Combining Ability and Parent-Offspring Correlation of Maize (Zea may L.) Grain β-Carotene Content with a Complete Diallel

Journal of Integrative Agriculture

January 2013

2013, 12(1): 19-26

RESEARCH ARTICLE

Combining Ability and Parent-Offspring Correlation of Maize (Zea may L.) Grain β-Carotene Content with a Complete Diallel LI Run, XIAO Lan-hai, WANG Jing, LU Yan-li, RONG Ting-zhao, PAN Guang-tang, WU Yuan-qi, TANG Qilin, LAN Hai and CAO Mo-ju Maize Research Institute, Sichuan Agricultural University/Key Laboratory of Crop Genetic Resource and Improvement, Ministry of Education/Key Laboratory of Maize Biology a n d G e n e t i c B re e d i n g o n S o u t h w e s t , M i n i s t r y o f A g r i c u l t u re , C h e n g d u 6 111 3 0 , P.R.China

Abstract Vitamin A deficiency has become a worldwide problem. Biofortified foods can potentially be an inexpensive, locally adaptable, and long-term solution to dietary-nutrient deficiency. In order to improve the β-carotene content in maize grain by breeding and minimize vitamin A deficiency, a complete diallel cross was designed with eight inbred lines of maize, and 64 combinations were obtained in this study. The experimental combinations were planted in Yunnan and Sichuan provinces, respectively, with a random complete block design. The β-carotene contents in the grains of the experimental materials were analyzed by high-performance liquid chromatography. Among the tested materials, the effect difference of general combining ability of the β-carotene content was significant; however, the effect difference of the special combining ability and the reciprocal effect were not significant. The β-carotene content of maize grain was not influenced significantly by the cross and the reciprocal cross. There was a significant correlation about the β-carotene content in the maize grains between the F 1 and their parents. The combinations with high β-carotene content were obviously influenced by the environment, and the mean value of β-carotene content for the experimental materials planted in Ya’an of Sichuan was higher than that planted in Yuanjiang of Yunnan, with the results being significant at the 0.01 level. Key words: maize, β-carotene content, complete diallel cross, combining ability

INTRODUCTION Vitamin A is one of the essential micronutrients, that plays a very important role in human health. Vitamin A deficiency can lead to visual impairment and even blindness; moreover, it contributes to predisposition to several major diseases, such as anemia, diarrhea, measles, malaria, and respiratory infections (Sommer and West 1996; Shankar et al. 1999; Villamor and Fawzi 2000; West 2000). More than 250 million people in the world experience deficiency of vitamin A (Sommer and Received 19 December, 2011

West 1996). Thus, vitamin A deficiency has become a worldwide problem. Diet diversification, in combination with food fortification and supplementation, has been used to combat deficiencies of dietary micronutrients. Biofortified foods can potentially be an inexpensive, locally adaptable, and long-term solution to dietary-nutrient deficiency (Taylor and Ramsay 2005; Aluru et al. 2008; Harjes et al. 2008). Plants usually do not contain vitamin A but can produce carotenoids which are the precursors of vitamin A and are called provitamin A. The International Center for Tropical Agriculture and

Accepted 16 March, 2012

Correspondence CAO Mo-ju, Mobile: 13882439529, Fax: +86-835-2882154, E-mail: [email protected], [email protected]

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60201-4

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the International Food Policy Research Institute have sponsored a program called HarvestPlus. The aim of HarvestPlus is to improve the content of essential micronutrients of the main food crops by crop breeding. After, the studies about carotenoids in crops were spread across rice, maize, wheat, and so on (Hoisington et al. 2002; Paine et al. 2005; Chander et al. 2008; Diretto et al. 2010; Ziliæ et al. 2012). Maize is a staple crop that provides food for a majority of the world’s population. The major carotenoids identified in maize grain are β-carotene, α-carotene, β-cryptoxapthin, and the xanthophylls lutein and zeaxanthin (Howe and Tanumihardjo 2006). The compounds β-carotene, βcryptoxapthin, and α-carotene can be transformed into vitamin A directly. Among these, β-carotene is the main source of vitamin A (Hui 2005). Therefore, it is very critical to improve the β-carotene content of food crops. Studies on the carotenoid biosynthetic pathway in plants have achieved significant progresses (DellaPenna and Pogson 2006; Menkir et al. 2008; Li et al. 2008a, b; Vallabhaneni and Wurtzel 2009; Zhou et al. 2012). In maize, some molecular markers have been selected for identifying the plants with high β-carotene content (Chander et al. 2008). In China, the populations with vitamin A deficiency are mainly distributed in areas far away from cities in China, living predominantly on plantbased foods; therefore, increasing the concentration of provitamin A, such as β-carotene, in staple food crops could potentially improve the situation of vitamin A deficiency (Hui 2005). Thus, enhancing the β-carotene content in maize is the most practical way for the HarvestPlus program in China. Combining ability is an important criterion for evaluation of inbred lines during the development of hybrids. Diallel cross has been used for the combining ability analysis for the contents of four carotenoids and two tocopherols in maize grain by high-performance liquid chromatography (HPLC) (Egesel et al. 2003; Senete et al. 2011). Till now, although some progress has been made in this field, the knowledge about carotenoids in plants is not very comprehensive. For example, βcarotene mainly exists in the endosperm of the maize grain, whether the cytoplasmic background has any effect on the β-carotene content is not clear yet. A complete diallel cross that contains the crosses and the reciprocal crosses simultaneously is suitable for the

