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Genomic insights into historical improvement of heterotic groups during modern hybrid maize breeding

Abstract

Single-cross maize hybrids display superior heterosis and are produced from crossing two parental inbred lines belonging to genetically different heterotic groups. Here we assembled 1,604 historically utilized maize inbred lines belonging to various female heterotic groups (FHGs) and male heterotic groups (MHGs), and conducted phenotyping and genomic sequencing analyses. We found that the FHGs and MHGs have undergone both convergent and divergent changes for different sets of agronomic traits. Using genome-wide selection scans and association analyses, we identified a large number of candidate genes that contributed to the improvement of agronomic traits of the FHGs and MHGs. Moreover, we observed increased genetic differentiation between the FHGs and MHGs across the breeding eras, and we found a positive correlation between increasing heterozygosity levels in the differentiated genes and heterosis in hybrids. Furthermore, we validated the function of two selected genes and a differentiated gene. This study provides insights into the genomic basis of modern hybrid maize breeding.

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Fig. 1: Population structure of 1,604 inbred lines.
Fig. 2: Morphological trait improvement and accumulation of favourable alleles in the FHGs and MHGs during modern hybrid maize breeding.
Fig. 3: Atlas of selection signatures in the FHGs and MHGs.
Fig. 4: Identification and functional validation of ZmEMF1L1.
Fig. 5: Identification and functional validation of ZmKW10.
Fig. 6: Genomic differentiation and effect of differentiated genes between the FHGs and MHGs on grain yield heterosis.
Fig. 7: Validation of ZmKOB1 as a differentiated gene between the PA and SPT heterotic groups.

Data availability

The raw sequencing data were deposited in the Genome Sequence Archive (https://bigd.big.ac.cn/gsa) under the accession code PRJCA009749 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA009749). The phenotype dataset reported here is available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

The custom codes used in this study are available at https://github.com/jasongit0311/maize_for_Li.

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Acknowledgements

The work was supported by the National Key Research and Development Program of China (grant nos 2016YFD0100103 to T.W., Yu Li and C. Li; 2021YFD1200700 to T.W.; 2016YFD0100303 to Yu Li; and 2020YFE0202300 and 2021YFF1000300 to H.W.), the Major Program of Guangdong Basic and Applied Research (grant no. 2019B030302006 to H.W.), the National Natural Science Foundation of China (grant no. 31971891 to C. Li) and the CAAS Innovation Program. J.R.-I. is supported by NSF grant no. 1546719 and USDA Hatch project no. CA-D-PLS-2066-H. We thank H. Lu, M. Yang and L. Huang from the Novogene Bioinformatics Institute for bioinformatics support and Z. Lin (Peking University) for his suggestions on data analysis. We thank Y. Yang of the Beijing Academy of Agricultural & Forestry Science for verifying the maize hybrids. We thank H. Li from the Chinese Academy of Agricultural Sciences and H. Zhou from Huazhong Agricultural University for their suggestions on the calculation of the dominance effect. We also thank Y. Xu and C. Xu from Yangzhou University for their advice on the statistical analyses.

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Authors and Affiliations

Authors

Contributions

T.W., Yu Li, H.W. and C. Li conceived and designed the experiments. T.W., Yu Li, C. Li, Yongxiang Li, Y. Shi and Y. Song participated in the germplasm preparation. C. Li, X.J. and C.J. performed bioinformatics analyses of the data. H.G., Yaoyao Li and C. Li carried out the validation of gene function and gene expression analysis. B.W., H.G., Yaoyao Li, Yongxiang Li and X.L. participated in the data analysis. C. Li, D.Z., C. Liu, X.X., H.Z., Y.W., J.L., P.Z., G.H., G.L., S.L., D.S., X.W., Y. Shi and Y. Song performed the phenotypic measurements. C. Li, H.W., J.R.-I., Yu Li and T.W. analysed the data and wrote the manuscript.

Corresponding authors

Correspondence to Chengzhi Jiao, Jeffrey Ross-Ibarra, Yu Li, Tianyu Wang or Haiyang Wang.

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The authors declare no competing interests.

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Nature Plants thanks Jianming Yu, Klaus Mayer, Xuehui Huang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Geographic distribution and population structure analyses of the 1,604 inbred lines.

a, Geographic distribution of all resequenced inbreds according to their origin. Bar graphs show the number of inbreds released in Era I and Era II in the US and China. The orange and blue pie charts show the proportions of inbreds released in Era I and Era II, respectively. The map was drawn using the R ggmap package (http://maps.stamen.com/, map tiles by Stamen Design, under CC BY 3.0. Data by OpenStreetMap, under ODbL). b, The marginal likelihood begins to plateau at a K = 11, which was considered as the optimal number of subgroups based on the fastStructure analysis. c, Population structure of the panel analyzed from K = 2 to 15. K = 11 clearly divided the panel into eleven subgroups, including PA1, PA2, PB, Lancaster1, Lancaster2, Zi330, Iodent, Amargo, BSSS, SPT1 and SPT2.

