Localization

The experiment was performed in plastic pots during the fall-winter of 2014-2015 and the spring-summer of 2015 at Colegio de Postgraduados, Texcoco, Estado de México (19° 29’ N, 98° 54’ W, 2,250 masl). The climate is humid subtropical (Cb(wo)(w)(i´)g), with rains during summer, annual precipitation of 637 mm, and temperature of 15 °C¹¹.

Plant material

It was used a total of 200 half-sib families (HSF) from the commercial varieties of San Miguel and Oaxaca (certified seeds from Casa Cobos S.A. de C.V., Central de Abastos, Ciudad de México, Mexico) and 200 HSF from the segregating populations of San Miguel (female progenitor) x Oaxaca (male progenitor) (direct cross) and Oaxaca (female progenitor) x San Miguel (male progenitor) (reciprocal cross). The seeds from segregating populations were obtained by the genetic cross between the San Miguel and Oaxaca varieties (direct cross) and the Oaxaca and San Miguel varieties (reciprocal cross), using pollinator insects (bees) under field conditions (Figure 1). The San Miguel and Oaxaca varieties were chosen for the crossings due to their notorious durability under field conditions in an experiment designed to study the productive behavior of five commercial varieties of alfalfa (San Miguel, Oaxaca, Moapa, Valenciana, and Cuf-101) during March 2000¹², this experiment was conducted until the winter-spring cycle of 2006 at Colegio de Postgraduados, Montecillo, Texcoco, Estado de México.

Figure 1: Genetic crossing system between two alfalfa varieties using pollinator insects under field conditions. Montecillo, Municipio de Texcoco, Estado de México 

Seed production took place in the period from January to May 2006. Seeds were harvested from all the plants in each cross's population and used to establish the first family selection cycle⁶. A styrofoam seed tray with 100 cells was used for each population. Five seeds of the same size were sown I n each cell. The soil used had a sandy loam texture with 41.6 % of field capacity (FC), 28.2 % of permanent wilting point (PWP), 9.3 % of organic matter , 0.019 % of nitrogen, 4.8 ppm of phosphorus, 4 mmol L^-1 of potassium, 1.5 dS m^-1 of electric conductivity, and pH of 6.9. Seeds were sown on June 6, 2014; when the seedlings presented the first trifoliate leaf (15 days after sowing, das), the most vigorous seedling was chosen from each cell, and it was transplanted into a plastic pot with a soil capacity of 3 kg. The HSF of each population were randomly assigned to the plastic pots in a completely randomized experimental design. It was fertilized with the 60-140-00 dose at 15 and 240 das, with urea as a nitrogen source and calcium triple superphosphate as a phosphorus source. At 98 das, a uniformization cut was made in the plants. During the experiment, soil humidity was maintained close to FC by applying water every other day.

Variables

Cuts were made every five weeks in the fall-winter 2014-2015 period and every 4 wk in the spring-summer 2015 period from day 98; the cuts were made 5 cm above ground level. The plant's morphological composition was evaluated in a subsample of four complete secondary tillers from each plant and cut in all the HSF, separating the leaves (leaflets and petioles) from the tillers. The subsample and the rest of the secondary tillers were dried at 65 °C until constant weight and were then weighted.

Plant height (PH, cm) was measured in all the HSF before the cutting with a 1 m long wooden ruler (graduated in cm) from the soil surface to the stem apex. The number of secondary tillers (NT) per plant was determined in each cut in all the HSF. The dry matter yield (DMY, g of DM plant^-1) or aerial biomass was obtained by adding the dry weight of the subsample of the four stems and the dry weight of the rest of the plant. The leaf:stem ratio (L:S) was calculated with the dry weight data from the leaves (LDW) and secondary tillers (TDW) (L:S = LDW / TDW). The absolute growth rate (AGR, g of DM plant^-1 d^-1) was calculated by dividing the DMY by the number of days (t) elapsed between one cut and the next (AGR = DMY/t). The radiation use efficiency (RUE, g of DM MJ^-1) was calculated by dividing the DMY by the amount of photosynthetically active radiation (PAR, MJ MJ m^-2 d^-1) accumulated (PAR= incident global radiation (cal cm^-2 d^-1) x 0.5 x 0.04148) between one cut and the next (RUE= DMY / PAR)¹³. The incident global radiation data were obtained from the meteorological station of the Universidad Autónoma Chapingo, Chapingo, Estado de México, located 4 km away from the experiment location.

