dry matter digestibility (IVDMD) was estimated according to AUFRÈRE and MICHALET-DOREAU (1983). Cell wall digestibility was investigated according to STRUIK (1983) and DOLSTRA and MEDEMA (1990). *In vitro* NDF digestibility (IVNDFD) was thus computed assuming that the non-NDF part of plant material was completely digestible [IVNDFD = 100 x (IVDMD - (100 - NDF)) / NDF]. Cell wall digestibility was also estimated in topcross experiments as the *in vitro* digestibility of the “non starch, non soluble carbohydrates, and non crude protein” part (DINAGZ, ARGILLIER et al., 1995, BARIÈRE et al., 2003) and computed assuming these three constituents are completely digestible [DINAGZ = 100 x (IVDMD - ST - SC - CP) / (100 - ST - SC - CP)] where ST, SC and CP are starch, soluble carbohydrates, and crude protein contents, respectively. All components were estimated using near infrared reflectance spectroscopy (NIRS, NIRS system 6500 spectrophotometer) and the robust calibrations available from the Centre de Recherches Agronomiques in Libramont (Belgium).

<table><caption>TABLE 1 - List of investigated origins as genetic resources for forage maize breeding.</caption><thead><tr><td>Geographical origin</td><td>Landrace or cultivar</td></tr></thead><tbody><tr><td>Argentina and Chili</td><td>BBC143, BBC325, BBC448, Chacao, Chacao estero, Curaco de Velez, Dalcahue, Puerto, Quemchi, Quemchi delgan, Villarica</td></tr><tr><td>Canada and USA</td><td>Beacon, Comptons early flint, Dakota white flint, Falconer, Gehu, Manalia, Parker flint, Polar dent, Quebec28, Rainbow flint, Salzer's white flint, Saskatchewan white flint, Tama</td></tr><tr><td>France</td><td>Argelès, Cassagnabère, Laval-Luzège, Saint-Engrace, Sarrancolin, Val d'Aran</td></tr><tr><td>Germany</td><td>Chiemgauer, Dr Delilles, Gelber Badisher Landmais, Mecklenburger, Pautzfelder, Rottaler silomais</td></tr><tr><td>The Nederlands</td><td>Baanbreker, Goudster, Foliant CIV6, Noordlander VC145, Gelderland VC150, VC175, Vroege Gele CB3-04</td></tr><tr><td>Portugal and Spain</td><td>Alto, Amarello colorado AldeiaMato, Amarello de Peroviseu, Cedovem senhora Conceicao, Grelha de Lebre, Millariega</td></tr><tr><td>Russia</td><td>Datnuva1, Sibiriacka, Zakarpatskaja Zeltaja Zubovidnaja</td></tr></tbody></table>
Analyses of variance were carried out following the standard procedure of a fixed model with genotype, year (or environment), block, sub-block and interaction effects, as $Y_{ijkl} = \mu + E_i + B_k \cdot E_j + SB_l \cdot B_k \cdot E_j + G_i + G_i \cdot E_j + \varepsilon_{ijkl}$, with $Y_{ijkl}$ = observed value for a given trait, $\mu$ = grand mean, $E_i$ = environment effect, $B_k \cdot E_j$ = block within environment effect, $SB_l \cdot B_k \cdot E_j$ = sub-block within block and environment effect, $G_i$ = genotype effect, $G_i \cdot E_j$ = genotype x environment interaction, and $\varepsilon_{ijkl}$ = residual error ($= \sigma_i^2$), using Modli (Least Square Means; KOBILINSKY *et al.*, 1983) and Splus Software (VENABLES and RIPLEY, 1994). Pearson's phenotypic correlations between traits were computed on the mean basis over years. These correlations were significantly different from zero for absolute values higher than 0.30 (P = 0.01).

Neutral markers (55 SSR) have been used to evaluate the genetic structure of maize diversity in a collection of 375 lines representative of the European and American genetic variation by CAMUS-KULANDAIVELU *et al.* (2006), with primer pairs chosen on the basis of their ability to detect a single locus, their broad genome coverage, and their good reproducibility in allele size determination. Based on this work and further investigations at INRA le Moulon, it was established for estimating the genetic lineages of these new lines a six-group structure including BSSS, Lancaster, Oh43 and Minnesota13, Pyrenees and Galicia flint, and low-land exotics including Eto and Tuxpeño related germplasms (VEYRIERAS, 2009, software Locus Miner 1.0). Oh43 and Minnesota13 groups have a close genetic relationship and this fact is at least related to the W8 ancestry of Oh43, given that W8 was bred in Minnesota13.

