The maize germplasm genetic base has been

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1 Published October 27, 2015 Agronomic Application of Genetic Resources Use of the Suwan1 Heterotic Group in Maize Breeding Programs in Southwestern China X.-M. Fan,* Y. Q. Bi, Y. D. Zhang, D. P. Jeffers, W. H. Yao, H. M. Chen, L. Q. Zhao, and M. S. Kang abstract Broadening the maize (Zea mays L.) genetic base is essential for improving grain yield. Suwan1, developed from a broad base of tropical germplasm in Thailand, has recently been identified as a heterotic group different from Reid and non-reid heterotic groups. The objectives of this study were (i) to evaluate the grain yield (GY) of crosses to see if there is value to utilizing Suwan1 as a heterotic group in maize breeding, and (ii) to investigate the general combining ability and specific combining ability to determine if Suwan1 shows good combining ability with Reid and non-reid heterotic groups for GY. Three testers, one from each of the three heterotic groups, were crossed with 20 elite lines collected from across China. Eighteen lines improved via introgression of exotic germplasm and two lines improved via pedigree selection were crossed with TRL02 (non-reid), YML46 (Suwan1), and TRL211 (Reid). Grain yield, ear length, ear diameter, kernel rows per ear, kernels per row, and 100-kernel weight of crosses and a check were evaluated at three locations. The results suggested that the Suwan1 temperate crossing system was a very useful heterotic pattern. Suwan1 improved all five yield components in crosses and demonstrated good combining ability for GY with Reid and non-reid heterotic groups. We have also determined that the appropriate percentage of introgressed germplasm should be between 6.25 and 12.5%. The maize germplasm genetic base has been narrowing throughout the world because most of the new maize inbred lines and hybrids or cultivars have been derived from existing elite materials (Goodman, 2005; Reif et al., 2010; Hallauer and Carena, 2014). For example, in China, two hybrids (Zhengdan958 and Xianyu335) and a large number of hybrids developed from inbred lines derived from these two hybrids constitute the majority of the cultivars grown in China, with more than 85% of the maize germplasm used in China s commercial market being related to these two hybrids (Dai and E, 2010). A narrow genetic base may cause serious maize production problems. A famous example is the southern corn leaf blight epiphytotic of 1970 in the US Corn Belt, which destroyed a large part of the US corn crop (Gregory et al., 1980). Another example is the outbreak of gray leaf spot that occurred in 2003 on 55% of the total maize production area in Yunnan province of China, which caused a 17% yield loss (Sun et al., 2007b). Researchers across the world have raised concern and suggested the introduction and incorporation of exotic germplasm into locally adapted germplasm for broadening the maize genetic base and for creating new superior inbred lines for hybrid maize development (Albrecht and Dudley, 1987; Zhang et al., 2000; Goodman, 2005; Fan et al., 2008b, 2009; Holland, 2004). Godshalk and Kauffmann (1995) indicated that there was considerable potential for exploiting exotic germplasm to improve temperate material, especially the Mo17- and Oh43-derived lines. Although good progress has been achieved in introducing exotic germplasm across the world (Godshalk and Kauffmann, 1995; Goodman, 2005; Fan et al., 2008b, 2009; Hallauer and Carena, 2014), efforts toward utilization of exotic germplasm have been inadequate, and so far only a small percentage of exotic germplasm has been used. For example, the Maize Gene Bank at the International Wheat and Maize Improvement Center (CIMMYT) holds more than 27,000 accessions of >250 races of maize, but only about 10% of these accessions have been utilized by breeders Published in Agron. J. 107: (2015) doi: /agronj Received 11 Mar Accepted 27 July 2015 Copyright 2015 by the American Society of Agronomy 5585 Guilford Road, Madison, WI USA All rights reserved X.-M. Fan, Y.Q Bi, W.H. Yao, H.M. Chen, and L.Q. Zhao, Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan, , China; X.-M. Fan, Y.Q Bi, Y.D. Zhang, W.H. Yao, and H.M. Chen, Yunnan Tian Rui Seed Company; D.P. Jeffers, CIMMYT Yunnan Office/Institute of Food Crops, Yunnan Academy of Agricultural Sciences; and M.S. Kang, Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS *Corresponding author (xingmingfan@163.com). Abbreviations: ED, ear diameter; EL, ear length; GCA, general combining ability; GY, grain yield per plant; KR, kernels per row; KW, 100-kernel weight; RE, kernel rows per ear; SCA, specific combining ability; SCH, standard check heterosis; SS, sum of squares. Agronomy Journal Volume 107, Issue

