Development of Chromosome Segment Substitution Lines and Genetic Dissection of Grain Size Related Locus in Rice

Aamir Riaz, Wang Huimin, Zhang Zhenhua,Workie Anley Zegeye, 2, Li Yanhui, Wang Hong, Xue Pao, Peng Zequn, Shen Xihong, Cheng Shihua, Zhang Yingxin

Letter

Development of Chromosome Segment Substitution Lines and Genetic Dissection of Grain Size Related Locus in Rice

Aamir Riaz1, #, Wang Huimin1, #, Zhang Zhenhua1,Workie Anley Zegeye1, 2, Li Yanhui1, Wang Hong1, Xue Pao1, Peng Zequn1, Shen Xihong1, Cheng Shihua1, Zhang Yingxin1

(State Key Laboratory of Rice Biology / National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China; Department of Plant Science, University of Gondar, Gondar 196, Ethiopia; These authors contributed equally to this work)

To investigate the genetic bases of grain weight (GW), a set of 76 chromosome segment substitution lines (CSSLs, BC4F5)were developed from the cross between Xieqingzao B (XQZB)and Zhonghui 9308 (ZH9308) using a marker-assisted selection (MAS). All substituted segments represented by 120 markers in those lines covered 96.7% of the donor parent (ZH9308). Consequently, two QTLs (and) for 1000-grain weight (TGW) were preliminarily mapped on chromosomes 1 and 6, respectively. The line CSSL7was selected for further mapping of.As a result,was validated and narrowed to a 1.4 Mb interval between markers InDel15 and RM11872 using secondary F2, and narrowed to a 500 kb in F2:3populations between markers RM11824 and RM11842. In F4:5secondary population,using homozygous recombinant strategy,was finally fine-mapped to a 286.4 kb region between markers D-12 and TG-57. In addition, a near-isogenic line (NIL) harbouringwas developed using MAS approach, which showed enhanced grain length compared with ZH9308 without changing other traits. In summary, these results lay a foundation for the genetic isolation ofand molecular breeding in rice.

TGW is one of the key factor of rice yield and is mainly determined by grain size, which is the combination of grain length (GL), GW and grain thickness (GT). In addition, grain size always affects the appearance quality in rice, which is one of the important factors determining the market value (Fitzgerald et al, 2009). To date, more than 400 QTLs for GW have been mapped across rice genome. Among these, 20 QTLs regulating grain size have been functionally cloned (Li et al, 2018; Wang A et al, 2019; Ruan et al, 2020). These genes function through different biological signaling pathways to regulate the formation of grain size. For instance, the large-effectnegatively regulates grain size, which has a major influence on GL,GW and grain length-width ratio (LWR), but a minor effect on GT (Fan et al, 2009). Recently, Wang A et al (2019) reported thatpositively controls GL and grain number by regulating cell number in young panicle and seed hull. CSSL population has been widely used for mapping and identification of yield- related QTLs in rice (Madoka et al, 2008; Qi et al, 2017, 2018). Here, we reportedthe development of the CSSLs and identificationof grain size-related QTLs, followed by fine mapping ofin rice.

To develop CSSLs, 173 recombinant inbred lines(RILs), derivedfrom the cross between XQZB and ZH9308, were randomly selected and backcrossed with the recurrent parent ZH9308, followed by backcrossing to BC4F1and selfing to BC4F7from 2012 to 2015 (Fig. S1). For MAS, a total of 87 simple sequence repeat (SSR) and 33 Insertion/Deletion (InDel) polymorphic markers were utilized in CSSL development (Table S1). In the process, there were 173, 346, 102 and 102 lines in BC1F1, BC2F1, BC3F1and BC4F1progeny, respectively. Subsequently, 102 BC4F1, 102 BC4F2, 76 BC4F3and 76 BC4F4lines were obtained. As a result, in 2014, a total of 76 BC4F5CSSLs were developed, each of which harboured a major segment inherited from XQZB, along with a variable number of additional segments (Fig. S2 and Table S2). All substituted segments in 76 lines can cover 96.7% of the XQZB genome with only two small missing regions on chromosomes 3 and 4.

There were significant differences between two parents for all studied traits (Fig. S3). Most significantly, TGW of XQZB was 27.7% higher than that of ZH9308, indicating that the two parents were suitable for investigating QTL for grain weight.Two QTLs for TGW in both environments, temporarily namelyand, were mapped on chromosomes 1 and 6 in CSSL population, respectively (Table S3). With benefit allele from XQZB,was located between InDel15 and RM12276 with 15.36% of phenotypic variation explained (PVE). Inherited from ZH9308,was mapped between the interval from InDel190 to RM20069, with 10.48% of PVE. To confirm, a CSSL, named CSSL7, was crossed with ZH9308 to develop secondary F2population. According to the comparison between CSSL7 and ZH9308, there were significantdifferences for TGW, GL and GW (Fig. S3), with no significant differences for the other agronomic traits (Fig. S4).