## RESULTS AND DISCUSSION

At least one line was obtained in each investigated genetic resource, except for the Chili origin Puerto. Because several S1 families gave less than three viable progenies, only 109 lines were obtained from 51 origins. Moreover, due to their low vigor and/or sterility, progenies of Quemchi, Chacao, Villarica, and Rottaler-silomais could not be self-pollinated successfully beyond the S2 stage. In this case, evaluations were based on a mix of S2 grains. In addition, for the same reasons, only 73 lines gave enough seeds for top-cross experiments.

## Mean and genetic variation analysis

**Lines per se experiments** - Average line DM content ranged from 33 to 43% according to years, with an average value equal to 37%, corresponding to high maturity stages which are convenient for a relevant investigation of lignification traits, even if cell wall digestibility was possibly slightly under-estimated in lines with the highest DM contents. Average silking date was July 16th, corresponding to early lines, but with a 30 days range between the earliest and the latest flowering lines. Genotype effects were highly significant for all investigated traits (P < 0.001), and much higher than genotype x year (GY) interaction effects for lignin and cell wall digestibility traits (Table 2). Conversely, GY interaction effects were nearly equal to genotype effects for Hcell and Cell contents, likely because ranges of genetic variation were low for these two traits. Among the 109 investigated lines, those with highest cell wall digestibility values had values equal to the bm3 control line while lines with the lowest cell wall digestibility had values close the lowest known ones. As previously observed for large ranges of variation in the two traits, cell wall digestibility was related to lignin content with r² value equal to 0.77 between ADL/NDF and IVNDFD.

Based on *per se* experiments, new germplasm of interest in forage maize breeding includes first the Gelderland-VC150 origin, and especially the line F7106 which had an IVNDFD *per se* value nearly equal to that of F7026bm3 with a similar earliness as INRA F2 (Table 3). Lines derived from Zakarpatskaja Zeltaja Zubovidnaja (F7114), Sibiriacka (F1113), and Comptons early (F7121) could also be considered as new resources for forage maize breeding. Two very early lines were obtained from the VGCB3-04 origin, but their cell wall digestibility was only equal