LI Run et al.

analysis of reciprocal effect. The objective of this study was to realize the inheritance feature of β-carotene content in maize grains by a complete diallel cross. In our previous study, the β-carotene contents of 31 inbred lines were initially assayed by column chromatography in combination with spectrophotometry, and eight inbred lines were selected as parents both according to their β-carotene contents and taking account of their practical application in maize breeding. A complete diallel cross based on Griffing’s Method I was adopted. The β-carotene content was measured by HPLC, and the inheritance effects and correlations were analyzed. The results could be helpful for improving the β-carotene content in maize grains by breeding and minimizing vitamin A deficiency.

RESULTS Combining ability analysis One set of data from Yunnan Province and another set of data from Sichuan make up the two replications. Variance analysis of the β-carotene content in the 64 combinations was conducted, and the results are shown in Table 1. The difference of the β-carotene contents among combinations was significant. The results of the variance analysis on the combining ability of β-carotene content are shown in Table 2. The difference in the general combining ability (GCA) was significant; however, the special combining ability (SCA) and the reciprocal cross effect were not significant. The GCA value was calculated and the difference was tested, the results are listed in Table 3. The GCA values of the eight inbred lines ranged from -0.6522 to 1.5195. The GCA values for 9636, 5311, Dongdan 54-3-1-1-1, and 698-3 were positive and significantly higher than those of the other parents at the 0.01 level, indicating that these parents contribute to Table 1 Variance analysis of β-carotene content among the 64 combinations Source Replication Crosses Error Total

df

Square sum

Mean square

F-value

P-value

1 63 63 127

6.0317 134.8729 17.4787 158.3832

6.0317 2.1408 0.2774

7.7164

3.058×10 -14

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Combining Ability and Parent-Offspring Correlation of Maize (Zea may L.) Grain β-Carotene Content with a

increasing the cumulative effect and the β-carotene content in the hybrids. The β-carotene content of 9636 was significantly higher than those of 5311, Dongdan 54-3-1-1-1, and 698-3 at the 0.01 level. However, there was no significant difference between 5311 and Dongdan 54-3-1-1-1, Dongdan 54-3-1-1-1, and 698-3. The GCA values for R08, Huangzaosi, Mo 17, and 48-2 were negative, indicating that these parents contribute to a decrease in the β-carotene content of the hybrids. The GCA effect estimated from the combining ability analysis indicated that 9636 made the greatest contribution to an increase in the β-carotene content of the hybrids.

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tained and simple correlation were analyzed for each experiment location respectively. The parent-offspring correlation analysis of the β-carotene content from the cross combinations and the reciprocal cross combinations showed that there was a significant correlation between parents and offspring in the two different environments at the 0.01 level (Table 4). It means that the β-carotene content of the F1 generation could be predicted based on the β-carotene content of the respective parents to some extent. The paired t-test was adopted to analyze the difference in β-carotene contents between the cross combinations and the reciprocal cross combinations at the two locations. The t-value of materials planted in Ya’an, Sichuan Province, China, was 1.1560 (t0.01, 23=2.807) and the t-value of materials planted in Yuanjiang was 0.2539 (t0.01, 27=2.771). Thus, no significant difference between the cross combinations and the reciprocal cross combinations was found at the two different experimental locations, indicating that the genetic effect of the β-carotene content was not influenced significantly by the cytoplasmic background. Based on the β-carotene content, the eight parents were divided into two groups: the relative high-content group with positive GCA value, labeled with high, and the relative low-content group with negative GCA value, labeled with low (Table 5). The results of the β-carotene content of combinations planted at two experimental locations were listed in Table 6. In order to

