Isogenic Japonica Rice Koshihikari Integrated with Late Flowering Gene Hd16 and Semidwarfing Gene sd1 to Prevent High Temperature Maturation and Lodging by Typhoon

We developed semidwarf and late-maturing isogenics of Koshihikari to stabilize high yield and avoid high temperature maturation. Whole-genome analysis (WGS) was conducted to examine the transitional changes in the entire genome, the size of DNA fragments integrated with the target gene, and genes accompanying the target gene owing to the progress of backcrossing. In both Koshihikari Hd16 (BC7F4) and Koshihikari sd1Hd16 (BC8F2), an SNP from adenine to guanine was detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC8F2), an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. From BC7 to BC8, the size of the DNA fragment integrated with Hd16 decreased by 5871 bp. Koshihikari sd1Hd16 flowered 12.1 days later than Koshishikari or Koshihikari sd1 did and was 14.2 cm (15%) shorter than Koshihikari. The yield in Koshishikari sd1Hd16 (63.2 kg/a) was 7.0% higher than that of Koshihikari. This is a new germplasm designed to avoid heat damage at ripening during high-temperature summer periods by late maturation owing to Hd16 as well as to avoid lodging by autumn typhoons by semidwarfness owing to sd1.


Introduction
Climate change because of global warming causes damage to crops globally [1,2]. In Japan, disastrous rainfall and floods, such as the "Heavy Rain in July, Heisei 30" [3], and large typhoons with wind speeds over 54 m/s, including Jebi and Trami, which were comparable to the worst typhoon in Japan's history (Isewan Typhoon) [4], have been occurring frequently every year [3][4][5][6][7]. These extreme weather phenomena have caused marked damage to agriculture, forestry, and fisheries (totaling 436.5 billion yen) [8]. Under these climate crises, rice must be robust and resistant to lodging [2,9].
A moderate reduction in crop height, namely, semidwarfism, improves lodging resistance to wind and rain at the full-ripe stage, light-interception properties, and nitrogen responsiveness [10]. The development of rice semidwarf varieties enhanced their adaptability to heavy manuring and markedly increased global rice productivity by up to double between 1960 and 1990 [11]. This has been referred to as the "Green Revolution", and is considered as the greatest agricultural contribution in the history of humankind. The gene contributing to "Green Revolution" in rice was identified as sd1.The sd1 alleles, on the long arm of chromosome 1 [12][13][14], encode a defective C20-oxidase in the gibberellin (GA) biosynthesis pathway (GA 20-oxidase, OsGA20ox2) [15][16][17] and mutations in the GA20oxidase gene lead to disruptions at a late stage of the GA pathway [15]. No detrimental effect on grain yield are conferred by sd1 [18][19][20].
The japonica rice Koshihikari is a leading variety in Japan, accounting for 33.7% of rice acreage in the country. Koshihikari is globally valued and produced, including in

Development of Koshihikari sd1Hd16
Koshihikari Hd16 is a late-maturing Koshihikari-type isogenic line developed by integrating the 2 weeks late-maturing gene (Hd16) derived from Isehikari with the genetic background of Koshihikari via six continuous backcrosses to a recurrent parent Koshihikari using a late-maturing segregant in F 2 of Koshihikari × Isehikari segregated as a nonrecurrent parent [40]. In the present study, the seventh backcross with Koshihikari was conducted using Koshihikari Hd16 (BC 6 F 3 ) as the pollen parent, and the Koshihikari × 7/ [(Koshihikari × Isehikari) F 2 ] BC 7 F 2 plants were tested ( Figure 1). The Hd16 homozygous plant (BC 7 F 2 ) was selected by gene diagnosis using the single sequence repeat (SSR) marker RM16089, and BC 7 F 3 , the BC 7 F 4 progeny of the Hd16 homozygous plant (BC 7 F 2 ) was tested. For all plants, we investigated ear emergence day, culm length, and morphological traits and conducted gene diagnosis of Hd16 using RM16089. Koshihikari Hd16 is a late-maturing Koshihikari-type isogenic line developed by integrating the 2 weeks late-maturing gene (Hd16) derived from Isehikari with the genetic background of Koshihikari via six continuous backcrosses to a recurrent parent Koshihikari using a late-maturing segregant in F2 of Koshihikari × Isehikari segregated as a nonrecurrent parent. Koshihikari sd1 was crossed with the late-maturing Koshihikari-type isogenic gene line (Koshihikari Hd16 (BC 6 F 2 )), and the F 2 (equivalent to BC 7 F 2 ) was tested to develop a semidwarf late-maturing Koshihikari-type isogenic line (Koshihikari sd1Hd16), which combines the semidwarf gene sd1 and the late-maturing gene Hd16 (Figure 2). For the Life 2022, 12, 1237 4 of 15 sd1 allele, the genotype was determined by culm length, and the genotype of Hd16 was determined using RM16089 near the Hd16 allele. The eighth backcrossing to Koshihikari was conducted using the Hd16Hd16Sd1sd1 plant (BC 7 F 2 ) segregated in BC 7 F 2 , and the Koshihikari/Koshihikari sd1/Koshihikari × 6/[(Koshihikari × Isehikari) F 2 ] BC 8 F 2 generation was tested. The ear emergence day, culm length, and morphology were investigated, and genetic diagnosis was conducted using RM16089. Whole genome analysis by nextgeneration sequencing was conducted for Koshihikari Hd16 (BC 7 F 4 ) and Koshihikari sd1Hd16 (BC 8 F 2 ). Koshihikari sd1 was crossed with a late-maturing Koshihikari-type isogenic gene line (Koshihikari Hd16 (BC6F2)) to develop a semidwarf late-maturing Koshihikari-type isogenic line (Koshihikari sd1Hd16), which combines the semidwarf gene sd1 and the latematuring gene Hd16.