2 for germplasm improvement across the world. This has probably happened because most breeders usually choose to work with Elite Elite germplasm involving a few good traits to avoid risk (Walter Trevisan, personal communication, 2014; accessed 28 June 2015). During the past 20 yr, in Yunnan, many tropical and subtropical germplasm have been introduced and intensively studied for adaptation to local conditions, combining ability, and their potential for broadening the genetic base of Chinese elite maize germplasm (Chen et al., 2000a, 2000b; Fan et al., 2001, 2002b, 2003a, 2003b, 2004, 2005, 2008a, 2008b, 2009). Suwan1, a cultivar developed in Thailand from Thai Composite no. 1 at Suwan Farm, was released in After the official release of Suwan1, it has been widely used in breeding programs in many developing countries because of its broad adaptation, stable yield performance, and high level of disease resistance (Sriwatanapongse et al., 1993). Before exotic germplasm can be productively used in any breeding program, pre-breeding is usually conducted, which may include evaluation of exotic germplasm and selection for adaptation and desirable agronomic traits (Hallauer and Carena 2014). Two major methods, including introduction and introgression, are frequently applied in utilizing exotic germplasm in plant breeding programs (Dowling and Secor, 1997; Goodman, 2005). Suwan1 was introduced into China 20 yr ago and was subjected to selection for adaptation and desirable agronomic traits. The inbred line YML46, used in the current study, resulted from such selection within Suwan1 at the Yunnan Academy of Agricultural Sciences (Fan et al., 2002a, 2002b, 2003a). YML46 was obtained via intensive selection, especially for photoperiod insensitivity and resistance or tolerance to lodging, drought stress, downy mildew, and head smut. Studies have shown that Suwan1 possesses wide genetic variation for GY and good combining ability for five yield components (ear length [EL], ear diameter [ED], kernel rows per ear [RE], kernels per row [KR], and 100-kernel weight [KW]) (Fan et al., 2002a, 2003a;Yan et al., 2007). For effective utilization of improved lines from Suwan1, classification and determination of their heterotic group have been studied (Fan et al., 2002a, 2002b, 2003a, 2008a). Fan et al. (2008a) studied the general combining ability (GCA) of parental lines and the specific combining ability (SCA) of crosses between 25 elite temperate maize lines and four tropical inbred lines, which led to the identification of Suwan1 temperate as a new heterotic pattern. Molecular methods have also confirmed Suwan1 to be a new maize heterotic group, different from Reid and Lancaster (Fan et al., 2003a, 2008a). Breeding practice in China has shown that the Suwan1 temperate system has great potential in improving local elite lines and hybrids (Chang and Jing, 2000; Sun et al., 2007a; Fan et al., 2009, 2014). Fan et al. (2002a) discovered Suwan1 Reid and Suwan1 non-reid heterotic patterns by crossing four elite domestic temperate maize inbred lines with 25 tropical and semi-tropical inbred lines. Sun et al. (2007a) studied the combining ability of 25 temperate maize lines and four tropical maize lines, and concluded that the Suwan1 temperate crossing system was a viable heterotic pattern for improving maize yield. Fan et al. (2014) recently established that a tri-heterotic group consisting of Suwan1, Reid, and non-reid was useful in improving maize breeding efficiency. The line tester design is one of the frequently used methods for estimating GY, GCA, and SCA (Menkir et al., 2004; Barata and Carena, 2006; Fan et al., 2008a). Menkir et al. (2004) used a line tester design and classified 23 of 38 inbred lines into two heterotic groups based on SCA and GY. Using the line tester method, Barata and Carena (2006) classified 13 elite North Dakota maize inbred lines into currently used US Corn Belt heterotic groups. Fan et al. (2008a) successfully assigned inbred lines to correct maize heterotic groups by use of a line tester design. Heterosis refers to the phenomenon in which hybrid offspring of two inbred lines have characteristics that lie outside the parental range (Shull, 1908). Hybrid offspring may be larger, possess a faster growth rate, and have other improved traits compared with their inbred parents (Flint-Garcia et al., 2009). Three different computational methods, mid-parent heterosis, high-parent heterosis, and standard check heterosis (SCH, which refers to hybrid performance being better than a check cultivar performance), are widely used for estimating heterosis ( perid=3&topicid=1779). In breeding practice, SCH may be the most meaningful measurement for evaluating heterosis because only hybrids with positive SCH value are commercially useful. A Suwan 1 representative line, YML46, has been intensively used for studying combining ability, disease resistance, and heterotic classification and also utilized in broadening the genetic base and developing new hybrids (Fan et al., 2002a, 2008a, 2009, 2014). YML46 was first assigned to the Suwan1 heterotic group in 2008 (Fan et al., 2008a), and it constituted one of the 12 parents of a diallel cross used in studying GCA, SCA, and reciprocal effects for GY; the 12 