Correlation between parents and their offspring In this study, the coefficient of parent-offspring correlation were calculated as follows: each inbred line while used as female parent, it must mated with other seven inbred lines and seven hybrids were produced, thus the mean value of β-carotene content of the seven hybrids were regarded as the content of offspring. According to this strategy, we can calculate out the β-carotene contents for every offspring corresponding to each inbred lines while used as female parent. On the other hand, the β-carotene contents for every offspring corresponding to each inbred lines while used as male parent can also be calculated out. So eight pairs of cross data and eight pairs of reciprocal cross data were obTable 2 Variance analysis of combining ability for β-carotene content Source

df

Square sum

GCA SCA Reciprocals Error

7 28 28 63

60.6903 4.2657 2.4805 8.7393

Mean square 8.6700 0.1523 0.0886 0.1387

F-value

P-value

62.5004 1.0982 0.6386

0.0001 0.3696 0.9038

Table 3 The GCA value of β-carotene content and the significance of difference Parents 9636 5311 Dongdan 54-3-1-1-1 698-3 R08 HuangzaoSi Mo 17 48-2 **

GCA

9636

5311

Dongdan 54-3-1-1-1

1.5195 0.4078 0.2612 0.0299 -0.4887 -0.4932 -0.5843 -0.6522

1.1116 ** 1.2582 ** 1.4895 ** 2.0082 ** 2.0127 ** 2.1038 ** 2.1717 **

0.1466 0.3779 ** 0.8965 ** 0.9010 ** 0.9922 ** 1.0600 **

0.2313 0.7499 ** 0.7544 ** 0.8456 ** 0.9134 **

698-3

0.5186 ** 0.5231 ** 0.6143 ** 0.6821 **

R08

0.0045 0.0956 0.1635

Huangzaosi

0.0911 0.1590

Mo 17

0.0679

, a significant difference in the GCA value of the β-carotene content between the two paired inbred lines at the 0.01 level.

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LI Run et al.

Table 4 The parent-offspring correlation analysis of the β-carotene content at the two experimental locations Location YunnanYuanjiang Sichuang Ya’an

Cross

Reciprocal cross

0.984 ** 0.971 **

0.931 ** 0.955 **

** , the parent-offspring correlation value arrives at significant standard at the 0.01 level by t-test.

realize the relationship between the β-carotene content of hybrids and the parents composition, the top 15 combinations (corresponding to the highest β-carotene content) and the bottom 15 combinations (corresponding to the lowest β-carotene content) were analyzed. The range of the average β-carotene content for the top 15 combinations was from 1.9090 to 3.8783 µg g-1. Among these, ten were high×high combinations (accounting for 66.7%), five were high×low combinations (accounting for 33.3%), and there was no low×low combination. The range of the average β-carotene content for the bottom 15 combinations was from 0.4559 to 1.0133 µg g-1. Among these, eleven were just low×low combinations (accounting for 73.3%), four were high×low combinations (accounted for 26.7%), and there was no high×high combination. As for the 10 top and 10 bottom combinations, high×high combinations accounting for 80% among the 10 top and low×low combinations accounting for 90% among the 10 bottom. So we can find that combinations with high β-carotene content usually come from the parents with high βcarotene content.

Effects of environment on the β-carotene content The paired t-test was adopted to analyze the significance of the difference in the β-carotene contents between the same combinations which grown at the two different locations, and the t-value was 4.4942 (t0.01, 51=2.678). It implied that the mean of the β-carotene content of the

plants grown in Ya’an of Sichuan was higher than that of the plants grown in Yuanjiang of Yunnan, with significance at the 0.01 level. The results showed that the environment had an effect on the β-carotene content of maize grain. From Table 6, we can observe that the β-carotene contents of some combinations, such as 9636×Huangzaosi, 698-3×9636, 5311×9636, 9636×5311, 9636×Dongdan 54-3-1-1-1, 698-3×5311, 48-2×Dongdan 54-3-1-1-1, 5311×Dongdan 54-3-1-1-1, 9636×698-3, and R08×9636, were higher planted in Ya’an than planted in Yuanjiang. Analyzing the parents composition of these combinations, it was found that the combinations rich in β-carotene content were influenced by environment obviously, and that at least one of the inbred lines 5311 or 9636 was used as a parent, except for 48-2×Dongdan 54-3-1-1-1.

