Selection of Nuclear Genotypes Associated with the Thermo-inducibility of Owen-type Cytoplasmic Male Sterility in Sugar Beet (Beta vulgaris L.)

Hiroaki Matsuhira (  ma2hira@affrc.go.jp ) Hokkaido Agricultural Research Center, National Agricuture and Food Research Organization https://orcid.org/0000-0002-5940-5964 Kazuyoshi Kitazaki Research Faculty of Agriculture, Hokkaido University Katsunori Matsui Research Faculty of Agriculture, Hokkaido University Keisi Kubota Research Faculty of Agriculture, Hokkaido University Yosuke Kuroda Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization Tomohiko Kubo Research Faculty of Agriculture, Hokkaido University


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Hybrid seed production of some crops relies on heritable male sterility (MS) because it converts a 55 hermaphroditic plant into a seed parent that never self-pollinates (Budar et al. 2006). On the other hand, 56 using male-sterile plants for hybrid seed production at a commercial scale is costly because the system is 57 complicated, in part, due to problems with how seed parents are propagated. MS can be caused by a

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In our previous study, we genetically analyzed maintainer lines that were developed outside procedure is called a test cross). We found a major recessive rf1 allele that predominates among many maintainer lines used by the world's breeders (Ohgami et al. 2016). In addition, several minor rf1 alleles 93 that could condition the male-sterile phenotype but varied in their gene organization were identified. It 94 was puzzling that these minor rf1 alleles were favored by a specific breeding station (Ohgami et al. 2016).

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We also observed that some lines recorded as 'maintainers' in the Gene Bank gave rise to fertility-96 restored F1 plants, indicating that these lines were not maintainer genotypes in our experimental growing     126 Seeds were sown in paper pots filled with soil, and seedlings were grown until four to five leaves 127 developed. Plantlets were transplanted to beds in a greenhouse in Memuro, Hokkaido, Japan, in early 128 May. A closed room of this greenhouse was equipped with a kerosene heater; the room was heated from 129 10:00 to 16:00 daily, from late June to early September, and the temperature did not exceed 40˚C.

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Temperatures were recorded once every hour by an Ondotori RTR507B data logger (T&D Corporation,

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Matsumoto, Japan) in each room (Fig. 1).    (Table 1), and we observed that these plants had band patterns 1 (hereafter abbreviated as p1), 171 p3, p4, and p5. We selected seven plants composed of the four s17 band patterns (Table 2). Because p2 172 was missing from our selections, we added two plants from NK-305, a sugar beet line that was previously 173 shown to contain p2 (Arakawa et al. 2019) ( Table 2). The nine plants were crossed with the cytoplasmic 174 male-sterile line TA-33BB-CMS, whose s17 marker type was homozygous at p4 (hereafter p4p4) to 175 obtain F1 seeds. These F1 selections were grouped into 15 genotypes according to their parentage and s17 176 band patterns (Table 2).

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Fertility restoration without supplemental heating 179 The F1 plantlets were divided into two groups to grow in two different environmental conditions. One

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The remaining 81 plants had restored pollen fertility (Table 2). Their phenotypes were either 7 The other F1 group was planted in a greenhouse room with a heater that operated only during the daytime.

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The heating protocol was started just before stalk elongation and ceased at the end of flowering (

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Of these F1 plants, the phenotypes of all p4p4 plants were W (   (Table   205 2). Note that the admixture phenotype was not present in plants receiving no supplemental heat (Table 2).

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On the other hand, plants with the TA-30-32 p1 and TA-36-32 p3 alleles had restored pollen fertility 207 when grown in the heated room, as was also seen in plants not receiving supplemental heat, indicating 208 that they were phenotypically stable.

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Purity of hybrid seed from thermo-induced male-sterile plants

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We tested whether heat-induced male-sterile plants had the potential to be seed parents for hybrid seed 212 production. We chose three F1 genotypes with the TA-37-19-p3, TA30-37-p5 and TA-36-3-p5 alleles as 213 seed parents because these genotypes produced flowers of the W phenotype at high frequency (Table 2).

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Seedlings with these alleles were selected from F1 populations by screening with the s17 marker and 215 planted in the greenhouse room with supplemental heat.

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Pollen fertility phenotypes are summarized in Table 3. All 24 plants with the TA-37-19-p3 217 allele exhibited the W phenotype, whereas plants with either the TA30-37-p5 or TA-36-3-p5 alleles 218 exhibited W and an admixture that consisted of S, G and W flowers ( Table 3). Note that anthers did not Plants with the TA-37-19-p3, TA30-37-p5 and TA-36-3-p5 alleles set seeds that were 223 harvested to test whether they were hybrids with TA-8. Deep red pigmentation in the hypocotyls and leaf 224 veins was a unique characteristic of TA-8 and this phenotype is governed by a dominant gene, as is also 225 documented in garden beet (Hatlestad et al. 2012). We sowed the seeds to determine the ratio of seedlings 226 with deep red pigmentation. The emerged seedlings exhibited deep red pigmentation that was clearly 227 distinguishable from sugar beet plants with pink hypocotyls and unpigmented veins, phenotypes seen in 228 some sugar beet cultivars (Fig. 2). Of 1111 seedlings derived from the seed parents with the TA-37-19-p3 229 allele, 1110 seedlings were intensely pigmented. All 371 seedlings from seed parents with the TA30-37-230 p5 allele and 2316 seedlings from seed parents with the TA-36-3-p5 allele were highly pigmented.  Table 3), the PCR products were 239 cloned into plasmid vectors to sort the DNA fragments. To eliminate clones with copies of TA-33BB-

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CMS Rf1-Oma1, we focused on the length of the first intron; TA-30-37, TA-36-3 and TA-37-19 had 241 exclusively short introns, whereas TA-33BB-CMS had a long intron as shown using 20L-int, a DNA 242 marker targeting the intron (Fig. 3). Therefore, we selected and sequenced plasmids with short first 243 introns and compared the nucleotide sequences with those of known Rf1 alleles.

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As such, we concluded that the TA-37-19-p3 allele was the same as PI 615522 rf1.

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Female fertility of our thermo-inducible male sterile plants was normal in those receiving the 293 supplemental daytime heat treatment. The purity of hybrid seeds from induced male-sterile plants was 294 over 99.9%, indicating that male sterile plants can be seed parents for hybrid seed production. Our 295 supplemental heating protocol increased the daytime temperature only and did not alter the nighttime 296 temperature. Therefore, we favor the notion that daytime temperature is critical for MS induction.

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Temperature conditions similar to that in our heated room can be seen in the southern part of Japan, i.e.,            Temperature uctuations in closed greenhouse rooms where sugar beets were grown. Vertical and horizontal axes depict the temperature in degrees Celsius and the date, respectively. Red and blue lines denote greenhouse room temperatures with and without supplemental heating, respectively Phenotypes of sugar beet seedlings. a. Hybrid seedlings emerged from the seeds harvested from heatinduced male-sterile plants (TA-33BB-CMS x TA37-19). The pollen parent was TA-8. b. Seedlings of a sugar beet cultivar that segregate for the pink hypocotyl phenotype