parents belonged to Reid, non-reid, and Suwan1 heterotic groups (Yao et al., 2013; Fan et al., 2014). To further identify if Suwan1 can be used directly in a commercial hybrid breeding program and to determine if there is good combining ability among Suwan1, Reid, and non- Reid heterotic groups, three testers, one from each of the three heterotic groups, were crossed with 20 improved elite lines. The 20 elite lines used in this study were selected from among inbred lines widely used in Chinese commercial hybrids; then the inbred lines were improved either by introgressing exotic germplasm or by pedigree selection. The objectives of this study were: (i) to evaluate the GY of all crosses to determine if use of the Suwan1 heterotic group would be beneficial in maize breeding; and (ii) to investigate the GCA and SCA to determine if Suwan1 has good combining ability with Reid and non-reid heterotic groups for GY. MATERIALS AND Methods Experimental Materials Twenty improved inbred lines were selected from among the elite inbred lines widely used in commercial hybrids in China. Of the 20 improved lines, 18 were obtained from elite inbred lines following introgression of different exotic germplasm possessing favorable traits not available in the elite lines. Selection was conducted in different backcross generations, and the best-performing line from each of the 18 backcrosses was selected. The other two inbred lines were obtained by 2354 Agronomy Journal Volume 107, Issue

3 adaptation testing and selection after they were introduced from northern China to southwestern China. The 20 improved lines were then crossed with three testers (TRL02 [non-reid], YML46 [Suwan1], and TRL211 [Reid]) (Fan et al., 2014; unpublished data, 2013). These three testers are typically used in maize breeding programs in southwestern China. Information on the pedigrees of the improved elite lines, their parents, and the heterotic group of the parental lines is given in Table 1. Experimental Design The three testers were used as female parents and crossed with the 20 improved elite inbred lines (male parents) according to the line tester design (Kaushik et al., 1984; Menkir et al., 2004) in the summer of 2012 at Mile (24 41 N, E) and in the winter of 2012 at Jinghong (22 0 N, E) in Yunnan Province. The resulting 60 crosses, along with a commercial check (Yunrui 88), were planted in early May 2013 at three preselected locations (fixed effect): Kunming (25 2 N, E, 1960 m above sea level), Wenshan (23 30 N, E, 1260 m above sea level), and Dehong (24 50 N, E, 877 m above sea level). A randomized completeblock design with three replications was used at each location. Each experimental unit was a two-row plot (row length = 5 m, row spacing = 0.75 m), with a distance of 0.23 m between two adjacent plants within a row. The population density was 58,334 plants ha 1. Ten consecutive plants were selected from the middle of a plot for measuring all traits. Data on EL, ED, RE, KR, KW, and GY were recorded after the grain was dried to a constant moisture of 130 g kg 1. Statistical Analyses Data analyses (PROC GLM, PROC REG, etc.) were conducted in SAS (SAS Institute, 2005). The ANOVA was done using the following statistical model: Y =m+a + b ( a ) +u + ( au ) + e ijkl l kl ij ijl ijkl u ij = li + t j + ltij where Y ijkl is the observed value from each experimental unit; m is the population mean; a l is the location effect; b(a) kl is the replication effect within each location; u ij is the F 1 hybrid effect = l i + t j + lt ij, where l i is the ith exotic line effect, t j is the jth tester effect, and lt ij is the interaction effect between the ith exotic line and jth tester; (au) ijl is the interaction effect between the ijth F 1 hybrid and the lth location; and e ijkl is the residual effect. Because the three locations selected for this experiment were not a random sample of all possible locations within Yunnan, we treated locations as a fixed factor. Hybrid or cross effect and, consequently, line and tester effects were also regarded as fixed effects. Only replication was considered as a random factor. Therefore, the significance of the location variance was tested against replications within locations. For all other significance tests, the experimental error term was used (Table 2). Standard check heterosis was calculated according to the following formula: SCH = (F 1 Check)100/Check. To test if the SCH differences among the three testers were statistically significant, an ANOVA was conducted and Bonferroni multiple comparisons were used for testing differences between means. Forward selection was used in regression analysis for selecting yield component traits for analysis. Table 1. Sources of the 20 temperate maize inbred lines. Line Inbred Parents and pedigree of the line Line s heterotic group Parent s heterotic group Introgression type Y1 YML340 Dan340/CML161 (BC3) NR NR/NR I Y2 YML6 Suwan1611/K22 (BC3) ?S S/R II Y3 YML15 HuangC/CML171 (BC3) R R/NR II Y4 YML56 478/CML147 (BC2) R R/Trop White III