Rice Research Institute,Academy of Agricultural Sciences,Southwest University,Chongqing 400715,P.R.China

Abstract Length of grain affects the appearance,quality,and yield of rice.A rice long-grain chromosome segment substitution line Z744,with Nipponbare as the recipient parent and Xihui 18 as the donor parent,was identified.Z744 contains a total of six substitution segments distributed on chromosomes (Chrs.)1,2,6,7,and 12,with an average substitution length of 2.72 Mb.The grain length,ratio of length to width,and 1 000-grain weight of Z744 were significantly higher than those in Nipponbare.The plant height,panicle number,and seed-set ratio in Z744 were significantly lower than those in Nipponbare,but they were still 78.7 cm,13.5 per plant,and 86.49%,respectively.Furthermore,eight QTLs of different traits were identified in the secondary F2population,constructed by Nipponbare and Z744 hybridization.The grain weight of Z744 was controlled by two synergistic QTLs (qGWT1 and qGWT7)and two subtractive QTLs (qGWT2 and qGWT6),respectively.The increase in the grain weight of Z744 was caused mainly by the increase in grain length.Two QTLs were detected,qGL1 and qGL7-3,which accounted for 25.54 and 15.58% of phenotypic variation,respectively.A Chi-square test showed that the long-grain number and the short-grain number were in accordance with the 3:1 separation ratio,which indicates that the long grain is dominant over the short-grain and Z744 was controlled mainly by the principal effect qGL1.These results offered a good basis for further fine mapping of qGL1 and further dissection of other QTLs into single-segment substitution lines.

Keywords:rice,chromosome segment substitution line,grain length,QTL

1.lntroduction

Rice,wheat,and corn are the three major food crops,with rice being the staple food of about half of the world’s population.Yield and quality have always been the main targets of rice breeding,with grain size and grain weight being important factors that affect these desiderata (Xuet al.2002;Fanet al.2006;Songet al.2007;Wanget al.2018).Grain size is controlled by multiple quantitative trait loci (QTLs)and can be assessed in terms of length,width,and thickness.However,by using special genetic means and biological techniques,such as the construction of chromosomal segment substitution lines or near-isogenic lines by means of molecular marker technology,the major quantity genes can be decomposed into a single Mendelian factor.A number of the genes that control grain size have been cloned.Studies have shown that brassinolide (BR)has an important effect on grain development (Zhuet al.2015).For example,DWARF11andDWARF11alleleCLUSTERED PRIMARY BRANCH 1(CPB1),which encode cytochrome P450 proteins in the BR synthesis pathway,regulate grain size positively (Tanabeet al.2005;Wuet al.2016).GW5encodes a calmodulin-binding protein that regulates the expression of BR-responsive genes positively and increases grain width and grain weight significantly (Liuet al.2017).Gibberellin also affects the formation of rice grain size.TheShort Grain Length(SGL)gene encodes a kinesin-like protein with transcriptional activation activity,and thesglmutant has a shorter grain length and shows a significant change in the expression of gibberellin pathway-related genes (Wuet al.2014).In addition,ubiquitin has an important influence on the development of rice grains.GW2encodes a RING-type protein with E3 ubiquitin ligase activity,and its loss of function increases cell number,thereby increasing grain width and weight (Songet al.2007).GIF1(GRAIN INCOMPLETE FILLING 1)encodes a cell wall invertase that promotes grain filling to make the grain fuller (Wanget al.2008).GS3encodes a transmembrane protein that interacts directly with the conserved keratin-like domain of the MADS transcription factor,acting as a cofactor,enhancing the transcriptional activity of OsMADS1,and acting synergistically to transactivate a common target gene,which in turn regulates grain size (Fanet al.2006;Maoet al.2010;Liuet al.2018).In a preliminary investigation,Yanet al.(2011)revealed the molecular regulation mechanism ofGL5,GW2,GIF1,andGS3in regulating grain size.GW2andGW5regulateGS3positively,GW2expression downregulatesGW5expression,GW5regulatesGIF1expression positively,GW2andGS3show negative regulation of the expression ofGIF1,and all these genes interact with each other to jointly regulate grain development (Yanet al.2011).It will be evident that the molecular mechanism of rice grain development is extremely complicated;many known genes are involved and it is necessary to identify more QTLs.