<table><caption>TABLE 2 - Variance analysis and trait means for lines in per se value experiments (genotype and genotype x year effects are significant at P < 0.001; silking dates are expressed as days in July, NDF = Neutral Detergent Fiber, Cell = cellulose, Hcell = bemicelluloses, ADF = Acid Detergent Lignin, IVNDFD = in vitro NDF digestibility, DINAGZ = cell wall digestibility according to ARGILLIER *et al.*, 1995, all components are expressed as percentages).</caption><thead><tr><th>Traits</th><th>genotype<br>MS</th><th>gen x year<br>MS</th><th>residual<br>MS</th><th>mean</th><th>mini</th><th>maxi</th><th>F2</th><th>F7025</th><th>F7026<br>bm3</th></tr></thead><tbody><tr><td>Silking date</td><td>221.6</td><td>22.8</td><td>14.9</td><td>16.1</td><td>3.7</td><td>35.3</td><td>12.9</td><td>23.4</td><td>21.5</td></tr><tr><td>NDF</td><td>65.7</td><td>15.8</td><td>6.0</td><td>45.0</td><td>38.0</td><td>54.7</td><td>43.8</td><td>40.9</td><td>42.1</td></tr><tr><td>Cell/NDF</td><td>7.0</td><td>4.7</td><td>1.3</td><td>39.2</td><td>37.1</td><td>41.9</td><td>39.6</td><td>38.8</td><td>38.7</td></tr><tr><td>Hcell/NDF</td><td>6.8</td><td>8.4</td><td>1.8</td><td>39.1</td><td>36.3</td><td>42.5</td><td>38.9</td><td>37.2</td><td>39.0</td></tr><tr><td>ADL/NDF</td><td>2.27</td><td>0.38</td><td>0.17</td><td>5.7</td><td>4.4</td><td>8.3</td><td>5.9</td><td>6.1</td><td>4.7</td></tr><tr><td>IVNDFD</td><td>47.6</td><td>9.6</td><td>5.2</td><td>25.8</td><td>15.6</td><td>32.8</td><td>26.8</td><td>25.2</td><td>31.5</td></tr></tbody></table>
<table><caption>TABLE 3 - Average values of a set lines with highest and lowest cell wall digestibility values in per se value experiments (Lines are ranked for their per se decreasing IVNDFD cell wall digestibility values. Silking date is expressed as days in July, IVNDFD and DINAGZ are cell wall digestibility traits, and ADL/NDF is lignin content in the cell wall, all expressed as percentage. LSD is Least Significant Difference at P = 5%).</caption><thead><tr><th>Landrace or hybrid origins of lines</th><th>INRA name</th><th>Silking date</th><th>IVNDFD</th><th>ADL/NDF</th></tr></thead><tbody><tr><td>Argentina-BBC143-5</td><td>F7103</td><td>35.3</td><td>32.8</td><td>4.4</td></tr><tr><td>Argentina-BBC325-3</td><td>F7104</td><td>27.8</td><td>30.9</td><td>4.5</td></tr><tr><td>Zakarpatskaja-Zeltaja-Zub.-2</td><td>F7114</td><td>25.5</td><td>30.8</td><td>5.1</td></tr><tr><td>Gelderland-VC150-1</td><td>F7106</td><td>13.0</td><td>30.5</td><td>4.8</td></tr><tr><td>Comptons-Early-3-2</td><td>F7121</td><td>20.5</td><td>30.5</td><td>4.7</td></tr><tr><td>Sibiriacka-2</td><td>F7113</td><td>15.5</td><td>29.8</td><td>5.1</td></tr><tr><td>Argentina-BBC448-1</td><td>F7105</td><td>16.5</td><td>29.4</td><td>4.8</td></tr><tr><td>Noordlander-VC145-1</td><td>F7107</td><td>19.5</td><td>29.4</td><td>5.3</td></tr><tr><td>Gelderland-VC150-3</td><td>F7108</td><td>14.0</td><td>29.1</td><td>5.2</td></