Y5 YML719 Dan340/S37 (BC2) ?S NR/S II Y6 YML756 Dan599/Mohuang9 (BC2) ?T NR/T II Y7 YML911 Ye107/CML171 (BC3) R R/NR II Y8 TRML26 Mohuang9/81219 (BC3) S T/NR II Y9 YML479 Chang7 2/CML161 (BC3) NR NR/NR I Y10 YML946 K12/SW1(S)C BBB (BC3) NR NR/NR I Y11 YML13 E28/P28C BBB-2-1 (BC3) NR NR/NR I Y12 YML402 E28/SW1(S)C9-S (BC2) S NR/S II Y13 YML161 Qi205/SW1(S)C B-B (BC2) S S/S I Y14 TRML725 K22/CML147 (BC4) R R/Trop White III Y15 YML122 Tie R R na Y16 TRML229 Zhong3/CML171 (BC5) NR NR/NR I Y17 YML26 Dan340/SW1(S)C9-S (BC2) S NR/S II Y18 Zheng58 Ye478 variation plant R R na Y19 YML556 Zi330/CML145 (BC3) NR NR/T II Y20 YML12 Mo17/P21C5HC84-F3-#-5-BBBB-##-B-B-B-B(BC3) NR NR/T II The inbred line above the slash (/) represents the recurrent parent in a backcross. R, Reid; NR, non-reid; S, Suwan1; T, Tuxpeño heterotic groups;? indicates that the line s heterotic group classification was grouped into a suggested group without strong support from statistically significant specific combining ability effects. From Fan et al. (2002a, 2003b) and Wu et al. (2007). Type I, introgression with germplasm (lines) from same heterotic group; Type II, introgression with germplasm (lines) from different heterotic groups; Type III, unknown; na, not applicable. Agronomy Journal Volume 107, Issue

4 Table 2. Analysis of variance for yield (g per plant) of 60 testcrosses tested across three locations in Yunnan province (2013). Source df Sum of squares Mean squares F value Locations (Loc) ** Replication(Loc) Crosses ** Lines ** Testers ** Lines testers ** Crosses Loc ** Lines Loc ** Testers Loc ** Lines testers Loc ** Error ** Significant at the a = 0.01 level. Loc: Locations; Rep(Loc.): replications within locations. results Analysis of Variance for Grain Yield per Plant for 60 Crosses Analysis of variance for GY at three locations is given in Table 2. The results showed that replications within locations was not significant; all other sources of variation were statistically significant. Because mean squares (MS) for lines and testers are mainly attributable to GCA effects, the significance of MS for lines and MS for testers suggested that GCA variances among lines and testers were significant. Similarly, MS for line tester interaction was mainly attributable to the SCA effect, indicating that SCA variances among crosses were significant. In addition, the MS for lines locations interaction, testers locations interaction, and lines testers locations interaction were statistically significant, suggesting that GCA and SCA effects were influenced by environments, which implied that multiple locations are needed to accurately measure these effects. The most interesting results from Table 2 are that the MS for crosses and testers were statistically significant, which laid the foundation for further analysis of differences relative to SCH, GCA, and SCA among crosses with the three testers from different heterotic groups. Differences in Standard Check Heterosis among Crosses The GYs from the 60 crosses averaged across the three testers (TRL02 [non-reid], YML46 [Suwan1], and TRL211 [Reid]), the SCHs, and SCAs of the 60 crosses are given in Table 3. The numbers of crosses with SCH > 0 (i.e., cross GY is larger than that of the check) were: 6 from non-reid, 16 from Suwan1, and 3 from Reid heterotic groups. More crosses with SCH > 0 were found when the 20 elite lines were crossed with the tester from the Suwan1 heterotic group. These results imply that had the Suwan1 group not been identified, a total of 16/( )100% = 64% of the superior hybrids with SCH > 0 would have been missed. Such a loss of opportunity to identify superior hybrids would not be acceptable to any commercial maize breeding company. The ANOVA showed that the differences among the three testers were statistically significant (data not shown). The Bonferroni multiple comparisons test showed that the mean SCH of the crosses with YML46 was 8.879, significantly higher (a = 0.05) than the mean SCHs for the crosses with TRL02 ( 3.051) and TRL221 ( ). Crosses with SCH ³ 5% of all 60 crosses are listed in Table 4. According to SCA effects, we could classify the 20 elite lines into the three different heterotic groups to which the three testers belong (Table 1). When heterotic groups of 19 crosses with Table 3. Mean grain yield for three testers (TRL02 [non-reid], YML46 [Suwan1], and TRL211[Reid]) across crosses, standard check heterosis (SCH), and specific combining ability (SCA) of the 60 crosses. Standard error for SCA = 5.864l. TRL02 YML46 TRL211 Line Yield SCH SCA Yield SCH SCA Yield SCH SCA g/plant g/plant g/plant Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Agronomy Journal Volume 107, Issue