Air temperature and rain

The maximum (TM) and minimum (Tm) air temperature data were recorded daily at 0800 h with a Six's thermometer (Taylor, model 5458P) placed 2 m above ground level. The TM ranged from 23.5 to 29.9 °C and the Tm from 4.0 to 12.6 °C during the experiment. Daily rainfall data were determined with a weekly accumulation rain gauge placed next to the plants. The total rain precipitation was 1,042 mm; the lowest levels occurred from November 2014 to January 2015; this did not affect plant growth as plants were irrigated as necessary.

Statistical analysis

The analysis of variance for all variables was performed using the Windows software SAS, Version 9.1¹⁴ and the statistical model:

Y _ijk = µ + Pop _i + Fam(Pop) _ik + Cutoff _j + (Pop*Cutoff) _ij + Cutoff*Fam(Pop) _ijk + E _ijk ;

Where:

Y _ijk represents the value of the response variable in the population i of the cutoff level j and k of the HSF;

µ is the general mean, Pob _i is the Population effect at level i = 1, 2, 3, and 4;

Fam(Pop) _ik is the effect of population nested half-sib families at level i and k;

Cutoff _j is the effect of the cutoff date at level j = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11;

(Pop*Cutoff) _ij is the effect of the Population x Cutoff interaction at level i and j;

Cutoff*Fam(Pop) _ijk is the effect of the Cutoff x population nested HSF interaction at level i, j, k;

E _ijk is the experimental error.

The Cutoff*Fam(Pob) _ijk component could not be separated from the error because there was no source of variation for replications in the analysis of variance.

Genetic and environmental effects

Genetic and environmental effects were calculated through phenotypic variance (σf2) and its components, genetic (σg2) and environmental variance (σe2), where the total or phenotypic variance is the sum of σg2 and σe2 (σf2 = σg2 + σe2)¹⁵. Variances were estimated with the mean squares of each population based on the analysis of variance obtained with the statistical model Y _ijk = µ + Fam(Pop) _ij + E _ijk , where Y _ijk represents the value of the response variable in the population i at level j of the HSF; µ is the general mean; Fam(Pop) _ij is the effect of the population nested HSF at level i, j; and E _ijk is the experimental error. Broad-sense heritability was also estimated (h ² _b = σg2 / σf2= σg2/(σg2+σe2)) ^15,16. The statistically superior HSF were selected in the segregating populations using the selection intensity value (i= D / σ_f), calculated by dividing the standardized selection differential (D) and the phenotypic standard deviation (σ_f)¹⁵, and the value of the selected proportion or selection pressure (p)^15,17.

Selection parameters

Family selection increased 32, 31, 32, 6, and 36 % the DMY, AGR, RUE, NT, and PH in the San Miguel x Oaxaca population; and 30, 28, 30, and 34 % the DMY, AGR, RUE, and PH in Oaxaca x San Miguel compared to the progenitors’ mean. Meanwhile, the L:S ratio decreased 48 % in San Miguel x Oaxaca and 51 % in Oaxaca x San Miguel (Table 1). The gain in DMY observed in this study was higher than that reported in progeny testing; the HSF and CSF (full-sib families) derived from open pollination (OP₁) of a diallel cross (F₁) produced only 8 and 9 % higher yield of green matter and 5 and 12 % higher number of tillers per plant than the original varieties¹⁸. The results of familial selection versus mass selection in alfalfa have indicated similar percentages for DMY (16 and 14 %) and raw fiber (-1.6 vs -1.5 %)¹⁹.