Extended Data Fig. 2 Phenotypic changes in the FHGs and MHGs across different breeding eras.

a-c, phenotypic changes in the two female groups (PA and US_SS) and four male groups (PB, SPT, CN_NSS and US_NSS) from Era I to Era II. Group 1 (a), Group 2 (b) and Group 3 (c) include seven, eight and three traits, respectively. The P values of Era I versus Era II in six groups using two-sided Wilcoxon test (left to right, as plotted) are: DA, P = 6.36×10−6, 3.73×10−2, 1.34×10−5, 2.07×10−2, 8.83×10−6 and 2.09×10−2, respectively; DS, P = 7.40×10−8, 1.27×10−2, 5.83×10−5, 9.61×10−2, 1.00×10−6 and 1.83×10−2, respectively; ASI, P = 4.14×10−7, 1.58×10−1, 1.29×10−1, 4.64×10−2, 1.05×10−2 and 2.64×10−1, respectively; EH, P = 5.35×10−3, 3.14×10−4, 3.27×10−2, 6.25×10−1, 7.50×10−6 and 1.81×10−2, respectively; EP, P = 3.94×10−3, 2.93×10−4, 1.39×10−3, 3.71×10−1, 4.12×10−8 and 1.30×10−6, respectively; TBN, P = 1.07×10−7, 1.26×10−4, 6.45×10−1, 9.31×10−1, 2.95×10−12 and 2.62×10−10, respectively; KR, P = 3.55×10−1, 3.55×10−3, 2.58×10−3, 1.55×10−2, 2.21×10−4 and 9.67×10−5, respectively; GYPP, P = 3.67×10−1, 1.68×10−1, 9.67×10−3, 2.13×10−1, 3.26×10−8 and 3.12×10−6, respectively; KWPE, P = 7.93×10−1, 2.56×10−1, 1.76×10−3, 2.09×10−2, 1.46×10−7 and 2.15×10−5, respectively; EW, P = 9.48×10−1, 4.39×10−1, 4.85×10−3, 3.47×10−2, 1.42×10−6 and 4.72×10−4, respectively; KNPR, P = 5.77×10−1, 2.05×10−1, 3.37×10−1, 9.58×10−1, 6.71×10−1 and 1.66×10−4, respectively; HKW, P = 6.13×10−2, 4.09×10−1, 4.53×10−1, 2.59×10−1, 6.90×10−4 and 4.29×10−4, respectively; HKV, P = 2.15×10−2, 5.56×10−1, 2.78×10−2, 3.84×10−2, 8.49×10−7 and 9.94×10−4, respectively; KL, P = 6.53×10−1, 3.14×10−1, 5.53×10−5, 1.17×10−5, 2.71×10−8 and 5.75×10−5, respectively; KW, P = 2.50×10−1, 6.41×10−1, 9.32×10−1, 5.72×10−1, 2.85×10−2 and 2.07×10−4, respectively; ED, P = 1.19×10−2, 1.60×10−1, 7.03×10−3, 1.73×10−2, 2.97×10−2 and 2.99×10−1, respectively; KRN, P = 2.06×10−2, 9.68×10−1, 3.30×10−2, 1.78×10−1, 2.00×10−1 and 2.95×10−1, respectively; CW, P = 1.93×10−1, 1.38×10−1, 3.98×10−1, 4.32×10−2, 2.71×10−3 and 8.35×10−1, respectively. d, Phenotypic changes in the FHGs and MHGs and in the two female groups (PA and US_SS) and four male groups (PB, SPT, CN_NSS and US_NSS) from Era I to Era II for the three traits of Group 4. The P values of FHGsEra I versus FHGsEra II, MHGsEra I versus MHGsEra II, FHGsEra I versus MHGsEra I and FHGsEra II versus MHGsEra II using two-sided Wilcoxon test are presented in Supplementary Table 5. The P values of Era I versus Era II in six groups using two-sided Wilcoxon test (left to right, as plotted) are: PH, P = 1.97×10−1, 3.34×10−1, 8.63×10−2, 1.52×10−1, 1.57×10−1 and 6.20×10−1, respectively; TL, P = 2.87×10−1, 3.53×10−1, 3.45×10−1, 3.78×10−3, 8.49×10−1 and 5.09×10−2, respectively; EL, P = 9.79×10−1, 7.53×10−1, 2.03×10−1, 7.36×10−1, 5.80×10−1 and 9.33×10−5, respectively. For boxplots in a-d, the central lines show the median, the box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Exact sample size (n) in FHGsEra I, FHGsEra II, MHGsEra I and MHGsEra II (left to right, as plotted): n = 72, 253, 330 and 267. Exact sample size (n) in PAEra I, PAEra II, US_SSEra I, US_SSEra II, PBEra I, PBEra II, SPTEra I, SPTEra II, CN_NSSEra I, CN_NSSEra II, US_NSSEra I and US_NSSEra II (left to right, as plotted): n = 46, 161, 26, 92, 32, 42, 46, 78, 204, 86, 48 and 61. Significant differences are indicated: ** P < 0.01 and * P < 0.05.