Rice grain traits (TGW, GL, GW and LWR) in the secondary F2population (409 out of 899 individuals) showed continuous variation (Fig. S5). TGW and GW showed normal distribution, and only GL exhibited a probably bimodal distribution with a segregation ratio of 3:1 (χ2= 3.05 < χ20.05= 3.84). In addition, the correlation results showed that GL had a significantly positivecorrelation with TGW and GW, whereas LWR had a significantly positive correlation with GL, but a negative correlation with GW (Table S4). The above results indicated that the effect ofon GW is partly caused by the effect of GL. In view of this, GL was regarded as the trait for QTL dissection and fine mapping, andwas mapped as(Fig. 1-A). We utilized RICE8K array assay for scanning genetic of line CSSL7, and two small unexpected segments were observed on chromosomes 3L and 7L (Fig. S6). The target position of the substituted segment from XQZB in CSSL7 was from 32 032 333 to 43 269 728 bp, which overlapped the target/. For QTL validation and dissection, the two additional substituted segments in CSSL7 were eliminated in secondary F2and F2:3populations utilizing a MAS strategy.

Fig. 1. Genetic mapping, dissection and physical maps ofon chromosome l.

A,was primarily mapped to the region between InDel15 and RM3520 on chromosome 1. CSSL, Chromosome segment substitution line. B, Validation ofbased on the secondary F2populations. The locus was mapped to the region between InDel15 and RM11872. C, Dissection ofbased on the F2:3populations and selection of homozygous recombinants. D, Fine mapping of. The allele was mapped to 286.4 kb between D-12 and TG-57 in F4:5population. NIL, Near-isogenic line. * and ** indicate the significant differences at the 0.05 and 0.01 levels, respectively.

In secondary F2population, the targetwas delimited to the interval between InDel15 and RM11872 with the additive effect of 0.12 for grain length, explaining 38.24% of PVE (Fig. 1-B). Next, a total of 99 heterozygous recombinants were obtained in the secondary F2population to generate F2:3population containing 4752 (99 lines × 48plants each) individuals. In F2:3, 1386 individuals (14 individuals for each line) were screened using four polymorphic markers (Fig. 1-C and Table S1), andshowed the 33.29% of PVE and 0.81 of additive effect for grain length, indicating the stable existence. F3:4and F4:5mapping populationswere constructed by selecting of homozygous recombinants in F2:3and F3:4using markers InDel15 and RM11872. Additionally, a line in F3:4with the onlyminimum substituted segment from XQZB carryingwas chosen as NIL(). Compared with the recurrent ZH9308, NIL() showed significant longer grain, indicating that thecontributed stably to TGW and GL.

To further narrow the target region, using 3366 out of 4752individuals,was delimited to a 500.5-kb region between markers RM11824 and RM11842 (Fig. 1-C). Subsequently, 32 homozygous recombinant lines were selected, and evaluated phenotypically and genotypically for further mapping ofin F3:4and F4:5generations. The results showed that all homozygousrecombinants withalleles inherited from XQZB showed significant longer grains than the recurrent parent, while lines withalleles from ZH9308 shared the same performance with ZH9308. As a result, thewas finally restricted into a 286.4-kb region between markers D-12 and TG-57 (Fig. 1-D).

In the present study,was located in a 286.4-kbregion between markers D-12 and TG-57, flanking the segment from 35318329 to 35644057 bp. Zhang et al (2019) fine- mapped thewithin a region of 119 kb (from 33762178 to 33765481 bp) on chromosome 1. Singh et al (2012) fine- mappedwithin the interval from 38786976 to 38895287 bp on the same chromosome. Recently,was fine-mapped to 77.50 kb (from 33011637 to 33089264 bp) on the long arm of chromosome 1 (Wang W H et al, 2019). Two QTLs (and) were reported to regulate grain size neighboringon the same chromosome (Dong et al, 2018). In details, thewas limited to a 57.7-kb region (from 35240841 to 35182928 bp) with upstream of, whereas(from 35518354 to 35644119 bp) was collocated with. However, both ofandhad a minor effect on grain size, whilehad a medium effect. The above results showed thatwas located in a gene cluster responsible for grain size in rice. It is noteworthy that the recombinant line FY19PA7063 showed similar performance of GL but higher value of GW comparing with the recurrent parent ZH9308, which may be caused by the abundant existence of minor effect QTL for GW. Therefore, it is a sensible decision to take GL as the target trait for fine mapping.

Acknowledgements

This study was supported by the Natural Science Foundation of China (Grant No. 31961143016), the National Key Research and Development Program (Grant No. 2016YFD0101801), the Fundamental Research Funds of Central Public Welfare Research Institutions (Grant No. CPSIBRF-CNRRI-202102), and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (Grant No. CAAS-ASTIP-2013-CNRRI).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Scheme for population development in this study.

Fig. S2. Graphical representation of the chromosome segment substitution lines.

Fig. S3. Phenotypic comparison among Xieqingzao B, Zhonghui 9308, CSSL7 and NIL().

Fig. S4. Agronomic trait comparison between CSSL7 and Zhonghui 9308.

Fig. S5. Frequency distribution in secondary F2population.

Fig. S6. Graphical genotype of CSSL7 derived from Xieqingzao B/Zhonghui 9308.

Table S1. List of primers.

Table S2. Genotype of 76 chromosome segment substitution lines.

Table S3. QTLs detected in chromosome segment substitution lines for 1000-grain weight.

Table S4. Correlation coefficient among grain traits in F2population.

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20 July 2020;

13 November 2020

Copyright ? 2021, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/j.rsci.2021.05.003

Zhang Yingxin (zhangyingxin@caas.cn); Cheng Shihua (chengshihua@caas.cn)