Genome sequence of the model rice variety KitaakeX

Abstract

34 Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety 35 KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly 36 contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing 37 the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic 38 variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX 39 and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly 40 and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate 41 rice functional genomics. 42 Background 45 Rice (Oryza sativa) provides food for more than half of the world's population [1] and also serves 46 as a model for studies of other monocotyledonous species. Cultivated rice contains two major types of O. 47 sativa, the O. sativa indica/Xian group and the O. sativa japonica/Geng group. Using genomic markers, two 48 additional minor types have been recognized, the circum-Aus group and the circum-Basmati group [2]. 3The Kitaake cultivar (ssp. japonica), which originated at the northern limit of rice cultivation in 50 Hokkaido, Japan [3], has emerged as a model for rice research [4] because it is extremely early flowering, 51 easy to propagate, and short in stature [5]. Kitaake has been used to establish multiple mutant 52 populations, including an RNAi mutant collection [6], T-DNA insertion collections [4], [7], and a whole-53 genome sequenced mutant population of KitaakeX, a Kitaake variety carrying the Xa21 immune receptor 54 gene (formerly called X.Kitaake) [8, 9]. Kitaake has been used to explore diverse aspects of rice biology, 55 including flowering time [10], disease resistance [11], [12], [13], small RNA biology [14], and the CRISPR-56 Cas9 and TALEN technologies [15], [16]. 57The unavailability of the Kitaake genome sequence has posed an obstacle to the use of Kitaake 58 in rice research. For example, analysis of a fast-neutron (FN) induced mutant population in KitaakeX [8], 59 required the use of Nipponbare (ssp. japonica) as the reference. Additionally, CRISPR/Cas9 guide RNAs 60 cannot be accurately designed for Kitaake without a complete sequence. To address these issues, we 61 assembled a high-quality genome sequence of KitaakeX, compared its genome to the genomes of rice 62 varieties Nipponbare and Zhenshan97 (ssp. indica), and identified genomic variations. 63 Results 64Kitaake has long been recognized as a rapid life-cycle variety [17], but it has yet to be systematically 65 compared to other rice varieties. We compared the flowering time of KitaakeX with other sequenced rice 66 varieties under long-day conditions (14 h light/10 h dark). Consistent with other studies, we found that 67 KitaakeX flowers much earlier than other varieties ( Fig. 1a, 1b), heading at 54 days after germination. Other 68 rice varieties Nipponbare, 93-11 (ss...

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

“…Using the CRISPR-PLANT guide RNA design platform 27 , we selected seven specific sites near the five candidate GSHs in the Kitaake rice genome 28 , and designed gRNAs (A, B, C, D1, D2, E1, and E2) targeting each of these sites ( Supplementary Fig. 2a).…”

“…The carotenoid-enriched rice described in this study was generated in the Kitaake genetic background. KitaakeX, a rice genotype whose genome is avialbale 28 , carries an XA21 transgene in the Kitaake genetic background. We used the KitaakeX reference genome 28 as a close approximation of the Kitaake genome in this study.…”

“…Predicting potential Cas9 off-target mutation sites in 48A-7. We screened the KitaakeX genome 28 (https://phytozome.jgi.doe.gov/) for potential off-target mutation sites using the Cas-OFFinder 43 with default parameters. All potential Cas9 off-target sites with four or fewer nucleotide mismatches compared with the true target of gRNA B were called.…”

“…Small insertions/deletions simultaneously called by SAMtools and Pindel were kept; those called only by SAMtools were filtered at a QUAL score of 100, and those only called by Pindel were further filtered with the following criteria: (1) the variation site had a minimum 10 reads, (2) at least 30% of the reads supported the variation. From the variants between 48A-7 and KitaakeX, we subtracted the known variants between Kitaake and KitaakeX 28 . The remaining are the true variants between 48A-7 and its genetic background Kitaake.…”

“…To assess the occurrence of off-target mutations caused by Cas9 in the process, we further analyzed the whole-genome sequencing result for 48A-7. We used Cas-OFFinder 43 to predict potential Cas9 off-target sites in the KitaakeX genome 28 and identified ten candidate sites (Supplementary Table 3). Sequence analysis indicates that none of the ten predicted off-target sites is mutated in plant 48A-7 (Supplementary Table 3 and Supplementary Data 2).…”