Extended Data Fig. 3 Accumulation of favorable alleles in the female and male heterotic groups during modern hybrid maize breeding.

a, Profile of favorable allele frequencies changes at GWAS associated SNPs in the FHGs and MHGs. Orange indicates an increase, while green indicates a decrease in the frequency of a favorable allele from Era I to Era II during modern breeding. Each row represents an associated SNP. Blue and gray colors (in the first column) mark rows representing the associated SNPs at the threshold of P < 10−6 and 10−5, respectively. b, The number of favorable alleles accumulated in the FHGs and MHGs across different breeding eras. The P values of FHGEra I versus FHGEra II, MHGEra I versus MHGEra II, FHGEra I versus MHGEra I and FHGEra II versus MHGEra II using two-sided Wilcoxon test are: DA, P = 3.70×10−1, 3.71×10−2, 2.10×10−1 and 2.04×10−4, respectively; DS, P = 5.60×10−2, 3.62×10−4, 3.36×10−1 and 7.53×10−1, respectively; ASI, P = 1.73×10−1, 2.54×10−15, 1.42×10−13 and 9.62×10−7, respectively; EH, P = 4.12×10−1, 3.52×10−1, 7.55×10−1 and 2.63×10−2, respectively; EP, P = 5.15×10−1, 1.77×10−1, 8.08×10−1 and 1.50×10−1, respectively; TBN, P = 8.11×10−7, 2.68×10−8, 2.34×10−19 and 4.83×10−41, respectively; KWPE, P = 5.30×10−1, 9.80×10−18, 1.70×10−15 and 2.63×10−10, respectively; EW, P = 6.43×10−1, 3.22×10−15, 2.61×10−15 and 9.41×10−10, respectively; KNPR, P = 2.67×10−1, 3.74×10−15, 1.15×10−17 and 7.94×10−17, respectively; HKW, P = 6.51×10−2, 2.50×10−13, 9.47×10−5 and 3.28×10−3, respectively; HKV, P = 2.22×10−1, 1.44×10−13, 4.83×10−4 and 7.16×10−2, respectively; KL, P = 2.43×10−1, 8.35×10−23, 4.71×10−17 and 5.82×10−3, respectively; KW, P = 8.62×10−1, 3.82×10−7, 2.95×10−1 and 1.65×10−4, respectively; KRN, P = 2.81×10−2, 2.16×10−1, 1.19×10−8 and 4.32×10−4, respectively; CW, P = 9.11×10−1, 7.04×10−3, 1.09×10−5 and 1.20×10−5, respectively. For boxplots, the central lines show the median, the box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Significant differences are indicated: *** P < 0.001, ** P < 0.01 and * P < 0.05. Exact sample size (n) are 72, 253, 330 and 267 for FHGsEra I, FHGsEra II, MHGsEra I and MHGsEra II, respectively. c, Correlation between the number of accumulated favorable alleles and improved trait in the FHGs and MHGs. The six traits of Group 1 include DA (days to anthesis), DS (days to silking), ASI (anthesis to silking interval), EH (ear height), EP (relative ear height), TBN (tassel branch number); the seven traits of Group 2 include KWPE (kernel weight per ear), EW (ear weight), KNPR (kernel number per row), HKW (hundred kernel weight), HKV (hundred kernel volume), KL (kernel length) and KW (kernel width); and the two traits of Group 3 include KRN (kernel row number) and CW (cob weight). The Pearson correlation coefficient (r) and P value are presented.