In the study reported herein,Nipponbare was used as a recipient parent and Xihui 18 as a donor parent.We identified a six substitution segments of the longgrain chromosome segment substitution line Z744 and constructed a secondary F2population for QTL mapping of such important agronomic traits as grain length and other grain type traits.Our results suggested that further cloning and functional analysis of the major QTLs carried by Z744 will be of great significance.

2.Materials and methods

2.1.Experimental materials

The rice chromosome segment substitution line Z744 was used.Z744 was devived from continuous backcrossing and selfing between Nipponbare as recipient parent and Xihui 18 as donor,combined with phenotype-based selection and simple sequence repeat (SSR)marker selection.The QTL mapping population material came from the F2population crossed by Nipponbare and Z744.

2.2.Plant materials and field planting

Seeds of Z744,Nipponbare,and the F2population comprising 146 individuals were sown on 10 March 2018 at the experimental station of Southwest University,Chongqing,China.Thirty seedlings of each parent and all F2seedlings were transplanted to the field on 20 April 2018,with 10 individuals per row.The spacing between rows and individual plants was 26.4 and 16.5 cm,respectively.Conventional management practices were applied.

2.3.ldentification of substitution segments in Z744

Firstly,263 SSR markers polymorphic between Nipponbare and Xihui 18 were screened from 429 markers that covered the entire rice genome.Then,molecular marker selection and phenotype selection were performed from the BC2F1to F6(Nipponbare/BC2F4)population.Finally,a long-grain chromosome segment substitution line Z744 with six substitution segments was identified.The substitution segment identification method followed the method of Zhaoet al.(2016),and the estimated length of the substitution segment was calculated according to the method of Patersonet al.(1991).Namely,the distance of substitution markers from donor plus half of distance between boundary markers from Nipponbare and substitution markers were regarded as the estimated substitution length.

2.4.Agronomic trait assessment

After maturity,10 plants at the 3rd-7th hills of the middle two rows in Nipponbare and Z744 plots,and 146 individuals of F2populations were harvested.Then,the plant height,panicle number,panicle length per panicle,number of primary branches per panicle,number of secondary branches per panicle,grain length,grain width and ratio of length-width,number of spikelets per panicle,number of filled grains per panicle,seed-set ratio,1 000-grain weight,and yield per plant were measured.Finally,t-test for these traits between Nipponbare and Z744 and simple statistical analysis such as the skewness and kurtosis in the F2population were conducted with intrinsic statistical functions in Excel 2003.

2.5.QTL mapping

The DNA of one plant each of Nipponbare,Xihui 18 and Z744 and 146 plants from the F2population was extracted using the CTAB method (McCouchet al.1988).PCR amplification,nondenaturing polyacrylamide gel electrophoresis,and rapid silver staining were performed according to the method of Zhaoet al.(2016).Nipponbare bands were scored as“-1”,Z744 bands were scored as“1”,heterozygous bands were scored as“0”,and the absence of marker bands was scored as“.”.The marker assignments of all 15 SSR markers on the substitution segments of Z744,together with the phenotypic values of each individual in the F2population,were used for QTL mapping.QTL mapping was performed using the restricted maximum likelihood (REML)method of the SAS Statistical Software HPMIXED Program atP＜0.05 (SAS Institute Inc.,Cary,NC,USA).

3.Results

3.1.ldentification of substitution segments in Z744

Fifteen polymorphic SSR markers in substitution segments of Z744,together with 24 other polymorphic SSR markers,were used to detect the substitution fragment and assess the homogeneity of the genetic background of Z744 using 10 plants of Z744.All the plants harbored six consistent substitution segments,and no additional chromosomal fragments derived from Xihui 18 were detected.The substitution segments of Z744 were located on chromosomes (Chrs.)1,2,6,7,and 12 (Fig.1).The total substitution length was 16.30 Mb and the average length was 2.72 Mb.