tr><tr><td>Amarelo-de-Poreviseu-2</td><td>F7119</td><td>27.2</td><td>29.0</td><td>5.0</td></tr><tr><td>Chacao-estero-2</td><td>F7120</td><td>19.2</td><td>28.9</td><td>5.2</td></tr><tr><td>Mecklenburger-2</td><td>F7112</td><td>12.7</td><td>28.5</td><td>5.4</td></tr><tr><td>Tama-2</td><td>F7116</td><td>23.5</td><td>27.9</td><td>5.5</td></tr><tr><td>Comptons-Early-3-1</td><td>F7117</td><td>22.3</td><td>27.9</td><td>5.3</td></tr><tr><td>Saint-Engrace-3</td><td>F7101</td><td>18.3</td><td>27.7</td><td>5.6</td></tr><tr><td>Vroege-Gele-CB3-04-1-2</td><td>F7109</td><td>8.5</td><td>27.7</td><td>5.6</td></tr><tr><td>Val-d'Aran-2</td><td>F7102</td><td>14.2</td><td>27.6</td><td>5.3</td></tr><tr><td>Baanbreker-1</td><td>F7110</td><td>3.7</td><td>27.6</td><td>5.7</td></tr><tr><td>Vroege-Gele-CB3-04-1-1</td><td>F7111</td><td>15.8</td><td>27.6</td><td>5.4</td></tr><tr><td>Quebec28-3</td><td>F7118</td><td>17.3</td><td>27.3</td><td>5.3</td></tr><tr><td>Goudster-2</td><td>F7127</td><td>14.7</td><td>26.9</td><td>5.3</td></tr><tr><td>Rottaler-silomais-1</td><td>F7128</td><td>11.8</td><td>26.4</td><td>5.5</td></tr><tr><td>Rainbow-flint-3</td><td>F7115</td><td>19.7</td><td>26.4</td><td>5.1</td></tr><tr><td>Gelber-Badisher-Landmais-2</td><td>F7122</td><td>14.2</td><td>25.8</td><td>5.4</td></tr><tr><td>Gelber-Badisher-Landmais-3</td><td>F7123</td><td>12.3</td><td>24.8</td><td>6.0</td></tr><tr><td>Curaco-de-Velez-1</td><td>F7124</td><td>16.0</td><td>20.6</td><td>6.9</td></tr><tr><td>Gehu-5</td><td>F7125</td><td>14.6</td><td>19.1</td><td>7.5</td></tr><tr><td>Baanbreker-2</td><td>F7126</td><td>16.0</td><td>15.6</td><td>8.3</td></tr><tr><td>F7026bm3</td><td>-</td><td>21.5</td><td>31.5</td><td>4.7</td></tr><tr><td>F2</td><td>-</td><td>12.9</td><td>26.8</td><td>5.9</td></tr><tr><td>F7025</td><td>-</td><td>23.4</td><td>25.2</td><td>6.1</td></tr><tr><td>LSD</td><td>-</td><td>3.1</td><td>1.7</td><td>0.3</td></tr></tbody></table>

to that of F2. Lines with high cell wall digestibility were also obtained from the three Argentinean origins BBC143 (F7103), BBC325 (F7104), and BBC448 (F7105), which all had low lignin contents. However, F7103 was significantly later silking than all other lines. The two progenies obtained from Gelber Badisher landmais (F7122 and F7123) had *per se* values close to that of INRA line F2. The only progeny obtained from Rottaler silomais had low cell wall digestibility and low vigor. The progeny obtained from Goudster also had low vigor, but a higher cell wall digestibility. Among investigated

French landraces, only one progeny of St Engrace, and at a lower level one progeny of Val d'Aran, gave lines with medium-high cell wall digestibility (F7101 and F7102). A few lines displayed high lignin content and low cell wall digestibility (F7124, F7125, and F7126), including a line obtained from the Baanbeker origin (F7126) while another progeny of the same variety (F7110) had medium values.