Whole Genome Sequence Analysis
Whole genome sequencing was conducted on both Koshishikari Hd16 (BC7F4) and Koshihkari sd1Hd16 (BC8F2), which were integrated with the late flowering gene Hd16 and semidwarfing gene sd1, by eight backcrosses into the genetic background of Koshihikari. The leaves were powdered using a mortar and pestle, and frozen in liquid nitrogen. Genomic DNA was extracted from each cultivar using the cetyltrimethylammonium bromide method. Genomic DNA was fragmented and simultaneously tagged so that the peak size of the fragments was approximately 500 bp using the Nextera ® transposome (Illumina Inc., San Diego, CA, USA). After purification of the transposome using DNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA, USA), adaptor sequences, including the sequencing primers, for fixation on the flow cell were synthesized at both ends of each Genetic material cultivation was conducted in a paddy field at Shizuoka University, Shizuoka, Japan, from 2013 to 2021. Genetic material BC n F 1 was grown from April to July, and BC n F 2 was grown from July to November. In other words, we accelerated the generation in a short period. Finally, the obtained genotypes were grown from May to October to test performance. Seedlings were individually transplanted into a paddy field in mid-July at a transplanting density of 22.2 seedlings/m 2 (one seedling per 30 × 15 cm). The paddy field was fertilized with 4.0 kg of basal fertilizer containing nitrogen, phosphorus, and potassium (weight ratio, nitrogen:phosphorus:potassium = 2.6:3.2:2.6), with 4.3 g/m 2 nitrogen, 5.3 g/m 2 phosphorus, and 4.3 g/m 2 potassium across the field. The heading date was recorded as the date on which the first panicle emerged from the flag leaf sheath for each plant. Culm length was measured as the length between the ground surface and the panicle base. For the yield test, after ripening, 10 plants typical of each genotype were sampled twice. The sampled plants were air-dried and assessed or measured for the following traits: panicle length, number of panicles, number of florets/panicles, proportion of fertile florets, total panicle number, and weight of unmilled rice/1000 grain. The yield of unpolished rice was calculated using the following equation: Yield of unmilled rice (g/m 2 ) = (number of panicles/m 2 ) × (number of florets/panicle) × (proportion of fertile florets) × (weight of unmilled rice/grain). Lodging degree was determined based on the inclination angle of plant; 0: standing, 1: almost 70, 2: almost 50, 3: almost 30, 4: almost 10, 5: lodged. Taste evaluation was based on a seven grade-organoleptic assessment by a panelist, and protein contents were determined using Infratec 1241(VOSS Japan Ltd., Tokyo, Japan). The means of traits were statistically compared using the t-test.
Koshihikari Hd16 is a late-maturing Koshihikari-type isogenic line developed by integrating the 2 weeks late-maturing gene (Hd16) derived from Isehikari with the genetic background of Koshihikari via six continuous backcrosses to a recurrent parent Koshihikari using a late-maturing segregant in F 2 of Koshihikari × Isehikari segregated as a nonrecurrent parent.