The values obtained for the selection parameters (D, σ_f, i, and p) indicated that 13 % of the HSF derived from San Miguel x Oaxaca and 17 % of the HSF derived from Oaxaca x San Miguel had higher DMY, AGR, and RUE per plant than the rest of the HSF (Table 1). The mean (µ₁) of DMY, AGR, and RUE for the 13 San Miguel x Oaxaca families and the 17 Oaxaca x San Miguel families was higher than the mean (µ₂) for the HSF derived from the original varieties of San Miguel and Oaxaca. However, the L:S ratio, number of tillers, and plant height behaved differently than the DMY. The mean L:S ratio of San Miguel x Oaxaca and Oaxaca x San Miguel HSF was lower than the mean of San Miguel and Oaxaca. The mean NT of the HSF derived from San Miguel x Oaxaca was greater than the mean of San Miguel and Oaxaca. The mean NT of Oaxaca x San Miguel HSF was similar to the mean of San Miguel and Oaxaca. The mean PH of the HSF derived from the two segregating populations was much higher than the mean in San Miguel x Oaxaca (Table 1).

The intrapopulation individual selection is effective in identifying superior genotypes using progeny testing⁶. The high average values of the L:S ratio, NT, and PH observed in the segregating populations did not allow the detection of outstanding HSF for these plant characteristics (Table 1).

Dry matter yield and its components

The analysis of variance detected 13 outstanding HSF, based on their DMY, AGR, and RUE, in the San Miguel x Oaxaca population (Table 2) and 17 HSF in the Oaxaca x San Miguel population (Table 3); this research also present data for the five families with the lowest DMY in these populations (Table 2 and 3). The 13 outstanding HSF of the San Miguel x Oaxaca population produced 53 % higher DMY than the average on the original varieties and 82 % higher DMY than the mean of the HSF with the lowest yield (Table 2). The outstanding HSF derived from the Oaxaca x San Miguel population produced 47 and 68 % higher DMY than the mean of the original varieties and the families with the lowest yield, respectively (Table 3).

The DMY of the outstanding HSF derived from the San Miguel x Oaxaca (direct cross) population (Table 2) was 11 % higher than the DMY of the outstanding HSF derived from the Oaxaca x San Miguel (reciprocal cross) population (Table 3). However, the San Miguel x Oaxaca HSF with the lowest yield produced 30 % lower DMY (Table 2) than the Oaxaca x San Miguel HSF with the lowest yield (Table 3).

The different genetic behavior, regarding DMY and its components, between the HSF derived from direct (San Miguel x Oaxaca) (Table 2) and reciprocal (Oaxaca x San Miguel) (Table 3) crosses could indicate the presence of DNA molecules in cytoplasmic organelles (chloroplasts and mitochondria), also known as cytoplasmic inheritance or maternal genetic effects, that express in reciprocal crosses by showing different results with respect to the direct cross¹⁶. The selection of the outstanding progeny derived from parental populations with cytoplasmic genetic effects on DMY inheritance is important to maximize genetic gains in productivity²⁰.

The outstanding San Miguel x Oaxaca HSF exhibited a higher AGR= 0.2 g of DM d^-1 than the progenitors’ mean and 0.3 g of DM d^-1 than the HSF with the lowest DMY (Table 2). The outstanding Oaxaca x San Miguel HSF showed a higher AGR= 0.15 g of DM d^-1 than the mean of San Miguel and Oaxaca and an AGR= 0.22 g of DM d^-1, higher than the mean of the HSF with the lowest DMY (Table 3). The AGR differences between the outstanding HSF and those with the lowest DMY were similar to the ones reported in other studies¹². The AGR is an important component of the DMY. In a previous study, the San Miguel, Oaxaca, and Moapa varieties produced a higher seasonal AGR and DMY than the Cuf-101 and Valenciana varieties in Montecillo, Texcoco, Edo. de México¹². Additionally, AGR is significantly related to the accumulation of dry matter and the leaf area index under field conditions; this is not the case for the number of leaves per stem²¹.

The RUE of the outstanding San Miguel x Oaxaca HSF was 52 % higher than the average of the original varieties and 82% higher than the mean of the HSF with the lowest DMY (Table 2). Similarly, the RUE of the outstanding Oaxaca x San Miguel HSF was 47 % higher than the progenitors’ mean and 78 % higher than the mean of the HSF with the lowest DMY (Table 3). Radiation use efficiency (RUE) determines the plant's capacity to capture solar radiation and transform it into biomass. Several studies have determined that the RUE of alfalfa is approximately 1.13 g of DM MJ^-1 in biomass production²². The average RUE increased linearly from 0.60 to 1.60 g of DM MJ^-1 as the air temperature increased from 6 to 18 °C²³.