**Topcross experiments** - Average hybrid DM content was equal to 36% and corresponded to maturity stages convenient for relevant investigation of
<table><caption>TABLE 4 - Variance analysis and trait mean for lines in topcross experiments (** = P < 0.001, * = P < 0.01, ° = P < 0.05, ns = non significant; NDF = Neutral Detergent Fiber, Cell = cellulose, Hcell = bemicelluloses, ADF = Acid Detergent Lignin, IVNDFD = in vitro NDF digestibility, DINAGZ = cell wall digestibility according to ARGILLIER et al., 1995; yield is given as t/ha, silking dates as days in July, starch and NDF as DM%, other traits as percentages).</caption><thead><tr><th>Traits</th><th>genotype<br>MS</th><th>gen x year<br>MS</th><th>residual<br>MS</th><th>mean</th><th>mini</th><th>Maxi</th><th>Nexxos</th><th>Anjou304</th></tr></thead><tbody><tr><td>Yield</td><td>61.7 **</td><td>3.6 *</td><td>2.2</td><td>14.3</td><td>8.4</td><td>20.1</td><td>22.1</td><td>19.7</td></tr><tr><td>Dry matter</td><td>29.6 **</td><td>2.8 *</td><td>1.5</td><td>36.2</td><td>31.2</td><td>42.6</td><td>33.5</td><td>34.4</td></tr><tr><td>Silking date</td><td>53.5 **</td><td>2.3 *</td><td>1.5</td><td>15.2</td><td>8.3</td><td>21.0</td><td>21.8</td><td>20.0</td></tr><tr><td>Starch</td><td>35.5 **</td><td>5.9 °</td><td>4.0</td><td>32.7</td><td>23.4</td><td>38.4</td><td>31.9</td><td>30.0</td></tr><tr><td>NDF</td><td>8.7 **</td><td>2.2 ns</td><td>2.0</td><td>38.4</td><td>35.5</td><td>42.3</td><td>39.6</td><td>37.0</td></tr><tr><td>Cell/NDF</td><td>1.9 **</td><td>0.5 °</td><td>0.3</td><td>48.7</td><td>47.1</td><td>49.6</td><td>49.7</td><td>47.8</td></tr><tr><td>Hcell/NDF</td><td>2.2 **</td><td>0.7 °</td><td>0.4</td><td>45.0</td><td>43.8</td><td>46.5</td><td>44.2</td><td>46.5</td></tr><tr><td>ADL/NDF</td><td>0.20 **</td><td>0.04 ns</td><td>0.03</td><td>6.3</td><td>5.8</td><td>6.7</td><td>6.2</td><td>5.8</td></tr><tr><td>IVNDFD</td><td>6.8 **</td><td>1.4 ns</td><td>1.0</td><td>22.3</td><td>19.7</td><td>25.3</td><td>23.5</td><td>24.6</td></tr><tr><td>DINAGZ</td><td>9.5 **</td><td>1.7 ns</td><td>1.2</td><td>45.2</td><td>42.3</td><td>50.5</td><td>45.6</td><td>49.4</td></tr></tbody></table>

agronomic and cell wall related traits (Table 4). Average silking date was July 15th, nearly five days earlier than the two control hybrids, illustrating the earliness of this set of genotypes. A 13 days range of variation between the earliest and the latest flowering hybrids was observed, a value reduced by more than 50% in comparison to the range between lines *per se* evaluation. Genotype effects were highly significant for all investigated traits (P < 0.001), and much higher than genotype × year (GY) interaction effects. No significant genotype × year interaction was observed for lignin content and the two cell wall digestibility traits. As it was observed in lines, ranges of variation and genotype effects were low for Hcell and Cell contents. Average yield of top-crossed lines was nearly 7 t/ha lower than the average value of the three control hybrids and only one hybrid (F7104 x UH002) had yield and earliness equal to those of control hybrids. This low value was related to the fact that, contrarily to check hybrids, investigated lines were derived from old resources that did not benefit from intense breeding conducted during the last 60 years. This low value is also related for many lines to the use of a flint male in top-crosses and consequently to lower heterosis than what could be obtained with a more genetically distant male. Average starch content of the set of top-crossed lines was equal to the value observed in experimental hybrids.