Whole Genome Sequence Analysis
Whole genome sequencing was conducted on both Koshishikari Hd16 (BC 7 F 4 ) and Koshihkari sd1Hd16 (BC 8 F 2 ), which were integrated with the late flowering gene Hd16 and semidwarfing gene sd1, by eight backcrosses into the genetic background of Koshihikari. The leaves were powdered using a mortar and pestle, and frozen in liquid nitrogen. Genomic DNA was extracted from each cultivar using the cetyltrimethylammonium bromide method. Genomic DNA was fragmented and simultaneously tagged so that the peak size of the fragments was approximately 500 bp using the Nextera ® transposome (Illumina Inc., San Diego, CA, USA). After purification of the transposome using DNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA, USA), adaptor sequences, including the sequencing primers, for fixation on the flow cell were synthesized at both ends of each fragment using polymerase chain reaction. The DNA fragments were then subjected to size selection using AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Finally, qualitative checks were performed using a Fragment Analyzer™ (Advanced Analytical Technologies, Heidelberg, Germany) and quantitative measurements using Qubit ® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to prepare a DNA library for next-generation sequencing. Sequencing was conducted in paired-end 2 × 100 bp on a HiSeq X next-generation sequencer, according to the manufacturer's protocol (Illumina Inc., San Diego, CA, USA). Illumina reads were trimmed using Trimmomatic (version 0.39) [40] (Figure 3). Sequencing adapters and sequences with low quality scores on the 3 ends (Phred score [Q], <20) were trimmed. The raw Illumina whole genome sequence reads were quality checked by performing quality control using FastQC (version 0.11.9; Babraham Institute, Cambridge, UK). Mapping of reads from Koshihikari Hd16 and Koshishikri sd1Hd16 to the Koshishikri genome as a reference was conducted using Burrows-Wheeler Aligner software (version bwa-0.7.17.tar.bz2; Appirits, Tokyo, Japan) [41]. Duplicated reads were removed using Picard (version 2.25.5; GitHub Inc., CA, USA) and secondary aligned reads were removed using SAMtools (version 1.10.2; SourceForge, CA, USA) [42]. To identify genetic variations among strains, single nucleotide variant detection (variant calling) and single nucleotide variant matrix generation were performed using GATK (version 4.1.7.0; Broad Institute, Cambridge, MA, USA) [43].
The relationship between culm length and heading date in BC8F2, in which the Hd16Hd16Sd1sd1 plant segregated in BC7F2 was crossed as the pollen parent with Koshihikari as the mother, is shown in Figure 6. In BC8F2, sd1 homozygous plants, whose culm lengths were 54.5-59.1 cm, similar to that of Koshihikari sd1 with short, thick and dark green flag leaves, intermediate plants, and the Sd1 homozygous plants whose culm lengths were 65.6-71.5 cm, similar to that of Koshihikari with long and thin color flag leaves, were segregated in a ratio of 10:25:12 ≈ a single gene inheritance theoretical ratio of 1 [sd1 homozygous]:2 [intermediate type]:1 [Sd1 homozygous] (χ 2 = 0.36, 0.80 < p < 0.90). From BC8F2, four sd1 homozygous plants were arbitrarily selected based on culm length and genetically diagnosed using RM16089. One plant was Hd16 homozygous, one was heterozygous, and two were hd16 homozygous (Figure 7). The semidwarf late-maturing Koshihikari isogenic line with sd1 and Hd16 homozygotes was acquired in BC8F2. Koshihikari sd1Hd16 flowered 12.1 days later than Koshishikari or Koshihikari sd1 did, and was 14.2 cm (15%) shorter than Koshihikari, with a characteristic deep green color (Figure 8). The yield of Koshishikari sd1Hd16 (63.2 kg/a) was 7.0% higher than that of Koshihikari (Table 1). Figure 6. Relationship between heading date and culm length in Koshihikari/Koshihikari sd1/Koshihikari × 6/[(Koshihikari × Isehikari) F2] BC8F2 and BC7F4 progeny of its parent. In BC8F2, sd1 homozygous plants, whose culm length were 54.5-59.1 cm, similar to that of Koshihikari sd1 with Figure 6. Relationship between heading date and culm length in Koshihikari/Koshihikari sd1/Koshihikari × 6/[(Koshihikari × Isehikari) F 2 ] BC 8 F 2 and BC 7 F 4 progeny of its parent. In BC 8 F 2 , sd1 homozygous plants, whose culm length were 54.5-59.1 cm, similar to that of Koshihikari sd1 with short, thick and dark green flag leaves, intermediate type plants, and the Sd1 homozygous plants whose culm length were 65.6-71.5 cm, similar to that of Koshihikari with long and thin color flag leaves, were segregated in a ratio of 10