DISCUSSION The GCA is mainly related to the additive effects of genes, and the SCA is affected by many factors, including dominance, epistasis, and the interaction between genotype and environment (Rojas and Sprague 1952). Combining ability analysis plays an important part in guiding maize breeding. According to the results obtained by this study, the GCA of the β-carotene content can be inferred to be the major source of variation; however, the SCA and the reciprocal effects are not significant, indicating that the additive effect is the main factor that affects the β-carotene content in maize grain. The result is in accordance with those obtained previously in sweet potato and maize (Egesel et al. 2003; Xie et al. 2004, 2006). The GCA value of the eight inbred lines in the context of the β-carotene content displayed a wide diversity. Some excellent germ plasm resources, such as 9636 and 5311, were identified and can be used for the breeding of improving the

Table 5 The pedigree and the β-carotene contents of the parent inbred lines Inbred 9636 5311 Dongdan 54-3-1-1-1 698-3 R08 48-2 Huangzaosi Mo 17

Pedigree Suwan C11 derived from Guizhou native maize variety derived from hybrid Dongdan 54 derived from US hybrid 78698 derived from Pioneer hybrid 78641 derived from synthetic hybrid Tangsipingtou Lancaster

Content of β-carotene (µg g-1) 5.2576 2.7545 2.2447 1.4302 0.7241 0.6139 0.6476 0.4533

High High High High Low Low Low Low

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Combining Ability and Parent-Offspring Correlation of Maize (Zea may L.) Grain β-Carotene Content with a

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Table 6 The β-carotene contents of combinations planted at two experimental locations No. 1 2

Combination

Yuanjiang (µg g-1)

Ya’an Average content (µg g-1) (µg g-1)

Type

No.

Yuanjiang (µg g-1)

Combination

Ya’an Average content (µg g-1) (µg g-1)

Type

3.3098 3.0373

4.4467 4.6749

3.8783 3.8561

High×High High×High

27 28

R08×5311 R08×698-3

1.2514 1.2718

1.4868 1.3792

1.3691 1.3255

Low×High Low×High

3

9636×698-3 9636×Dongdan 54-3-1-1-1 9636×Huangzaosi

2.2596

5.3553

3.8075

High×Low

29

0.7185

1.8938

1.3062

Low×High

4

5311×9636

2.8127

4.6739

3.7433

High×High

30

1.3496

1.2116

1.2806

High×Low

5 6 7 8

9636×5311 698-3×9636 R08×9636 Dongdan 54-3-1-1-1×698-3 Dongdan 54-3-1-1-1×5311 5311×Dongdan 54-3-1-1-1 9636×R08 9636×Mo 17

2.7703 2.2785 1.8734 2.0268

4.5428 4.1879 2.9936 2.6496

3.6565 3.2332 2.4335 2.3382

High×High High×High Low×High High×High

31 32 33 34

48-2×Dongdan 54-3-1-1-1 Dongdan 54-3-1-1-1× Mo 17 698-3×R08 698-3×Huangzaosi 48-2×698-3 5311×R08

1.4214 1.0732 0.8631 0.8486

0.9384 1.2353 1.4239 1.3787

1.1799 1.1543 1.1435 1.1137

High×Low High×Low Low×High High×Low

2.0023

2.5524

2.2774

High×High

35

Huangzaosi×698-3

1.2312

0.9626

1.0969

Low×High

1.6823

2.8394

2.2609

High×High

36

698-3×48-2

0.8746

1.2364

1.0556

High×Low

2.2146 1.6746

2.2587 2.4797

2.2367 2.0772

High×Low High×Low

37 38

0.7578 1.0417

1.3023 0.9850

1.0301 1.0133

High×Low High×Low

698-3×Dongdan 54-3-1-1-1 9636×48-2 5311×698-3 Huangzaosi×9636 48-2×9636 698-3×5311

1.7204

2.1235

1.9219

High×High

39

1.0450

0.8523

0.9486

High×Low

1.7909 1.5536 2.0880 1.6644 1.1509

2.0521 2.2643 1.7226 2.1045 2.6153

1.9215 1.9090 1.9053 1.8845 1.8831

High×Low High×High Low×High Low×High High×High

40 41 42 43 44

1.0160 0.9011 0.8818 0.7219 1.0061

0.8002 0.8818 0.7990 0.8867 0.5908

0.9081 0.8915 0.8404 0.8043 0.7985

Low×Low Low×High Low×Low Low×Low Low×High

1.6757 1.6094 1.2497 1.6640 1.4811

1.8887 1.6312 1.9764 1.5465 1.5843

1.7822 1.6203 1.6131 1.6053 1.5327

High×Low Low×High Low×High Low×High Low×High

45 46 47 48 49

698-3×Mo 17 Dongdan 54-3-1-1-1×48-2 Dongdan 54-3-1-1-1× Huangzaosi R08×Mo 17 Mo 17×698-3 Mo 17×R08 Mo 17×Huangzaosi Huangzaosi×Dongdan 54-3-1-1-1 Huangzaosi×Mo 17 R08×48-2 R08×Huangzaosi 48-2×Huangzaosi Huangzaosi×R08