In topcross experiments, the mini-maxi range in IVNDFD was equal to 5.4 percent points while it was 3-fold higher, equal to 17.2 percent points, in *per se* experiments. The two cell wall traits were

correlated with r² value between IVNDFD and DINAGZ equal to 0.42, corroborating the fact that each of these two traits does not estimated identically the cell wall digestibility characteristics (MÉCHIN et al., 2001; ROUSSEL et al., 2002). Several top-crossed lines had similar quality trait values than the control hybrid Anjou304, while the F7106 x UH002 hybrid was little higher, strengthening the interest this Gelderland-VC150 progeny (Table 5). A high cell wall digestibility was observed with the F7127 x UH002 hybrid, which was higher than expected from the F7127 (Goudster) line *per se* value. Because several lines had *per se* values higher than the control line F2, cell wall digestibility of the whole set of progeny is probably underestimated as a consequence of the use of UH002 line as top-cross male, as this line is of low cell wall degradability. Range of variation of lignin content was low, slightly lower than 1%, but correlations between lignin content and cell wall digestibility were nevertheless significant with r² values between ADL/NDF and IVNDFD and DINAGZ equal to 0.39 and 0.53, respectively. Correlations between *per se* and top-cross values were low with r² values equal to 0.35 for both ADL/NDF and IVNDFD traits. These values corresponded to the lowest observed ones as previous values were shown to range from 0.38 to 0.88 for cell wall digestibility traits, and from 0.39 to 0.76 for lignin content (BARRIÈRE et al., 2003).

## Line genetic structure

Genetic structure was investigated for lines with the highest IVNDFD cell wall digestibility values
<table><caption>TABLE 5 - Average values of a set of lines with highest and lowest cell wall digestibility values in top-cross experiments (Lines are ranked for their per se decreasing IVNDFD cell wall digestibility values. NDF = Neutral Detergent Fiber, ADF = Acid Detergent Lignin, IVNDFD = in vitro NDF digestibility, DINAGZ = cell wall digestibility according to ARGILLIER et al., 1995. Yield is given as t/ha, and other traits as percentages, na = not available data. LSD = Least Significant Difference at P = 5%).</caption><thead><tr><th>Landrace or hybrid origins of lines</th><th>INRA name</th><th>Yield</th><th>DM%</th><th>ADL/NDF</th><th>IVNDFD</th><th>DINAGZ</th></tr></thead><tbody><tr><td>Argentina-BBC143-5</td><td>F7103</td><td>13.8</td><td>29.3</td><td>6.0</td><td>23.7</td><td>46.8</td></tr><tr><td>Argentina-BBC325-3</td><td>F7104</td><td>20.1</td><td>33.4</td><td>6.3</td><td>22.4</td><td>44.7</td></tr><tr><td>Zakarpatskaja-Zeltaja-Zub.-2</td><td>F7114</td><td>16.4</td><td>34.8</td><td>6.3</td><td>22.0</td><td>46.2</td></tr><tr><td>Gelderland-VC150-1</td><td>F7106</td><td>15.0</td><td>35.8</td><td>6.1</td><td>25.3</td><td>47.1</td></tr><tr><td>Comptons-Early-3-2</td><td>F7121</td><td>15.1</td><td>33.7</td><td>6.0</td><td>24.5</td><td>46.3</td></tr><tr><td>Sibiriacka-2</td><td>F7113</td><td>13.8</td><td>34.0</td><td>6.3</td><td>23.2</td><td>45.2</td></tr><tr><td>Argentina-BBC448-1</td><td>F7105</td><td>15.7</td><td>33.8</td><td>5.9</td><td>23.5</td><td>47.0</td></tr><tr><td>Noordlander-VC145-1</td><td>F7107</td><td>14.9</td><td>36.4</td><td>6.2</td><td>23.0</td><td>45.5</td></tr><tr><td>Gelderland-VC150-3</td><td>F7108</td><td>13.8</td><td>37.8</td><td>6.2</td><td>23.7</td><td>45.5</td></tr><tr><td>Amarelo-de-Poreviseu-2</td><td>F7119</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Chacao-estero.4-2</td><td>F7120</td><td>14.8</td><td>37.2</td><td>6.4</td><td>22.7</td><td>45.1</td></tr><tr><td>Mecklenburger-2</td><td>F7112</td><td>14.6</td><td>39.2</td><td>6.1</td><td>24.5</td><td>45.8</td></tr><tr><td>Tama-2</td><td>F7116</td><td>15.5</td><td>34.2</td><td>6.4</td><td>21.3</td><td>45.1</td></tr><tr><td>Comptons-Early-3-1</td><td>F7117</td><td>13.3</td><td>33.6</td><td>6.3</td><td>21.8</td><td>45.2</td></tr><tr><td>Saint-Engrace-3</td><td>F7101</td><td>14.3</td><td>36.6</td><td>6.4</td><td>21.8</td><td>45.5</td></tr><tr><td>Vroege-Gele-CB3-04-1-3</td><td>F7109</td><td>12.5</td><td>37.7</td><td>6.5</td><td>22.5</td><td>44.9</td></tr><tr><td>Val-d'Aran-2</td><td>F7102</td><td>14.4</td><td>37.0</td><td>6.1</td><td>23.8</td><td>46.2</td></tr><tr><td>Baanbreker-1</td><td>F7110</td><td>11.1</td><td>40.0</td><td>6.3</td><td>23.5</td><td>44.7</td></tr><tr><td>Vroege-Gele-CB3-04-1-1</td><td>F7111</td><td>14.3</td><td>39.5</td><td>6.6</td><td>21.8</td><td>44.9</td></tr><tr><td>Quebec28-3</td><td>F7118</td><td>14.7</td><td>36.9</td><td>6.6</td><td>21.4</td><td>42.2</td></tr><tr><td>Goudster-2</td><td>F7127</td><td>11.1</td><td>32.6</td><td>5.8</td><td>25.3</td><td>50.5</td></tr><tr><td>Rottaler-silomais-1</td><td>F7128</td><td>15.1</td><td>37.3</td><td>6.5</td><td>22.8</td><td>44.2</td></tr><tr><td>Rainbow-flint-3</td><td>F7115</td><td>11.7</td><td>36.4</td><td>6.2</td><td>23.9</td><td>44.9</td></tr><tr><td>Gelber-Badisher-Landmais-2</td><td>F7122</td><td>15.1</td><td>40.0</td><td>6.2</td><td>23.2</td><td>45.4</td></tr><tr><td>Gelber-Badisher-Landmais-3</td><td>F7123</td><td>14.4</td><td>38.4</td><td>6.5</td><td>21.1</td><td>43.7</td></tr><tr><td>Curaco-de-Velez-1</td><td>F7124</td><td>13.7</td><td>38.4</td><td>6.6</td><td>19.9</td><td>42.5</td></tr><tr><td>Gehu-5</td><td>F7125</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Baanbreker-2</td><td>F7126</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Nexxos</td><td>–</td><td>22.9</td><td>33.5</td><td>6.2</td><td>23.5</td><td>45.6</td></tr><tr><td>Anjou304</td><td>–</td><td>19.7</td><td>34.4</td><td>5.8</td><td>24.6</td><td>49.4</td></tr><tr><td>PR38V12</td><td>–</td><td>18.0</td><td>33.5</td><td>6.5</td><td>20.9</td><td>45.2</td></tr><tr><td>LSD</td><td>–</td><td>1.2</td><td>0.9</td><td>0.2</td><td>0.8</td><td>0.9</td></tr></tbody></table>