Whole Genome Sequencing of Koshihikari sd1Hd16
The number of reads decoded by the next-generation sequencer was 41,630,793 for Koshihikari sd1Hd16 (BC 8 F 2 ). The obtained reads of Koshishikri sd1GW2 were mapped to the consensus sequence of Koshihikari as a reference, and the mean coverage was 23.72. After removing the secondary alignment and duplicate reads, the unique reads were 31,249,311. A total of 43,861 SNPs [homozygous 2044, heterozygous 41,817] were detected. In both Koshihikari Hd16 (BC 7 F 4 ) and Koshihikari sd1Hd16 (BC 8 F 2 ), SNPs from adenine to guanine were detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC 8 F 2 ), SNPs from thymine to guanine were detected in sd1 at 38,267,510 bp on chromosome 1. Except for the region around Hd16 and sd1, the number of SNPs was less than 10 per 0.1 Mb. The results indicated that a large portion of the 12 chromosomes in rice was substituted into the genome of Koshihikari (Figure 9) after continuous backcross targeting of these two genes.
After a single backcross, the total number of SNPs decreased from 725 in Koshihikari Hd16 (BC 6 F 2 ) to 348 in BC 7 . The size of the DNA fragment integrated into Hd16 was determined as the distance between both ends of an SNP cluster. In Koshihikari Hd16 (BC 6 F 2 ), it was 3,150,236 bp, from 31,239,632 bp to 34,389,868 bp in the short arm of chromosome 3. In contrast, in Koshihikari sd1Hd16 (BC 7 F 4 ), it was 3,144,365 bp, from 31,239,632 to 34,389,868 bp. Therefore, after one backcross from BC 6 to BC 7 , the size of the DNA fragment integrated with Hd16 decreased by 5871 bp (Figure 10). A total of 617 annotated genes were identified in the integrated DNA fragments. Among them, there were mutations in four genes, including the zinc finger protein gene (Table 2).  Figure 9. Causative SNP for Hd16 in Koshihikari sd1Hd16 (BC8F2). In both Koshihikari Hd16 (BC7F4) and Koshihikari sd1Hd16 (BC8F2), SNPs from adenine to guanine were detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC8F2), SNPs from thymine to guanine were detected in sd1 at 38,267,510 bp on chromosome 1. Figure 9. Causative SNP for Hd16 in Koshihikari sd1Hd16 (BC 8 F 2 ). In both Koshihikari Hd16 (BC 7 F 4 ) and Koshihikari sd1Hd16 (BC 8 F 2 ), SNPs from adenine to guanine were detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC 8 F 2 ), SNPs from thymine to guanine were detected in sd1 at 38,267,510 bp on chromosome 1. The size of the DNA fragment integrated into Hd16 was determined as the distance between both ends of an SNP cluster. After a single backcross from BC7 to BC8, the size of the DNA fragment integrated with Hd16 decreased by 5871 bp.

Discussion
The threat of strong typhoons, rainfall, and floods caused by global warming causes serious lodging [44], resulting in yield loss and grain quality deterioration in rice production [2]. The first author developed Koshihikari sd1, designated as Hikarishinseiki [21,23], and registered it under the Plant Variety Protection Act in Japan and the United States [23,25]. Koshihikari sd1 was approximately 20 cm shorter than Koshihikari, and its genome consists of more than 99.8% of the genome of Koshihikari, except for sd1 derived from Jukkoku [20,21]. However, Koshihikari also suffers from poor filling and yield reduction, caused by high-temperature maturation. To avoid high-temperature damage in the hot summer, shifting rice ripening to early autumn is an effective solution. In this study, first, the late-maturing gene Hd16 from Isehikari was integrated into Koshishikari by seven backcrosses with Koshihikari as the recurrent parent using a late-maturing plant as a non-recurrent parent that was segregated in F2 of Koshihikari × Isehikari. Then, the late maturing isogenic Koshishikri Hd16 was crossed with Koshihikari sd1 to combine the semidwarf gene sd1 and Hd16 into the genetic background of Koshihikari, and eight backcrosses to the genetic background of Koshihikari were completed to build isogenic Koshihikari integrating both Hd16 and sd1. Through the backcross process, Hd16 allele was diagnosed by SSR marker RM16089 near the Hd16 allele, and sd1 homozygotes were successfully selected by their phenotype in each relativity-limited BCnF2 population. Finally, whole genome sequencing of Koshihikari sd1Hd16 showed that an SNP from adenine to guanine was detected at 32,996,608 bp in Hd16 on chromosome 3, and an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. The size of the DNA fragment integrated with Hd16 was determined to be 3,144,365 bp in Koshihikari