Regarding PH, NT, and L:S ratio, it is important to consider their importance for DMY and forage quality in the selection of new varieties since, in this study, the selection by high DMY resulted in tall plants with a low L:S ratio and NT. However, it was identified some HSF with high DMY, L:S ratio (HSF-201), and NT (HSF-219, HSF-250, HSF-260, and HSF-282) in the San Miguel x Oaxaca population (Table 2) and high DMY and L:S ratio (HSF-652, HSF-673, and HSF-685), and NT (HSF-605, HSF-623, HSF-631, HSF-639, HSF-652, HSF-673, HSF-685, and HSF-690) in the Oaxaca x San Miguel population (Table 3). The selection by DMY and other plant morphological traits depends on the number of genes that control the trait of interest (additive, dominant, or epistatic genes); qualitative traits (controlled by one or few genes) are easier to select than quantitative traits (controlled by numerous genes). It is also important to consider the heritability level and negative genetic interactions that by improving one genetic trait inhibit the expression of another, as well as the appropriate general steps such as the aims, variability creation/collection, selection, evaluation, and release of cultivars, use of methods and techniques based on the species reproduction (autogamy, allogamy, or clonal propagation)²⁴.

Genetic, environmental, and heritability effects

The genetic variance, for DMY and its components, was higher in the direct cross (San Miguel x Oaxaca) than in the reciprocal cross (Oaxaca x San Miguel); the values of genetic variance for the San Miguel and Oaxaca varieties were intermediate between both crosses (Table 4). Genetic variance derives from the contribution of segregating genes and their interactions with other plant genes; therefore, the effective selection of genetically superior individuals requires that: 1) the phenotypic variation is appropriate in the original population, and 2) the heritability is sufficiently high for an effective selection. Overall, an increase in heritability and phenotypic variance is considered to increase genetic gain through selection²⁵.

The DMY, NT, and PH showed greater genetic variance than the other yield components, both in the crosses and in the progeny varieties. One of the major aspects of genetic improvement programs is the heritability (h ²) of useful plant traits present in the available genetic variability. In the direct cross, heritability ranged from moderate to high in DMY and its components and from low to moderate in the reciprocal cross. PH was the exception; this trait showed a greater heritability than the other traits (Table 4). Heritability measures the genotype contribution to the total phenotypic variance, which can theoretically vary from zero, when there is no genetic variation, to one, when the total variation is genetic in origin. However, the previously described broad-sense heritability (h ² _b) must be distinguished from narrow-sense heritability (h ² _n), which represents the quotient between the additive variance, instead of the total genetic variance, and the phenotypic variance²⁶.

The DMY and forage quality, represented by the L:S ratio, are primary objectives for alfalfa improvement. The heritability for DMY, L:S ratio, and the rest of the growth components was higher in San Miguel x Oaxaca than in Oaxaca x San Miguel. However, the data confirm genotypes with high DMY, L:S, and remaining growth components in both populations (Tables 2 and 3). These results differ from those obtained for DMY and L:S ratio in alfalfa HSF and lines (S₁), in which forage yield and L:S ratio were independent due to the lack of L:S variability in the materials selected because of their high DMY⁹. However, these results indicate that the selection allowed the identification of HSF with high values for DMY and other desirable traits, such as L:S ratio, NT, AGR, and RUE (Tables 2 and 3), which may favor a continuous response to selection²⁶.

The combination of the nuclear gene inherited traits that result from the additive genetic variance and the cytoplasmic inheritance or maternal genetic effects, due to the presence of DNA molecules in mitochondria and chloroplasts, can contribute to maximize the genetic gain in DMY and increase the selection efficiency in alfalfa and other cultivated species with maternal genetic effects expressed in the progenies phenotype. The breeder hardly needs to deal with genetic traits carried by one or the other of said organelles; however, sometimes, these organelles can be carriers of exceptional important traits for genetic improvements, such as the cytoplasmic male sterility that resides in the mitochondria²⁷.