0.4909 0.7241 0.8818 0.6620 0.7917

1.0366 0.7765 0.5784 0.7227 0.4329

0.7638 0.7503 0.7301 0.6924 0.6123

Low×Low Low×Low Low×Low Low×Low Low×Low

1.2810

1.7401

1.5106

High×Low

50

48-2×Mo 17

0.3700

0.7289

0.5495

Low×Low

1.4349 1.4173

1.5188 1.4479

1.4769 1.4326

High×Low Low×High

51 52

Huangzaosi×48-2 Mo 17×48-2

0.4978 0.2432

0.5552 0.6686

0.5265 0.4559

Low×Low Low×Low

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

5311×Huangzaosi 48-2×5311 Huangzaosi×5311 Mo 17×5311 Mo 17×Dongdan 54-3-1-1-1 Dongdan 54-3-1-1-1×R08 5311×Mo 17 R08×Dongdan 54-3-1-1-1

The average β-carotene contents are the average value of the two experimental locations in the combinations.

β-carotene content in maize grain. This suggested that it is promising and feasible for enhancing the β-carotene content of maize grain by breeding. The parent-offspring correlation of the β-carotene content between the parents and their F1 generation is significant at the 0.01 level, both in the crosses and the reciprocal crosses at the two different locations. This provides important information regarding how to select the parents in breeding programs aimed at increasing the β-carotene content in maize grain. There is no significant difference in the β-carotene content between the F1 plants of the cross and the reciprocal cross. This indicates that the inheritance of β-carotene content in maize grains may mainly be controlled by the nuclear background, with the effect of the cytoplasmic back-

ground on the β-carotene content not reaching statistically significant levels. Through analyzing the the parents composition of the top 15 and the bottom 15 combinations, it can be inferred that high-content combinations included at least one high-content inbred line as a parent, and that two low-content inbred-lines parents cannot produce highcontent combinations. This result is in accordance with the previous report (Brunson and Quackenbush 1962). The mean value of the β-carotene contents obtained in maize grains from the tested materials planted in Ya’an of Sichuan is significantly higher than that of the same tested materials planted in Yuanjiang of Yunnan. The result is not in accord with that in Abebe Menkir’s study, in which the environment does not have an obvious

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effect on the β-carotene content in the inbred-line grains of tropical maize (Menkir et al. 2008). Whether the environment can affect the β-carotene content in maize grains depends on many factors, such as the experimental locations of different ecotypes and the origins of the different experimental materials. If the experimental locations are not geographically or ecotypically different, or if the genetic diversity of the tested materials is relatively small, a different conclusion may be obtained. In this study, we find that there is an obvious difference in the β-carotene content between plants grown in Ya’an and Yuanjiang only for some combinations, whereas the others did not show a difference; morever, the β-carotene contents of maize grains are higher in Ya’an of Sichuan with the midsubtropical climate than those of maize grains in Yuanjiang of Yunnan with the southern subtropical climate. Combinations in which the β-carotene contents were affected obviously by the environment, were rich in β-carotene, and their parents, such as 5311, 9636, or Dongdan 54-3-1-1-1, had high β-carotene content. It seems that the higher the β-carotene content, the greater is the environmental effect. According to this study, the broad sense heritability for the β-carotene content in maize grain was 79.45%, the narrow sense heritability for the β-carotene content in maize grain was 78.88%, owing to the narrow sense heritability is relatively high, so selecting at early generation of breeding for increasing the β-carotene content in maize grain was more reliable and feasible.

MATERIALS AND METHODS Materials Eight inbred lines of maize, with a relatively broad range of β-carotene content, were used as parents; they are 5311, 9636, Dongdan 54-3-1-1-1, 48-2, R08, 698-3, Huangzaosi, and Mo 17 (Table 5). Sixty-four combinations were produced by complete diallel cross.