Discussion
The threat of strong typhoons, rainfall, and floods caused by global warming causes serious lodging [44], resulting in yield loss and grain quality deterioration in rice production [2]. The first author developed Koshihikari sd1, designated as Hikarishinseiki [21,23], and registered it under the Plant Variety Protection Act in Japan and the United States [23,25]. Koshihikari sd1 was approximately 20 cm shorter than Koshihikari, and its genome consists of more than 99.8% of the genome of Koshihikari, except for sd1 derived from Jukkoku [20,21]. However, Koshihikari also suffers from poor filling and yield reduction, caused by high-temperature maturation. To avoid high-temperature damage in the hot summer, shifting rice ripening to early autumn is an effective solution. In this study, first, the late-maturing gene Hd16 from Isehikari was integrated into Koshishikari by seven backcrosses with Koshihikari as the recurrent parent using a late-maturing plant as a non-recurrent parent that was segregated in F 2 of Koshihikari × Isehikari. Then, the late maturing isogenic Koshishikri Hd16 was crossed with Koshihikari sd1 to combine the semidwarf gene sd1 and Hd16 into the genetic background of Koshihikari, and eight backcrosses to the genetic background of Koshihikari were completed to build isogenic Koshihikari integrating both Hd16 and sd1. Through the backcross process, Hd16 allele was diagnosed by SSR marker RM16089 near the Hd16 allele, and sd1 homozygotes were successfully selected by their phenotype in each relativity-limited BCnF 2 population. Finally, whole genome sequencing of Koshihikari sd1Hd16 showed that an SNP from adenine to guanine was detected at 32,996,608 bp in Hd16 on chromosome 3, and an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. The size of the DNA fragment integrated with Hd16 was determined to be 3,144,365 bp in Koshihikari Hd16sd1, based on the distance between both ends of an SNP cluster. After backcrossing BC 7 to BC 8 , the size of the DNA fragment integrated with Hd16 decreased by 5871 bp.
The SNP found in Hd16 from Isehikari was the same as that found in Nipponbare. Hd16 of Nipponbare encodes casein kinase I [45,46]. Under long-day conditions, Hd16 acts upstream of the photosensitive floral repressor gene Ghd7 and phosphorylates the transcript of Ghd7, which is located upstream of the flowering gene Ehd1, to strengthen photosensitivity and delay flowering [46,47]. The introgression of Hd16 from Nipponbare into Koshihikari has been reported to cause a 10-day delay in maturation [46,[48][49][50]. In the present study, we developed an isogenic line via eight backcrosses and clarified the genome structure in which almost all sequences were replaced by the Koshihikari genome, except for the vicinity of Hd16 on chromosome 3 derived from Isehikari and sd1 derived from Jukkoku on chromosome 1. In the present study, the same nonsynonymous substituted Hd16 allele from Isehikari resulted in decreased photoperiod sensitivity to noticeably delay flowering time by 12 days, which was considered to be attained in the highly isogenic background. Twelve days of late-flowering Koshihikari owing to Hd16 will avoid flowering and ripening during the high-temperature period in the hottest summer period in August. Furthermore, it is one of the promising options of a regionally adaptive genotype to address the overuse of Koshihikari throughout Japan. Late-maturing Koshihikari is highly desired in the rice industry. The yield merit underpinning Hd16 has been previously reported [51]. Ministry of Agriculture, Forestry and Fisheriesof Japan (MAFF) has registered the late-maturing isogenic Koshihikari, which was integrated with Hd16, designated as a new plant variety 'Koshihikari Suruga Hd16 [52] under Japanese varietal protection. Furthermore, the author has applied for Japanese varietal protection for the late-maturing and semidwarf isogenic Koshihikari, which was integrated with both Hd16 and semidwarf gene sd1, designated as a new plant variety 'Koshihikari Suruga sd1Hd16 [53]. This is a new germplasm designed to avoid heat damage at ripening during high-temperature summer periods by late maturation owing to Hd16 as well as to avoid lodging by autumn typhoons by semidwarfness owing to sd1.

Conclusions
We developed a semidwarf and late-maturing isogenic Koshihikari sd1Hd16 (BC 8 F 2 ) strain to stabilize high yield and avoid high-temperature maturation. Whole genome analysis detected an SNP from adenine to guanine in Hd16 at 32,996,608 bp on chromosome 3, and an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. From BC 7 to BC 8 , the size of the DNA fragment integrated with Hd16 decreased by 5871 bp. Koshihikari sd1Hd16 flowered 12.1 days later than Koshishikari or Koshihikari sd1 did and was 14.2 cm (15%) shorter than Koshihikari. Koshishikari sd1Hd16, with a 7.0% higher yield than Koshihikari, is a new germplasm to avoid heat damage during ripening during high-temperature summer by Hd16 as well as to avoid lodging by autumn typhoons by sd1. Data Availability Statement: All the data generated in this study are present in the main manuscript.