5 Table 4. General combining ability (GCA) effects of three testers and lines, standard check heterosis (SCH), and specific combining ability (SCA) effects of 20 crosses with top grain yield (GY). Heterotic Line Tester pattern GY Rank SCH GCA_line GCA_tester SCA g/plant Y18 YML46 R S Y19 YML46 NR S Y18 TRL02 R S Y1 YML46 NR S Y6 YML46?T S Y9 YML46 NR S Y7 YML46 R S Y7 TRL02 R NR Y17 TRL211 S R Y3 YML46 R S Y6 TRL02?T NR Y10 YML46 NR S Y2 YML46?S S Y4 YML46 R S Y18 TRL211 NR R Y14 YML46 R S Y11 YML46 NR S Y16 YML46 NR S Y2 TRL02?S NR SE NR, non-reid; S, Suwan1; R, Reid heterotic groups.? indicates that the heterotic group assignment to the line needs to be confirmed. SCH ³ 5% (Table 4) were examined, we found that 14 of them were crosses either between Suwan1 and Reid or between Suwan1 and non-reid. Based on mean SCHs and the results from Tables 3 and 4, we can conclude that the Suwan1 heterotic group is a valuable maize heterotic group and offers greater opportunity than the other two heterotic groups to develop superior commercial hybrids in combination with lines from Reid and non- Reid heterotic groups. Furthermore, these results proved that Suwan1 temperate is a highly desirable heterotic pattern and could be effectively utilized in maize breeding programs. Differences in Mean and Sum of Squares of Yield Components among Crosses To determine which yield components were different among the crosses relative to the three different testers, the means for the yield components were computed and the results were plotted (Fig. 1). In this figure, we can see that all five yield components had higher means from crosses involving the YML46 tester than those from crosses involving the other testers (TRL02 and TRL211). Bonferroni multiple comparison results (Table 5) confirmed that the means for GY, ED, EL, Fig. 1. Mean comparison for five yield components of ear length (EL, in cm), ear diameter (ED), rows per ear (RE), kernels per row (KR), and 100-kernel weight (KW, in g). TRL02, YML46, and TRL211 are the three testers used in the experiment. Different letters on the histogram bars refer to significantly different means at a = Agronomy Journal Volume 107, Issue

6 RE, and KW from crosses with YML46 were all significantly higher than those from the crosses involving TRL02 and TRL211. The KR mean from the crosses involving YML46 was significantly higher than the means of the crosses involving TRL211 (Table 5). These results strongly suggested that YML46 improved all key yield components in all its crosses, which ultimately translated into high GY for all crosses involving YML46. The percentage of the sum of squares (SS%) usually measures how closely trait variation is related to a target trait in a general linear model (Bernardo et al., 1992; Kang, 1994). Regression analysis conducted using GY as a dependent variable and five yield component traits by forward selection, viz., EL, ED, RE, KR, and KW, as independent variables, revealed that the SS percentages for these five traits were quite different from those of the crosses grouped by the three testers (Table 6). The differential SS% values indicated the relative contribution of each trait to GY variance among the crosses (Bernardo et al., 1992). By examining the details relative to SS% for each trait grouped by the three testers (Table 6), we found that EL and ED were the largest and second largest contributors, respectively, to GY variance for the crosses with testers TRL02 and TRL211. In the YML46 crosses, however, the largest and second largest variance contributors were KW and ED. Large variance for a trait usually indicates that more genetic gain may be obtained for the trait if selection is practiced in a population (Hallauer and Miranda, 1988; Bernardo et al., 1992;Kang, 1994). Because the means of KW and ED in all the crosses involving YML46 were significantly higher than those from the crosses with TRL02 and TRL211 (Fig. 1; Table 5), it is certain that there would be a greater chance to develop an inbred line with high KW from crosses between elite lines and YML46 because of the largest expected genetic gain from their offspring. There will also be greater opportunity to develop high KR inbred lines from the crosses involving YML46 than from crosses involving the other two testers. For developing inbred lines with high ED, there would be no big difference among crosses from all three testers because the SS% for ED was the second largest contributor to GY variance in the crosses with all three testers. General Combining Ability Effects of Testers and Specific Combining Ability Effects for Crosses The GCA effects of the three testers and the SCA effects of the top 19 crosses with high GY are given in Table 4. Upon examination of the crosses, we found that all 19 crosses had either significantly positive high GCA effects or significantly positive high SCA effects or both, suggesting that the GCA of Suwan1 and the SCA of the crosses between Suwan1 and lines from the other two heterotic groups were major contributors to hybrids with superior GY. For example, the GCA effect for YML46 (14.49) was significantly higher than those for both TRL02 ( 1.18) and TRL211 ( 13.31) (LSD = 8.77, P = 0.01); the GCA effects for the latter two testers were negative. Table 5. Bonferroni test results for the means for ear diameter (ED), 100-kernel weight (KW), ear length (EL), kernels per row (KR), and kernel rows per ear (RE) from crosses with three different testers. Tester ED KW EL KR RE YML A A A A A TRL B B B A C TRL B B C B B SE Means followed by the same letter are not statistically significant at a = Table 6. Sum of squares (SS) and percentage of SS (SS%) for five yield component traits obtained via forward selection with regression on grain yield. Tester Trait Estimate t value Pr > F Type I SS SS% TRL02 EL < , ED < , RE KR < , KW Sum 57, YML46 EL , ED < , RE < KR < , KW < , Sum 95, TRL211 EL , ED < , RE < KR < KW < Sum 146, EL, ear length; ED, ear diameter; RE, kernel rows per ear; KR, kernels per row; KW, 100-kernel weight; GY, grain yield per plant Agronomy Journal Volume 107, Issue