Source data

Extended Data Fig. 4 Distribution and sharing of selective sweeps and profile of allele frequency change for the selected genes in the FHGs and MHGs.

a, The distribution ratio of selective sweeps located in genic and intergenic regions in the six heterotic groups. b, Distribution of the distances from intergenic selective sweeps to the nearest gene. c, Enrichment of shared genes (below the diagonal) and GO terms (above the diagonal) in the female heterotic groups (FHGs) and/or male heterotic groups (MHGs). Squares are marked with an asterisk (P < 0.05) and two asterisks (P < 0.01) if the observed number shared was significantly higher than the background number under permutation test conditions. d, Profile of allele frequency change of the 589 selected genes exhibiting co-directional change in allele frequencies between the FHGs and MHGs from Era I to Era II. e, Profile of allele frequency change of the 28 selected genes exhibiting anti-directional changes between the FHGs and MHGs from Era I to Era II. f, Profile of allele frequency change of the 400 selected genes exhibiting convergent increase or reduction in allele frequencies in the MHGs, but not in the FHGs, from Era I to Era II. For d-f, Orange indicates an increase of the reference allele frequency of nonsynonymous SNPs, whereas green indicates an increase of the alternative allele frequency from Era I to Era II. Each row represents a nonsynonymous SNP of the selected genes.

Extended Data Fig. 5 Representative selected genes related to plant growth and development and abiotic stress responses in at least two heterotic groups.

a-l, Genes functionally characterized in maize or their homologous genes have been functionally characterized in rice. Each gene includes three plots: XP-CLR plot, physical position of the gene (indicated by vertical gray dotted line), top 5% score of XP-CLR in individual heterotic group (shown by horizontal dotted line). Haplotype table plot, the type and number of haplotypes formed by nonsynonymous SNPs of gene were counted. The haplotypes with at least 20 inbred lines were listed in the table. Haplotype frequency bar plot, different haplotype frequency change from Era I to Era II was shown within each of the six heterotic groups.

Extended Data Fig. 6 Representative differentiated genes related to abiotic stress responses and plant growth and development.

a-k, Genes functionally characterized in maize or their homologous genes have been functionally characterized in rice. Each gene includes three plots: FST and allele frequency difference (AFD) plot, physical position of the gene (indicated by vertical gray dotted line), top 5% score of FST and AFD between the FHGs and MHGs were shown by horizontal orange dotted line and green dotted line, respectively. Haplotype table plot, the type and number of haplotypes formed by nonsynonymous SNPs of gene were counted. The haplotypes with at least 10 inbred lines were listed in the table. Haplotype frequency bar plot, different haplotype frequency change from Era I to Era II was shown in the female and male heterotic groups.

Extended Data Fig. 7 The relationships between heterozygosity levels and heterosis and between accumulated superior heterozygous alleles and heterosis for yield-related traits in four testcross populations.

a-d, Each plot group includes six plots for two traits (kernel weight per ear (KWPE) and ear weight (EW)): Left plot, correlation between heterozygosity levels of whole-genome nonsynonymous SNPs in non-divergent regions and better parent heterosis (BPH) of KWPE and EW in the testcross population. Middle plot, correlation between heterozygosity levels of nonsynonymous SNPs located in genes continuously selected in divergent regions and BPH of KWPE and EW in the testcross population. A total of 2,563 and 2,136 nonsynonymous SNPs contained in 478 and 375 genes continuously selected in PA × SPT and US_SS × US_NSS, were used to calculate the heterozygosity levels for the testcross populations from PA × SPT and SS × NSS heterotic patterns, respectively. Right plot, correlation between the number of accumulated superior heterozygous alleles and BPH of KWPE and EW in the testcross population. Four testcross populations include 88 hybrids derived from CNH3754 (a PA inbred) × 88 SPT inbreds, 91 hybrids derived from 91 PA inbreds × Jing2416 (a SPT inbred), 106 hybrids derived from Xunshi104-8 (a SS inbred) × 106 US_NSS inbreds, and 101 hybrids derived from 101 US_SS inbreds × F62 (a NSS inbred). The Pearson correlation coefficient (r) and P value are presented.

Source data

Extended Data Fig. 8 Identification of ZmKOB1 as a differentiated gene between the PA and SPT heterotic groups.

a, FST (above the axis) and allele frequency difference (AFD) of ZmKOB1. The candidate gene was visualized by green shadow. b, Gene structure and haplotype analyses of ZmKOB1. c, Box plots for kernel weight per ear (KWPE), kernel number per row (KNPR) and ear length (EL) for the four haplotypes. Center line, medium; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range. n indicates the number of inbred lines for each haplotype. The significance of difference was analyzed using two-sided Wilcoxon test. d, Haplotype frequency of ZmKOB1 in different breeding eras in the PA and SPT heterotic groups.

Supplementary information

Supplementary Information

Supplementary Tables 3–5, 8, 14 and 18–22.

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Supplementary Data 1

Supplementary Tables 1, 2, 6, 7, 9–13 and 15–17.

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Li, C., Guan, H., Jing, X. et al. Genomic insights into historical improvement of heterotic groups during modern hybrid maize breeding. Nat. Plants 8, 750–763 (2022). https://doi.org/10.1038/s41477-022-01190-2

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