Substitution Mapping of Two Closely Linked QTLs Controlling Grain Chalkiness and Grain Shape on Rice Chromosome 8

14 Background: Rice varieties are required to have high yield and good grain quality. Grain chalkiness 15 and grain shape are two important traits of rice grain quality. Low chalkiness slender grains are 16 preferred by most rice consumers. Here, we dissected two closely linked quantitative trait loci (QTLs) 17 controlling grain chalkiness and grain shape on rice chromosome 8 by substitution mapping. 18 Results: Two closely linked QTLs controlling grain chalkiness and grain shape were identified using 19 single-segment substitution lines (SSSLs). The two QTLs were then dissected on rice chromosome 8 20 by secondary substitution mapping. qPGC8.1 was located in an interval of 1382.6 kb and qPGC8.2 was 21 mapped in a 2057.1 kb region. The maximum distance of the two QTLs was 4.37 Mb and the space 22 distance of two QTL intervals was 0.72 Mb. qPGC8.1 controlled grain chalkiness and grain width. 23 qPGC8.2 was responsible for grain chalkiness and for grain length and grain width. The additive 24 effects of qPGC8.1 and qPGC8.2 on grain chalkiness were not affected by heat stress. 25 Conclusions: Two closely linked QTLs qPGC8.1 and qPGC8.2 were dissected on rice chromosome 8. 26 They controlled the phenotypes of grain chalkiness and grain shape. The two QTLs were insensitive to 27 high temperature.

In previous study, we fine-mapped two QTLs qPGC9 and qPGC11 for grain 82 chalkiness on rice chromosomes 9 and 11, which were sensitive to high temperature (Yang et al. 2021).

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In the present study, two closely linked QTLs for grain chalkiness on chromosome 8, qPGC8.1 and 84 qPGC8.2, were detected. The two QTLs were showed effect on grain chalkiness and grain shape, and 85 insensitive to high temperature. Dissection of the two closely linked QTLs laid a foundation for 86 revealing the genetic architecture of grain chalkiness and the relationship between grain chalkiness and 87 grain shape in rice.

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Two SSSLs 15-08 and 03-08 with lower grain chalkiness were selected from the HJX74-SSSL library 92 (Fig. 1a). The SSSLs were used to investigate grain chalkiness in consecutive 6 cropping seasons from 93 the first cropping season (FCS) of 2017 to the second cropping season (SCS) of 2019. On average, the 94 PGC of 15-08 and 03-08 was 11.9% and 10.3% respectively, which were significantly lower than 21.2% 95 of recipient HJX74 ( Fig. 1b and Additional file 1: Table S1). It meant that the two SSSLs each carried a 96 QTL for PGC on their substitution segments.

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Eight agronomic traits of 15-08 and 03-08 were investigated. Most traits of the SSSLs had no 102 significant difference with HJX74 ( Fig. 1a and Additional file 1: Table S4). However, the grain shapes 103 of 15-08 and 03-08 were significantly different with HJX74. For 15-08, the grain width was 104 significantly narrower than that of HJX74, the former was 2.62 mm and 2.45 mm and the later was  Table S4). It is noted that GW8 is outside the substitution 109 segment of 03-08 (Fig. 1c). Therefore, the difference of grain shape between 03-08 and HJX74 was not  NILs, NIL15-08-26 and NIL15-08-43, were as low as 15-08, while those of other two NILs, 117 NIL15-08-4 and NIL15-08-14, were as high as HJX74. Substitution segments of the two NILs with low 118 PGC overlapped in the region between markers RM4815 and RM23137, while substitution segments of 119 other two NILs with high PGC located outside the region. These results indicated that the QTL for 120 5 grain chalkiness, qPGC8.1, was located in the region between markers RM4815 and RM23137 with 121 the estimated interval length of 1382.6 kb ( Fig. 2a-b).

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However, the grain width segregated in the NILs. Two NILs, NIL15-08-26 and NIL15-08-43, with low NILs, NIL03-08-9, NIL03-08-15 and NIL03-08-19, were as low as 03-08, while those of other two 140 NILs, NIL03-08-14 and NIL03-08-55, were as high as HJX74. Substitution segments of the three NILs 141 with low PGC overlapped in the region between markers SNP8M54 and SNP8M28, while substitution 142 segments of other two NILs with high PGC located outside the region. These results indicated that the 143 QTL for grain chalkiness, qPGC8.2, was located in the region between markers SNP8M54 and 144 SNP8M28 with the estimated interval length of 2057.1 kb ( Fig. 3a-b).

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qPGC9 and qPGC11, by substitution mapping, and found that they had no effect on grain shape (Yang 213 et al. 2021). Therefore, the relationship of grain chalkiness and grain shape in rice remains unclear. In 214 the present study, two closely linked QTLs, qPGC8.1 and qPGC8.2, on chromosome 8 showed effects 215 on grain chalkiness and grain shape ( Fig. 2 and Fig. 3). In addition, it is noted that the fgr gene for

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During the seed filling period, the air temperature of FCS is usually higher than that of SCS. In 231 2017-2019, the mean temperature of FCS was 4.1℃ higher than that of SCS (Additional file 1: Table   232 S5). It led to grain chalkiness of all lines in FCS was significantly higher than that in SCS (Fig. 4).

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Obviously, the grain chalkiness of HJX74 and SSSLs was greatly affected by higher temperature 234 during the seed filling period.

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Recently, we mapped two QTLs for grain chalkiness, qPGC9 and qPGC11, and found that the additive 242 effects of qPGC9 and qPGC11 on chalkiness in SCS were almost twice of those in FCS. The additive 243 effects of qPGC9 and qPGC11 on chalkiness were decreased by heat stress in FCS. Therefore, the 244 qPGC9 and qPGC11 were sensitive to high temperature (Yang et al. 2021). In the present study, we 245 found that although grain chalkiness of HJX74 and SSSLs was greatly affected by higher temperature 246 during the seed filling period, the additive effects of qPGC8.1 and qPGC8.2 on PCG, PCA and PGC 247 were no significant difference in different cropping seasons (Fig. 5). The results showed that the Two SSSLs 03-08 and 15-08 with lower grain chalkiness were selected from the HJX74-SSSL library.

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The substitution segment of 03-08 was from the donor Zhong4188 and that of 15-08 was from the            Table S1. Phenotypes of chalky traits in SSSLs. Table S2. Markers developed to 538 detect the substitution segments of SSSLs. Table S3. Substitution segments of SSSLs. Table S4.

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Phenotypes of agronomic traits in SSSLs. Table S5. Average temperatures for 30 days after flowering