7 This indicates that a line from Suwan1 would have better GCA than lines from the Reid and non-reid heterotic groups. Furthermore, by checking the five crosses with statistically significant positive SCA effects (SCA ³ = 11.72), we found that four (or 80%) of them were the crosses between Suwan1 and Reid or Suwan1 and non-reid, suggesting that there was more favorable genetic interaction between Suwan1 and the two temperate heterotic groups. Thus, Suwan1 would offer commercial maize breeders a big opportunity to develop high-yielding hybrids. Discussion Heterotic Groups In North America, before the 1990s, most of the commercial maize hybrids belonged to two maize heterotic groups, i.e., Reid and Lancaster (non-reid) (Hallauer and Miranda, 1988). The introduction of tropical germplasm has broadened the genetic base of US maize (Holley and Goodman, 1988; Hallauer and Carena, 2014). Liu et al. (2003) used 94 DNA microsatellites to classify Suwan1 germplasm into tropical and semi-tropical groups that are different from Stiff Stalk and Non-stiff Stalk groups. Recently, based on principal component analysis of molecular-marker data, Romay et al. (2013) found that the population structure between Reid (Stiff Stalk)/non-Reid (Non-stiff Stalk) and tropical germplasm was distinct. Fan et al. (2014) argued that classifying maize germplasm into three heterotic groups, i.e., Suwan1, Reid, and non-reid, should greatly improve maize breeding efficiency, with little or no risk of losing hybrids with superior GY. This study, which utilized testers from the three heterotic groups crossed with widely used commercial inbred lines across China, supported our previous conclusion that the Suwan1 heterotic group, of tropical origin, was different from Reid and non-reid maize heterotic groups and that GCA among the three heterotic groups was the basis for the hybrids with superior GY. Maize breeding programs would benefit from making crosses between the Suwan1 heterotic group and Reid and/or non-reid heterotic groups. Impact of Introgression of Exotic Germplasm on Recurrent Lines Heterotic Grouping In our study, 18 out of the 20 improved lines had been developed via introgression of exotic germplasm into local adapted lines (Table 1). To determine the impact of exotic-germplasm introgression on recurrent parental lines heterotic grouping, the 18 elite lines used in this study were classified into Reid, non-reid, and Suwan1 heterotic groups based on SCA effects. Then the heterotic groups of the 18 lines were compared with those of their recurrent parental lines heterotic groups. The results showed that 13 (or 72.2%) of the improved lines fell in the same heterotic group as their recurrent parental lines (Table 1), while the remaining five lines could not be assigned to the same heterotic group as their recurrent parental lines. These results strongly suggest that introgression of exotic germplasm via backcrossing usually would not change the recurrent parent s original heterotic group. Interestingly, by carefully checking the five improved lines that did not fall in the same heterotic group as their recurrent parents, we found that they (i.e., Y5, Y6, Y8, Y12, and Y17) resulted from introgression of exotic germplasm between two different heterotic groups. These results suggest that introgression between germplasm from different heterotic groups might change recurrent lines original heterotic group. Thus, breeders may not be able to determine a new line s heterotic group based only on its pedigree information; re-assessing a line s heterotic group after exotic germplasm introgression, especially after introgression between germplasm from different heterotic groups, may be needed to effectively utilize the new improved germplasm. Furthermore, classification of Line Y8 is of special significance. It was definitively assigned to the Suwan1 heterotic group based on SCA ( 18.02) for Y8 YML46 (Suwan1). However, it had been developed via introgression of a non-reid inbred line into the Tuxpeño inbred line, implying that the genetic base of the Suwan1 heterotic group may be equal to the sum of the genetic bases of the non-reid and Tuxpeño heterotic groups. Further research is needed to confirm this finding. Tuxpeño and ETO are two important heterotic groups, and