De Novo Genome Assembly of a Foxtail Millet Cultivar Huagu11 Uncovered the Genetic Difference to the Cultivar Yugu1, and the Genetic Mechanism of Imazethapyr Tolerance

Abstract

Background: Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. Results: Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97 and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. Conclusions: A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was inferred that the Ser-626-Aln substitution in AHAS was responsible for the © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: [email protected]; [email protected]; [email protected] Jie Wang, Shiming Li and Lei Lan contributed equally to this work. 1 Genetic, Engineering Research Center, School of Life Sciences, Chongqing University, Chongqing 401331, China 2 BGI Institute of Applied Agriculture, BGI-Shenzhen, Shenzhen 518120, China 5 Key Laboratory of Crop Molecular Breeding, Xining 810008, Qinghai, China Full list of author information is available at the end of the article Page 2 of 11 Wang et al. BMC Plant Biol (2021) 21:271 Background Foxtail millet (Setaria italic) is a climate-resilient cereal crop domesticated in northern China more than 8000 years ago [1] and is mainly cultivated in arid and semi-arid regions. It also serves as a model crop for the study of C4 photosynthesis, stress tolerance and bioenergy traits, due to its small genome size and short lifecycle [2–6]. The genome assemblies of two foxtail millet strains, ‘Yugu1’ and ‘zhang gu’, were published in 2012, which have accelerated the research of biology and genetics of this species [7–9]. The ‘Yugu1’ genome assembly based on Sanger sequencing, has higher contiguity than the short-read genome assembly of ‘Zhang gu’ (contig N50: 126.3 kb versus 25.8 kb) and is currently used as a reference genome of foxtail millet. Although the ‘Yugu1’ reference genome is well-annotated and has been used in many studies, its total length of 401 Mb only covers ~ 80% of the estimated genome size (~ 510 Mb,) based on k-mer analysis and is distributed in 6,778 contigs [9]. The missing sequences are mainly long repeats with lengths of 5–10 kb, which are difficult to assemble at that time using Sanger or next generation sequencing strategy. Although the function of repetitive sequences is seldom researched in foxtail millet, they have proved to play important roles in the regulation of gene expression and genome evolution in other cereal crops [9]. The hard-to-sequence gaps in the scaffolds reside in the intergenic regions and may also contribute to the regulation of gene expression. As quantitative genetic analyses have revealed that many causal variations underlying phenotype changes are from regulatory regions, a complete and high-quality genome assembly is crucial to understand of the genetic mechanisms of climate-resilient features, genome evolution and important agronomic traits in foxtail millet. The herbicide-resistance trait is an important trait for foxtail millet. Foxtail millet had the relatively small grain, which made the weed problem worse. The weed seeds were similar to the foxtail millet, which was hard to clean the weed seed in seed production. Moreover, the small seed produce the small plant in the germination stage. It is easy to be covered by the weed. The production reduction will be very serious without weed controlling [10]. The use of herbicides is a major measure to control weeds effectively in modern agriculture. Through the original millet cultivars did not have the ability to resist the herbicides, several herbicide-resistance genes had been imported into the modern millet cultivars through the hybridization with wild related species, or chemical mutagenesis. The different herbicide-resistance genes had the resistance to the different herbicide, and the different resistance to the same herbicide [11]. It will be very helpful of understanding the molecular mechanism of the herbicide-resistance for agriculture production. In the breeding process, the breeder usually used several parents to obtain one cultivar with the herbicide-resistance, which lead to trace the resource of the herbicide-resistance gene hardly. The genome assembly of the cultivar will provide an easy way to uncover the mechanism of the herbicide-resistance. Here, we provide the assembly of a high-quality Huagu11 reference genome through three technologies: single-molecule real-time sequencing (SMRT) chromosome conformation capture sequencing (Hi-C) and next generation sequencing (NGS). Compared with former cultivars, Huagu11 was planted widely in western China, which should present a special ecotype of Foxtail millet. This new reference genome is helpful for further analysis and comparison of genomic diversity of intraspecific genomes in foxtail millet. By intensively aligning the Yugu1 and Huagu11 genomes, we identified 969,596 SNPs, 156,282 small insertions/deletions (indels, length shorter than 100 bp) and more than 19 Mb of presence/absence-variation (PAV, length longer than 500 bp) sequences between these two millet genomes. Interestingly, our comparative genomics analysis revealed a wide range of intraspecific gene-order variations: about 7.54% of genes were non-syntenic between these two genomes. The two cultivars had different phenotype in the grouting days, plant height, glume color, thousand grain weight, and the response to the imazethapyr (Table S1). Considering the amino acid substitution in AHAS could produce the resistance against the herbicide imazethapyr in several plants [12], the sequences of AHAS were aligned to understand its functional diversity in the two cultivars.