Supporting Information For
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Supporting Information for: ZmCCT9 enhances maize adaptation to higher latitudes Cheng Huanga, Huayue Suna, Dingyi Xua, Qiuyue Chena, Yameng Lianga, Xufeng Wanga, Guanghui Xua, Jinge Tiana, Chenglong Wanga, Dan Lia, Lishuan Wua, Xiaohong Yanga, Weiwei Jina, John F. Doebleyb,1, and Feng Tiana,1 aNational Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, Laboratory of Crop Heterosis and Utilization, Joint International Research Laboratory of Crop Molecular Breeding, China Agricultural University, Beijing 100193, China; and bDepartment of Genetics, University of Wisconsin, Madison, WI 53706 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. SI Materials and Methods Plant Materials. The population of 866 maize-teosinte BC2S3 RILs was obtained from the Maize Genetics Cooperation Stock Center (Maize COOP). This population was derived from a cross between W22, a typical temperate maize (Zea mays ssp. mays) inbred line, as the recurrent parent, and CIMMYT accession 8759, a typical accession of teosinte (Zea mays ssp. parviglumis), as the donor parent. Detailed information on this population has been previously reported (1-4). A maize association panel composed of 513 diverse maize inbred lines (5) was used in the association analysis of the 2.4-kb causative region for qDTA9. To analyse the evolutionary origin of the Harbinger-like element, a panel of 73 diverse teosinte lines (Zea mays ssp. parviglumis) (SI Appendix, Table S1), representing the genetic and geographic variability within extant populations of the progenitor species, was genotyped using primers specific to the Harbinger-like element. Another diverse panel, including 27 maize inbred lines and 19 teosinte accessions (Zea mays ssp. parviglumis), was used 1 to analyse nucleotide diversity across the 2.4-kb causative region for qDTA9. The panel was selected to maximize the genetic diversity of maize and represents a wide geographical distribution of wild teosinte germplasm (6-8). The 27 diverse maize inbred lines include the 26 founder lines of the maize NAM population and the Mo17 founder of the B73 x Mo17 (IBM) population. The 19 teosinte accessions include eight teosinte inbred lines (SI Appendix, Table S2). A total of 1,008 accessions of maize landraces (SI Appendix, Table S3) representing the entire pre-Columbian range of maize races native to the Americas was used to examine the geographic distribution of the Harbinger-like transposon insertion at ZmCCT9 and the CACTA-like transposon insertion at ZmCCT10. Passport data for these maize landraces can be found in previous studies (9, 10). F1 plants derived from crosses between the 25 NAM founders and the reference B73 were used to assay allele-specific expression. Phenotypic Effect Analysis of qDTA9. A pair of NILs that are homozygous for the maize and teosinte alleles across the qDTA9 region, designated NIL(maize) and NIL(teosinte), was developed from a HIF that was heterozygous only at qDTA9 (SI Appendix, Fig. S1). To examine the effect of qDTA9 on flowering time, NIL(maize) and NIL(teosinte) were grown in neighbouring rows with five replications under natural LD conditions (Beijing (39.9°N, 116.4°E), China) and SD conditions (Sanya (18.1°N, 109.3°E), Hainan Province, China). NIL(maize) and NIL(teosinte) were scored for four flowering-related traits, including days to anthesis (DTA), days to silking (DTS), plant height and leaf number. DTA is the number of days between planting and the time at which half of the central spike is shedding pollen. DTS is the number of days between planting and the time at which silks are visible on the ear. Plant height is the distance from the ground to the uppermost node on the stalk. To measure the number of leaves, the 5th and 10th leaves of each plant were marked. Fine Mapping of qDTA9. We previously detected qDTA9 between markers M114923 and M123776 on chromosome 9 using the maize-teosinte BC2S3 RIL population (2). To perform fine mapping of qDTA9, 5,394 F2 plants derived from selfing a HIF that is 2 heterozygous only at qDTA9 region (SI Appendix, Fig. S1) were planted in a winter nursery in Sanya, Hainan Province, China, in 2013. The two molecular markers M114923 and M123776 flanking qDTA9 were used to identify the recombinants from the F2 population. Ten additional markers were developed to further determine the recombination breakpoints of the F2 recombinants. A recombinant-derived progeny testing strategy (1-4) was used to delimit the region of qDTA9. Briefly, within each recombinant-derived F3 family, homozygous recombinant (HR) and homozygous non-recombinant (HNR) plants were identified using markers. The significance of flowering-time differences between the HR and HNR plants within each family was determined using Student's t-test. If a significant flowering-time difference between the HR and HNR plants was observed, the parental F2 recombinant was heterozygous for qDTA9; otherwise, the recombinant was homozygous for either parent. The phenotypic evaluation of the selected recombinant-derived F3 families