the heterotic pattern of ETO Tuxpeño was well studied by Vasal et al. (1992). For utilizing tropical germplasm, Fan et al. (2002b) studied the combining ability of five tropical heterotic groups and races (i.e., Suwan1, Antigua, Tuxpeño, ETO, and POP28). They found that Tuxpeño was closer to the Reid heterotic group, and POP28 was closer to a local non-reid heterotic group called Tang Si Ping Tou. Based on these studies, heterotic groups were assigned as listed in Table 1. To see the impact of the introgression of exotic germplasm into local adapted material, we grouped the 18 improved lines into three introgression types (Table 1): Type I, introgression with germplasm (lines) from the same heterotic group; Type II, introgression with germplasm (lines) from different heterotic groups; and Type III, unknown, i.e., at least one of the line s heterotic groups in the introgression is unknown. We counted the number of the improved lines within each type of introgression. The numbers of improved lines with Type I, Type II, and Type III introgression were: 6 (i.e., Y1,Y9, Y10, Y11, Y13, and Y16), 10 (i.e., Y2,Y3, Y5, Y6, Y7,Y8,Y12, Y17, Y19, and Y20), and 2 (i.e.,y4,and Y14), respectively. We grouped Lines Y4 and Y14 into an unknown group because Tropical White has not yet been classified into any of the three heterotic groups, i.e., Reid, non-reid, and Suwan1. If we examine lines involved in the 19 crosses with top GY (Table 4), five lines (i.e.,y1, Y9, Y10, Y11, and Y16) with Type I introgression and six lines (Y2, Y3, Y6, Y7, Y17, and Y19) with Type II introgression produced crosses with GY greater than that of the check and thus have potential of being utilized in commercial hybrid development. These results imply that exotic germplasm introgression can improve local germplasm either within the same heterotic group mating or in different heterotic group matings. Introgression for Utilizing Exotic Germplasm Introgression is used to improve local elite cultivars by backcrossing to integrate a few favorable genes into the local elite cultivars. In contrast, introduction, followed by selection, is used to develop new lines or cultivars that meet breeding targets and are adapted to a breeder s local or target environments. A majority of breeders are of the opinion that exotic germplasm provides benefits through its slow integration or introgression Agronomy Journal Volume 107, Issue

8 of favorable genes into elite local germplasm (Goodman, 2005). Some breeders strongly believe that to benefit from exotic germplasm, it should only be introgressed into locally adapted lines within the same heterotic group (Walter Trevisan, personal communication, 2014). Their opinion is supported by breeding practice in both the United States and China (Goodman, 2005; Chang and Jing, 2000; Chen et al., 2011). Thus, introgression seems to be a more effective technique than introduction for utilizing exotic germplasm in maize. The 20 improved elite inbred lines used in this study have been developed from selected elite lines widely used in China. They were developed via either introgression or introduction, followed by selection, in southwestern China. We counted the crosses with different mating designs and selection stages for the 20 improved elite lines and plotted the counts (see Fig. 2) and noticed that 18 of the improved lines were developed via introgression and two of them were developed by introduction followed by selection. Among the 18 lines developed by introgression, 6 and 10 lines were developed from BC2 and BC3 populations, respectively. These results strongly suggest that introgression was a very important method for utilizing exotic germplasm, and the best introgression proportion should be between 6.25% (i.e., BC3 has 93.75% genes from the recurrent parent and 6.25% from the exotic or donor parent) and 12.5% (i.e., BC2 has 87.5% genes from the recurrent parent and 12.5% from the exotic germplasm) when introducing exotic germplasm into local elite lines. With regard to what percentage of exotic germplasm should be integrated into local elite lines, different results have been reported. Albrecht and Dudley (1987) suggested that the population containing 25% tropical genetic background was better than a population containing >50% tropical genetic complement. In contrast, however, Hallauer and Miranda (1988) thought that 50% tropical background of a population was most appropriate. Tallury and Goodman (1999) reported that hybrids containing 10 to 60% tropical background had higher yield with improved agronomic traits, whereas the hybrids with tropical germplasm proportion >60% would lead to yield reduction along with inferior agronomic traits. Gouesnard et al. (1996) suggested that germplasm with 25% exotic background would be valuable in breeding programs. Chen et al. (2003) studied several traits, e.g., tassel, pollen, silk stage, and plant and ear height that related to photoperiod, and found that a BC1 generation that contained 25% tropical background had the weakest