and five lines with lower values (Table 6). Probably due to the choice of early genetic resources, derived lines showed significant northern flint ances- tries. Fifteen lines out of 26 have at least one third of northern flint lineage and only four are not related to this group. Similarly, 16 lines out of 26 are related to the Oh43/Minnesota13 group, strengthening this group as a resource of favorable allele in breeding for higher cell wall degradability. The F7106 (Gelderland-VC150-1) line, which is mostly related to northern flint and has also an Oh43/Minnesota13

lineage, is thus probably a new resource of allele of interest, in addition to lines previously bred which displayed a high cell digestibility in former studies (BARRIÈRE et al., 2009). The F7121 (Comptons-Early-3-2) line is as F7106 mostly related to the northern flint group, but with a relationship with the Pyrenees/Galicia flint group. The F7114 (Zakarpatskaja-Zeltaja-Zubovidnaja-2) appeared to be close to old Oh43/Minnesota13 dent germplasm used in the European hybrids of the 1970s, but with a cell wall digestibility value higher than what is currently
<table><caption>TABLE 6 - Genetic structure of lines based on six genetic groups (BSSS = Iowa Stiff Stalk Synthetic, Min13 = Minnesota13, Pyrenees Galicia Fl = Pyrenees and Galicia flint, na = not available data. Lines are ranked for their per se decreasing IVNDFD cell wall digestibility values).</caption><thead><tr><th rowspan="2">Lines</th><th rowspan="2">Origins of lines</th><th rowspan="2">BSSS</th><th rowspan="2">Lancaster</th><th colspan="3">Genetic relationships</th><th rowspan="2">Lowland Tropical</th></tr><tr><th>Oh43 Min13</th><th>Pyrenees Galicia Fl</th><th>Northern Flint</th></tr></thead><tbody><tr><td>F7103</td><td>Argentina-BBC143-5</td><td>2</td><td>0</td><td>4</td><td>0</td><td>38</td><td>56</td></tr><tr><td>F7104</td><td>Argentina-BBC325-3</td><td>0</td><td>23</td><td>0</td><td>0</td><td>0</td><td>77</td></tr><tr><td>F7114</td><td>Zakarpatskaja-Zeltaja-Zub.-2</td><td>6</td><td>0</td><td>85</td><td>0</td><td>0</td><td>9</td></tr><tr><td>F7106</td><td>Gelderland-VC150-1</td><td>2</td><td>0</td><td>11</td><td>0</td><td>86</td><td>0</td></tr><tr><td>F7121</td><td>Comptons-Early-3-2</td><td>0</td><td>0</td><td>0</td><td>13</td><td>87</td><td>0</td></tr><tr><td>F7113</td><td>Sibiriacka-2</td><td>10</td><td>47</td><td>17</td><td>8</td><td>18</td><td>0</td></tr><tr><td>F7105</td><td>Argentina-BBC448-1</td><td>0</td><td>0</td><td>70</td><td>0</td><td>25</td><td>5</td></tr><tr><td>F7107</td><td>Noordlander-VC145-1</td><td>6</td><td>15</td><td>38</td><td>5</td><td>19</td><td>18</td></tr><tr><td>F7108</td><td>Gelderland-VC150-3</td><td>0</td><td>26</td><td>0</td><td>1</td><td>73</td><td>0</td></tr><tr><td>F7119</td><td>Amarelo-de-Poreviseu-2</td><td>4</td><td>25</td><td>9</td><td>14</td><td>37</td><td>11</td></tr><tr><td>F7120</td><td>Chacao-estero-2</td><td>0</td><td>66</td><td>8</td><td>0</td><td>26</td><td>0</td></tr><tr><td>F7112</td><td>Mecklenburger-2</td><td>0</td><td>0</td><td>6</td><td>7</td><td>57</td><td>31</td></tr><tr><td>F7116</td><td>Tama-2</td><td>13</td><td>17</td><td>45</td><td>7</td><td>18</td><td>0</td></tr><tr><td>F7117</td><td>Comptons-Early-3-1</td><td>0</td><td>3</td><td>0</td><td>7</td><td>90</td><td>0</td></tr><tr><td>F7101</td><td>Saint-Engrace-3</td><td>0</td><td>0</td><td>0</td><td>71</td><td>0</td><td>29</td></tr><tr><td>F7109</td><td>Vroege-Gele-CB3-4-1-3</td><td>1</td><td>0</td><td>39</td><td>19</td><td>41</td><td>0</td></tr><tr><td>F7102</td><td>Val-d'Aran-2</td><td>0</td><td>6</td><td>0</td><td>77</td><td>0</td><td>18</td></tr><tr><td>F7110</td><td>Baanbreker-1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>0</td></tr><tr><td>F7111</td><td>Vroege-Gele-CB3-4-1-1</td><td>0</td><td>2</td><td>27</td><td>43</td><td>28</td><td>0</td></tr><tr><td>F7118</td><td>Quebec28-3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>0</td></tr><tr><td>F7127</td><td>Goudster-2</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>F7128</td><td>Rottaler-silomais-1</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>F7115</td><td>Rainbow-flint-3</td><td>2</td><td>0</td><td>26</td><td>9</td><td>46</td><td>17</td></tr><tr><td>F7122</td><td>Gelber-Badisher-Landmais-2</td><td>0</td><td>3</td><td>19</td><td>5</td><td>64</td><td>10</td></tr><tr><td>F7123</td><td>Gelber-Badisher-Landmais-3</td><td>5</td><td>0</td><td>10</td><td>17</td><td>52</td><td>16</td></tr><tr><td>F7124</td><td>Curaco-de-Velez-1</td><td>14</td><td>5</td><td>12</td><td>0</td><td>58</td><td>11</td></tr><tr><td>F7125</td><td>Gehu-5</td><td>0</td><td>1</td><td>0</td><td>15</td><td>85</td><td>0</td></tr><tr><td>F7126</td><td>Baanbreker-2</td><td>0</td><td>8</td><td>0</td><td>0</td><td>92</td><td>0</td></tr><tr><td>F2</td><td>Lacaune</td><td>0</td><td>0</td><td>0</td><td>100</td><td>0</td><td>0</td></tr><tr><td>UH002</td><td>Northern Flint</td><td>0</td><td>0</td><td>0</td><td>30</td><td>70</td><td>0</td></tr><tr><td>F7025</td><td>Iodent x F113bm3</td><td>69</td><td>0</td><td>30</td><td>0</td><td>1</td><td>0</td></tr><tr><td>F113</td><td>Reid Yellow Dent x Minnesota13</td><td>0</td><td>25</td><td>75</td><td>0</td><td>0</td><td>0</td></tr><tr><td>W117</td><td>Minnesota13 x Golden Krug</td><td>1</td><td>51</td><td>19</td><td>0</td><td>29</td><td>0</td></tr></tbody></table>