was performed at the Shangzhuang Experimental Station of China Agricultural University, Beijing, China, in the summer of 2014. By integrating the QTL location information from all recombinants, the causative region for qDTA9 was finally delimited to a 2.4-kb noncoding region between markers M115705 and M115707. Association Mapping. Three pairs of primers were used to sequence the 2.4-kb causative region for qDTA9 in the maize association panel containing 513 diverse maize inbred lines. This association population was previously phenotyped for flowering time in multiple environments (5). In this study, the DTA data scored in the typical natural long-day lengths in Beijing (mean day length = 13.7 h, summer 2012, Shangzhuang Experimental Station of China Agricultural University, Beijing, China) were used as the phenotype in the association analysis of 2.4-kb causative region for qDTA9. Sequencing reactions on PCR products were performed in both directions. Multiple sequence alignments were performed using BIOEDIT (v.7.0.9.0; North Carolina State University, Raleigh, NC, USA) and manually edited when necessary. A total of 41 sequence variants (SNPs and insertions and deletions) with MAF ≥ 0.05 were identified among these sequences, and their associations with DTA were tested 3 using a mixed linear model that accounts for familial relatedness (11) in Tassel 3.0 ((12)). The kinship matrix used in the model is the same as the previous study (5). A Bonferroni adjusted significance threshold (P ≤ 0.01/41 = 2.44 × 10–4) was used to identify significant associations. Pairwise linkage disequilibrium analysis was performed using R software (version 3.1.0). The association between the Harbinger-like element and the DTA phenotype scored in five other locations at different latitudes (5) was also tested using the same association analysis model. To minimize the effect of temperature differences among environments on the observed flowering times and obtain a comparable analysis across different environments, DTA were converted to growing degree days (GDD) following previous method (13), with 10 °C as TBASE and 30 °C as Tmax. GDD to anthesis represents the cumulative average daily heat units that a row of plants received from the time that seeds were sown in the field until anthesis (13). Generation and Analysis of CRISPR/Cas9 Knockout Lines. The CRISPR/Cas9 knockout vector was constructed according to previously described protocol (14). Briefly, the two target sites located at the first and second exons of ZmCCT9 were incorporated into PCR forward and reverse primers, respectively, for the assembly of two guide RNAs (gRNAs). A PCR fragment was amplified from the pCBC-MT1T2 vector using the two long primers. Subsequently, the purified PCR fragment, together with the pBUE411 vector, was used to set up restriction-ligation reactions using the BsaI restriction enzyme and T4 ligase (New England Biolabs). The resulting vector was confirmed by sequencing and introduced into the Agrobacterium tumefaciens strain EHA105. The Agrobacterium-mediated method (15) was used to transform young embryos of the maize inbred line ZC01 at China National Seed Group Co., Ltd (Wuhan, China). A total of 20 independent T0 transgenic plants were obtained. Genomic fragments covering each target site and both target sites were amplified from the T0 plants by PCR. The PCR products were separated on agarose gels and cloned in the pMD-18T vector (Takara). At least six clones per PCR product were sequenced. Three T0 plants with homozygous deletions at the target sites were identified (referred to 4 as KO#1, KO#2 and KO#3) (SI Appendix, Fig. S7). Homozygous mutations in edited target genes were readily found in T0 plants in previous studies (16-21). In addition to the non-homologous end joining (NHEJ) mechanism, the homology-directed repair(HDR) repair mechanism might also involve the production of the homozygous mutations (16, 22, 23). The three homozygous T0 plants (KO#1, KO#2 and KO#3) were self-pollinated to get T1 progenies. The resulting T1 progenies, together with the wild-type (ZC01), were planted in natural LD conditions. All T1 plants were further genotyped by PCR and sequencing to confirm the presence of mutations at the target sites. T1 and wild-type plants were scored for flowering-time traits (DTA and DTS). RNA Extraction and Quantitative Real-time PCR (qRT-PCR) Analysis. To examine the spatiotemporal expression patterns of ZmCCT9, NIL(maize) and NIL(teosinte) were grown under LD conditions in the field (summer 2015, Shangzhuang Experimental Station of China Agricultural University, Beijing, China). Various plant tissues were collected from the NILs at different development stages, with three biological replicates in each case. Total RNA was extracted using TRIzol (Invitrogen), treated with RNase-free DNase I (Takara) and purified using the RNAclean Kit (Tiangen). The RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and 2 μg of total RNA was reverse transcribed using a random primer and recombinant M-MLV reverse transcriptase (Promega) following the manufacturer's instructions.