sensitivity to photoperiod. Dudley (1982) suggested that backcrossing one or more times to the elite line was useful for selecting improved offspring. Santos et al. (2000) conducted a systematic study and found that the best exotic proportions were 6.25 and 12.5%. Our results from this study are highly consistent with the conclusion of Santos et al. (2000). The broad adaptation, yield stability, and high level of disease resistance of Suwan1 (Sriwatanapongse et al., 1993) make it an extremely valuable exotic germplasm for broadening the genetic base to improve temperate germplasm via introgression (Goodman, 2005; Fan et al., 2002a, 2002b, 2003b, 2009, 2014). However, the sensitivity of Suwan1 to photoperiod may cause excessive plant height, a greater leaf number, a longer growing season, weaker ear development, immature grains, and reduced ear and kernel weights in long-day environments. To integrate favorable genes from Suwan1 into Chinese local elite maize lines via introgression, Chinese breeders have made great efforts to decrease Suwan1 s photoperiod sensitivity and have successfully developed different lines that can flower and set seed from southern to northern China. Many lines with Suwan1 genetic background have been developed (Chang and Jing, 2000; Fan et al., 2002a, 2002b). Heterotic group classification results from the current study have shown that four improved elite lines (Y8, Y12, Y13, and Y17) can be assigned to the Suwan1 heterotic group (Table 1), suggesting that with several years of introgression and introduction efforts, the genetic base of some elite lines in the temperate region of China has been expanded. The Suwan1 heterotic group has high potential for developing superior hybrids with the Suwan1 temperate pattern in China and possibly globally, too. Breeding practice and results from China have led us to believe that the different results about the suitable Fig. 2. Number of inbred lines developed in different generations via a backcross introgression procedure. BC2, BC3, BC4, and BC5 are backcross populations with two, three, four, and five backcrosses Agronomy Journal Volume 107, Issue

9 introgression proportions of exotic germplasm in maize breeding might be the result of photoperiod sensitivity, the latitude and altitude where the materials were evaluated, and the number of favorable alleles that were examined. The best introgression proportion may be relatively higher (25 50%) in low latitude and altitude regions and relatively lower (<25%) in high latitude and altitude regions. In summary, the results from this study showed that many more crosses with GY > check were obtained from crosses between YML46 (a line representative of Suwan1) and 20 elite lines than those from crosses between Reid or non-reid lines and the 20 elite lines. These results suggest that Suwan1 is a new valuable maize heterotic group and may be used directly in hybrid maize development and that Suwan1 temperate is a very important heterosis-generating system that should be used in maize breeding programs. Had the Suwan1 heterotic group not been identified, most of the superior hybrids would have been missed in maize breeding programs in China. In addition, Suwan1 has improved all five yield component traits, i.e., EL, ED, RE, KR, and KW, and has shown very good combining ability with Reid and non-reid heterotic groups for GY. An improved line via introgression of exotic germplasm should usually fall into the same heterotic group as its recurrent parent s heterotic group when a backcross procedure is used. The most desirable introgression percentage should be between 6.25 and 12.5%. Furthermore, in addition to introgression and incorporation, introduction followed by selection could be another useful and effective way of utilizing exotic germplasm. This study was conducted in the subtropical region of China, and further evaluation of the top crosses at higher latitudes should provide more information on adaptation for the local target environment. AcknowledGMents We gratefully acknowledge the financial support from Yunnan Leading Talent Introduction Project (2014HA002), National Natural Science Foundation of China Project ( , ), Application of Key Product Project of Yunnan Province (2012BB012). We would like to give our great thanks to Suwan Farm in Thailand for kindly offering original Suwan population to us. We are also thankful for the cooperation and help provided by personnel from experimental sites in Wenshan and Dehong. REFERENCES Albrecht, B., and J.W. Dudley Evaluation of four maize populations containing different proportions of exotic germplasm. Crop Sci. 27: doi: /cropsci x x Barata, C., and M. Carena Classification of North Dakota maize inbred lines into heterotic groups based on molecular and testcross data. Euphytica 151: doi: / s y Bernardo, R., M. Bourrier, and J.L. Oliver Generation means analysis of resistance to head smut in maize. Agronomie 12: doi: /agro: Chang, H.Z., and S.L. 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