Field trial In the spring of 2007, the eight inbred lines were planted in Ya’an of Sichuan. Sixty-four combinations were obtained from the eight inbred lines by the complete diallel cross, based on Griffing’s Method I. In 2008, the 64 combina-

LI Run et al.

tions were planted in Yuanjiang of Yunnan (with southern subtropical climate) and Ya’an of Sichuan (with midsubtropical climate) separately. The combinations were arranged in a randomized complete block design, with two replications at both experimental locations. Each combination was grown in two rows with a 0.8-m interval between rows and 0.45 m interval between plants. Each row was 3.5 m long. Six plants were self-fertilized for each combination, and three or four ears were harvested separately, each ear was considered as one test sample for analyzing the β-carotene content.

Analysis of β-carotene content Maize grains were used for the assay of β-carotene content by HPLC. The extraction protocol was based on the method of Kurilich and Juvik (1999), as modified by Howe and Tanumihardjo (2006). Carotenoids were released from dried maize grains (0.6 g) by adding ethanol (6 mL) containing 0.1% butylated hydroxytoluene (v/v), mixed by vortexing for 20 s, and held in a water bath maintained at 85°C for 5 min. KOH (500 µL, 80% w/v) was added to the heated ethanol-maize mixture. The samples were mixed by vortexing, placed in a 85°C water bath for 10 min, and then immediately placed in ice. Cold distilled water (3 mL) and 200 µL of β-apo-8´-carotenal were added in succession. Carotenoids were extracted thrice with hexane (3 mL) using centrifugation (1 200×g) to separate the layers. The combined organic layers were washed with distilled water (5 mL), and the organic layer was removed to a new tube. The remaining aqueous layer was extracted twice more with hexane (3 mL). The combined organic layers were dried under nitrogen. The dried extract was dissolved in 500 µL of a mixture of methyl alcohol:ethylene dichloride (50:50, v/v). Subsequently, 30 µL of the aliquot was injected onto the HPLC system for β-carotene analysis. Based on the method of Rodriguez-Amaya and Kimura (2004) and Kimura et al. (2007), the experimental conditions of HPLC were modified. The chromatographic column used in this experiment was Shim-pack VP-ODS, 5 µm, 250 mm×4.6 mm. The mobile phase was acetonitrile: methanol:ethyl acetate (with 0.05% triethylamine), the initial proportion of 90:10:0 was increased to 60:20:20 within 20 min (concave gradient), maintained in this proportion for 22 min, then changed to 20:40:40 in 35 min (linear gradient) to remove the lipids, and maintained in this proportion for 40 min. The flow rate was 0.8 mL min-1 and reequilibration took 10 min. The absorbance of β-carotene was measured at 450 nm. One of the two replications from Yunnan and Sichuan location respectively was assayed for the β-carotene content. Standard β-carotene and the internal standard β-apo8´-carotenal were used for the calculation of β-carotene content in sample. Based on the measurement of the mixturie of standard β-carotene and the internal standard β-apo-8´-

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Combining Ability and Parent-Offspring Correlation of Maize (Zea may L.) Grain β-Carotene Content with a

carotenal assayed by HPLC, the correction factor (f) was calculated according to the peak area of standard β-carotene (Ar) and peak area of internal standard β-apo-8´carotenal (As), along with the concentration of standard β-carotene (Cr) and the concentration of internal standard β-apo-8´-carotenal (Cs), the formula was as f=(As/Cs)/(Ar/ Cr) Each extracted sample was mixed with a certain amount of the internal standard substance, then injected into HPLC. If Cx was used to stand for the value of β-carotene content in sample , Ax stand for the peak area of β-carotene content, As and Cs stand for the peak area and the concentration of the internal standard substance respectively, Cx can be calculated by the formula: Cx=f×Ax/(As/Cs)

Statistical analysis Statistical analysis of diallel data was based on the measurement value of β-carotene content in one plant. Data of β-carotene content from Yunnan and Sichuan were considered as two replications. The variance analyses, combining ability analysis and significance test were carried out according to Griffing’s Method I (Rong et al. 2003). In this study, combinations were considered as fixed effects, replications were considered as random effects. The simple correlations between parents and their F1 were calculated, the significance was tested. At the same time, the difference between the cross and the reciprocal cross, the difference between two locations were analyzed using paired t-test (Ming 2008). All the data were conducted with DPS software.

Acknowledgements This work was supported by the Harvest-Plus China Program, the National High-Tech R&D Program of China (2011AA10A103), and the Sichuan Maize Breeding Program in the 12th Five-Year Plan, China.

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