3.2.Phenotypes of Z744

Compared with the recipient parent Nipponbare,Z744 showed significantly greater grain length,ratio of lengthwidth,and 1 000-grain weight (Fig.2-A-D).Plant height,panicle number per plant,and seed-set ratio in Z744 were significantly lower than those of Nipponbare.However,the plant height and panicle number in Z744 was still 78.7 cm and 13.50,respectively,and the seed-set ratio in Z744 was still 86.49%.There were no significant differences for other traits between Z744 and Nipponbare (Table 1).

Fig.1 Substitution segments of Z744.Physical distances (Mb)are shown at the left of each chromosome (Chr.)and markers at the right.The estimated length of the chromosomal substitution fragment is marked on the right side of each Chr.The markers in each box included boundary markers and substitution markers.

3.3.Frequency distribution and genetic analysis of long grain and other traits in the F2population

Fig.2 Phenotype of Nipponbare and Z744.A,plant type of Nipponbare and Z744.Bar,20 cm.B,main panicle of Nipponbare and Z744.Bar,10 cm.C,grain type of Nipponbare and Z744.Bar,10 mm.D,50 brown rice of Nipponbare and Z744.

Table 1 Agronomic traits of Nipponbare,Z744 and the F2population

In the F2population of 146 strains,the skewness of 13 traits was -1.98 to 2.81,and the kurtosis was -0.2 to 18.1(Table 1).The skewness and kurtosis of the standard normal distribution are 0 and 3,respectively.Table 1 shows that the skewness and kurtosis of these traits were not all close to 0 and 3,which indicates that the distribution of these traits does not follow a standard normal distribution.The grain length traits are basically bimodal,with the short-grain peaks concentrated mainly between 7.15 and 7.64 mm for a total of 40 strains,and the long-grain peaks concentrated mainly between 7.65 and 8.94 mm for a total of 106 strains.A Chisquare test showed that the numbers of short-grain (40)and long-grain (106)strains correspond to a 1:3 separation ratio(χ2=0.45＜χ2(0.05,1)=3.84)(Fig.3).The results suggested that the grain length of Z744 was regulated mainly by a single main QTL.

3.4.QTL mapping of important agronomic traits carried by Z744

Z744 still has six substitution segments from donor parent Xihui 18 and many differences for agronomic traits compared to recipient parent Nipponbare.Thus,the distribution of these QTLs for agronomic traits on the six chromosome substitution segments in Z744 were determined.Consequently,the secondary F2population consisting of 146 individuals was constructed by crosses of Nipponbare and Z744 in order to map the QTLs.In addition,15 SSR markers located on the six substitution segments,noting that gene conversion only occurs in the six substitution regionals,were used to analyze linkage using the REML method in the HPMIXED procedure of SAS.Finally,a total of eight existing QTLs were detected atP＜0.05 as the threshold for QTL.Two QTLs,qGL1andqGL7-3affected the grain length,and were located on Chrs.1 and 7 and their additive effects could be increased by 0.15 and 0.12 mm,respectively,with contribution rates of 25.54 and 15.58%,respectively.Four QTLs affected grain weight,of whichqGWT1andqGWT7increased 1 000-grain weight by 0.66 and 0.53 g,respectively,and the contribution rates were 17.97 and 11.64%,respectively.Two QTLs,qGWT2andqGWT6,showed reduced efficiency,with a reduction in the 1 000-grain weight of 0.61 and 0.46 g,respectively,so the contribution rates were 15.19 and 8.53%,respectively.In addition,Z744 contained a QTL-qPH1that reduced plant height and a QTL-qPN12that reduced the number of effective panicles per plant.These were located on Chrs.1 and 12,respectively.Their additive effects were -3.11 cm and -0.92 panicles per plant,and their contribution rates were 11.89 and 5.24%,respectively (Table 2).

