Recombinant inbred lines and next-generation sequencing enable rapid identification of candidate genes involved in morphological and agronomic traits in foxtail millet

www.nature.com/scientificreports OPEN Recombinant inbred lines and next‑generation sequencing enable rapid identification of candidate genes involved in morphological and agronomic traits in foxtail millet Kenji Fukunaga1,7*, Akira Abe2,7*, Yohei Mukainari1, Kaho Komori1, Keisuke Tanaka3, Akari Fujihara1, Hiroki Yaegashi2, Michie Kobayashi2,6, Kazue Ito2, Takanori Ohsako4 & Makoto Kawase5 We constructed recombinant inbred lines (RILs) between a Japanese and a Taiwanese landrace of foxtail millet and employed next-generation sequencing, such as flexible ddRAD-seq and Nanopore sequencing to identify the candidate genes involved in the crop evolution of foxtail millet. We successfully constructed a linkage map using flexible ddRAD-seq with parents and RILs and detected major QTLs for each of three traits: leaf sheath colors, spikelet-tipped bristles (stb), and days to heading (DTH). (1) For leaf sheath colors, we identified the C gene on chromosome IV. (2) We identified a homeobox (HOX14) gene for stb on chromosome II, which shows homology with HvVrs1 in barley. (3) Finally, we identified a QTL with a large effect on DTH on chromosome II. A parent of the RILs from Taiwan and Yugu1 had a Harbinger-like TE in intron 3 of this gene. We also investigated the geographical distribution of the TE insertion type of this gene and found that the insertion type is distributed in the northern part of East Asia and intensively in South and Southeast Asia, suggesting that loss/reduction of function of this gene plays an important role in spreading into the northern part of East Asia and subtropical and tropical zones. Foxtail millet [Setaria italica (L.) P. Beauv.] is one of the oldest cereals in the Old World. It is characterized by diploidy with small chromosome numbers (2n = 2x = 18), small genome size (approximately 500 Mb), an inbreeding habit, and a relatively short growth habit; therefore, it has become an ideal model plant for genetic studies on panicoid grass species, including switchgrass and Napier grass, which are considered as biofuel sources, and other cultivated millet species such as pearl m illet1. This millet adapts to various environmental conditions, and its agronomic traits show large variation as a result of adaptation to local environments ranging from temperate to tropical climates, high- and low-altitude conditions, and cultivation under various cultural conditions. Several landrace groups are genetically differentiated and distributed in different geographical a reas2. The foxtail millet genome has been s equenced3,4 and recently, the genome of its presumed wild ancestor, S. viridis, was also determined5. Owing to its high variations in several agronomic traits, this millet will also be a good material for studying crop evolution in the context of adaptation to variable environmental conditions and human selection. 1 Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 5562 Nanatsuka‑cho, Shobara, Hiroshima 727‑0023, Japan. 2Iwate Biotechnology Research Center, 22‑174‑4 Narita, Kitakami, Iwate 024‑0003, Japan. 3NODAI Genome Research Center, Tokyo University of Agriculture, 1‑1‑1 Sakuragaoka, Setagaya‑ku, Tokyo 156‑8502, Japan. 4Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 74 Kitainayazuma, Seika‑cho, Kyoto 619‑0244, Japan. 5Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 243‑0034, Japan. 6Present address: Institute of Agrobiological Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 3‑1‑1 Kannondai, Tsukuba, Ibaraki 305‑0856, Japan. 7These authors contributed equally: Kenji Fukunaga and Akira Abe. *email: fukunaga@ pu-hiroshima.ac.jp; Scientific Reports | (2022) 12:218 | https://doi.org/10.1038/s41598-021-04012-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Characteristics of parental landraces. Maternal parent: JP73913 from Taiwan with green leaf sheath and spikelet-tipped bristles (stb) and heading early-middle August. Pollen donor: JP71640 from Miyazaki Prefecture, Japan, with red leaf sheath and wild type of bristles and heading early September. The map was created using the R package “maps” ver. 3.3.0 (https://CRAN.R-project.org/package=maps). Recently, next-generation sequencing (NGS) technology has become a powerful tool for genetic mapping and population genetics. Several methods for genetic mapping using NGS have also been developed, including genotyping by sequencing (GBS) 6,7, RAD-seq8,9, ddRAD-seq10,11, QTL-seq12, and GRAS-Di13,14. Several studies on gene and QTL mapping15–23 and genome-wide association studies (GWAS)24–26 in foxtail millet have been conducted using NGS technology, and several genes have been mapped and identified successfully. However, most studies on QTL mapping have been carried out using hybrid-derived populations from crosses within Chinese cultivars, Japanese cultivars, and between Setaria viridis and a Chinese cultivar. No population between distantly related landraces collected from different geographical conditions has been used for mapping. Consequently, we constructed an RIL population derived from a cross between a Japanese landrace (JP71640) and a Taiwanese landrace (JP73913) adapting to different latitudes and climates and mapped three traits: leaf sheath color, spikelet-tipped bristles (stb), and days to heading (DTH) (Fig. 1). JP 71640 and JP 73913 were chosen as parents because they are genetically distinct from each other in terms of morphological and physiological characteristics, rDNA genotypes, RFLP variations of genomic DNA, and intervarietal hybrid s terility2. Herein, we successfully identified major QTLs and identified candidate genes for the C gene controlling leaf sheath color, a homeobox (HOX14) gene for stb, and PRR37 for DTH. We also found allelic variation in the C gene and the homeobox gene in Yugu1 and naturally occurring mutants of S. viridis, respectively. The geographical distribution of the TE-insertion type of S. italica PRR37 (SiPRR37) will be discussed in terms of its adaptive significance. Results Segregation of traits. Stb and the green leaf sheath are recessive traits27, as we previously reported in the F2 population. Two types of leaf sheath colors, red and green, were observed in 49 and 41 lines of the RILs, respectively. This result fits the 1:1 ratio using the Chi-square test (P > 0.05). Common wild-type bristles and mutant stb (Supplementary Fig. S1) are observed in lines 43 and 47, respectively. This result also fits the 1:1 by Chi-square test (P > 0.05). Days to heading (DTH) showed a rather broad but bimodal distribution in all cultivations in 2018, 2019, and 2020, and a high positive correlation in DTH between different years was observed (Fig. 2). Construction of high‑density linkage maps. The genomes of all RILs for the F10 generation were subjected to flexible ddRAD-seq to detect genome-wide SNPs and Presen (...truncated)