known in lines of this group such as F113 or W117. This line is also related to lowland tropical germplasm. An original genetic background was shown in the F7104 (Argentina-BBC325-3) line of high cell wall degradability, which appeared mostly related to lowland tropical germplasm (77%) introgressed with US Lancaster dent, without any relationships with flint or northern flint origins. The F7103 (Argentina-BBC143-5) line was also shown to be a related to tropical germplasm (56%), but also

to the northern flint group (38%). The third Argentina line, F7105 (BBC448-1), was shown to be of a different origin, belonging mostly to Oh43/Minnesota13 and Northern flint groups, with only 5% of exotic lineage. F7101 (St-Engrace-3) and F7112 (Mecklenburger-2) progenies differed from other lines derived from northern Europe landraces by their relationships to exotic germplasm with 29 and 31% of lowland tropical related origin. This fact could be in agreement with the history of maize introduction in
the south of Europe (REBOURG *et al.*, 2003). Complex genetic ancestry within landraces was shown from the two F7122 and F7123 (Gelber Badisher Landmais) lines, even if nearly half of their lineages was related to northern flint germplasm. F7122 is typified by a relationship with the Oh43/Minnesota13 group while F7023 has a relationship with the Pyrenees/Galicia flint group, and both are for a part related to lowland exotics. Such an unexpected result should be confirmed, as it could also be consequence of out-crosses in the received Gelber Badisher Landmais landrace or during self-pollinations. Different genetic lineages were also observed between the two F7117 and F7121 (Comptons-Early) lines, and to a lesser extent between the two F7106 and F7108 (Gelderland-VC150) lines, illustrating a large genetic diversity within these landraces.

## CONCLUSION

Several lines displaying high or medium high cell wall digestibility in *per se* and top-cross experiments, representative of different and new germplasm could be selected in the set of investigated lines. F7106, F7103, F7114, F7104, and F7121 are likely among the more promising resources for improvement of cell wall digestibility in elite dent or flint germplasm, in addition to flint (F4, F7, F66, F286, F324) or dent lines (F113, W117, F7019, F7058, F7082). Moreover, these investigations have shown that new alleles of interest for cell wall traits can be searched for in exotic germplasm which has not currently been considered for this thematic up to now. The agronomic value of these newly released lines is lower than in the current elite germplasm, and strategies for their use have to be considered as for other genetic resources. Even if a large number of genes are involved in maize cell wall biosynthesis and assembly (GUILLAUMIE *et al.*, 2007; BARRIÈRE *et al.*, 2009), several investigations have pointed out the crucial role of a set of genes in the genetic variation of cell wall digestibility (GUILLET-CLAUDE *et al.*, 2004; ANDERSEN *et al.*, 2007; SHI *et al.*, 2007; GUILLAUMIE *et al.*, 2008; RIBOULET *et al.*, 2009; SONBOL *et al.*, 2009). Allele sequencing of genes involved in monolignol biosynthesis or in its regulation such as ZmPAL (phenylalanine ammonia lyase), ZmCOMT (caffeic acid O-methyltransferase), ZmCAD (cinnamyl alcohol dehydrogenase), ZmMYB, ZmNAC, etc, is likely a first way allowing a better characterization of these new lines. They

could also be characterized by a larger set of markers (SNP) available for association genetic panels. QTL investigations in progenies of a set of crosses such as F7106 x elite-line should contribute to give relevant information on the specific areas to be investigated in elite material.

In addition to lignin, maize and grass cell walls are also characterized by significant amounts of esterified *p*-coumaric (*pCA*), esterified and etherified ferulic (esterFA, etherFA) *p*-hydroxycinnamic acids. Even if its mode of action is unknown, *pCA* content, which is easily quantifiable, appears to be an efficient marker of cell wall digestibility. Along with lignin content, etherFA, which is involved in cell wall component cross-linkages, has a direct effect on cell wall digestibility. The newly obtained lines are probably also of interest as genetic resources of new alleles of genes involved in *pCA* biosynthesis and in ether linkage assembly in the cell wall, and the new best lines should also be characterized for these traits.

ACKNOWLEDGEMENTS - This work was funded by INRA and the breeding companies involved in the PROMAÏS - INRA network on maize cell wall lignification and digestibility (Advanta, Caussade Semences, Limagrain Genetics, MaïsAdour, Monsanto SAS, Pioneer Génétique, Pau Euralis, R2n RAGT Semences, SDME KWS France, Syngenta seeds). Thanks to Christiane Minault, Pascal Vernoux, and Jacques Laborde for their help in experimentation and seed production. We are grateful to Fabrice Dumas and Valérie Combes for their contribution to line genotyping. We are also grateful to Corinne Melin for the maize reference database management.

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