4.Discussion

4.1.Z744 is an important genetic material

Rice yield is determined mainly by three factors:effective panicle number,grain number,and 1 000-grain weight (Liuet al.2009).The 1 000-grain weight is determined mainly by grain length,grain width,and grain thickness.In our study,we identified a six-segmental chromosome segment substitution line Z744,with Nipponbare as the recipient parent and Xihui 18 as the donor parent.The increase in 1 000-grain weight of Z744 was due mainly to an increase in grain length.However,although Z744 has long grains,at 9.03 mm (the grain length of recipient Nipponbare is 7.40 mm),its 1 000-grain weight only increased by 1.51 g.This low increase of 1 000-grain weight in Z744 was mainly due to insufficient filling compared with Nipponbare,in which the brown rice in Z744 was not well-stacked (Fig.2-D).High temperature during the filling period has a certain impact on grain filling (Luet al.2013).In particular,this impact may be more serious for rice varieties with large grains as Z744 in Chongqing of China,for example,where the temperature was higher than 35°C during the middle 10 days of July,2018.

Fig.3 Distribution of grain length in the F2population from a cross of Nipponbare and Z744.

Table 2 QTLs identified for agronomic traits in Z744

4.2.Z744 carried six unreported QTLs previously

Z744 contains two QTLs for increasing grain weight and two QTLs for increasing grain length,which further confirms that the increase in Z744 grain weight is due to the increase in grain length.Moreover,grain length in Z744 is controlled by a major grain length QTL-qGL1(contribution rate 25%)and a micro-effect QTL,qGL7-3.In addition,grain length displayed a bimodal distribution for short grain and long grain in the F2population,and the Chi-square test showed that the grain length of Z744 is regulated dominantly byqGL1.Compared with mapped QTL and cloned genes,qGL1has still not been reported.On Chr.7,three QTLs for grain length were fine mapped or cloned,GL7(24.66 Mb)(Wang Y Xet al.2015),qGL7(RID711-RM6389)(Baiet al.2010),andqGL7-2(Indel1-RM21945)(Shaoet al.2010).GL7(Wang Y Xet al.2015)encodes a protein homologous toArabidopsis thalianaLONGIFOLIA proteins,which are located at the same locus asGW7(Wang S Ket al.2015).We mapped QTL for grain length on Chr.7,and it was not the same as the other three QTLs,thus,it was named asqGL7-3,linked with RM5672 (6.41 Mb).The QTLqPH1for plant height,linked with RM6950 (34.5 Mb)may be associated withERF3(33.77 Mb)(Zhaoet al.2015).ERF3interacting withWOX11,acts on the cytokinin response geneRR2to regulate the expression of cytokinin and promote the development of coronary roots,thereby affecting plant height (Zhaoet al.2015).Determining whetherERF3isan allele forqPH1would require DNA sequencing and functional confirmation.qGWT2for 1 000-grain weight,linked with RM6378 (5.48 Mb),may be associated withOsBT1(5.74 Mb).OsBT1encodes an ADP glucose transporter,which participates in starch synthesis in rice endosperm and increases the grain weight (Liet al.2017).To determine whether these genes are alleles of the located QTLs,further sequencing and functional complementation are needed.qPN12,qGWT1,qGWT6,andqGWT7have not been reported previously and our findings pertaining to them indicate that further dissection of these QTLs into different single-segment substitution lines will have important application values for their genetic mechanisms and molecular breeding.

5.Conclusion

We identified a long-grain chromosome segment substitution line Z744,with Nipponbare as the recipient parent and Xihui 18 as the donor parent.The average substitution length was 2.72 Mb.The Z744 substitution segment contains two QTLs that increase the grain length,two that increase the grain weight,two that reduce the grain weight,one that reduces the number of panicles,and one that reduces the plant height.The increase in grain weight of Z744 is due to the increase of grain length,and its grain length is regulated mainly by the main QTLqGL1,with a small contribution from QTLqGL7-3.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31871593),the Chongqing Science and Technology Commission Special Project,China(cstc2016shms-ztzx0032),and the Southwest University Innovation Team Project,China (XDJK2017A004).