Fine mapping and validation of a major QTL for grain weight on chromosome 5B in bread wheat

A major QTL QTgw.caas-5B for thousand grain weight in wheat was fine mapped on chromosome 5B, and TraesCS5B02G044800 was predicted to be the candidate gene. Thousand grain weight (TGW), determined by grain length and width, and is an important yield component in wheat; understanding of the underlying genes and molecular mechanisms remains limited. A stable QTL QTgw.caas-5B for TGW was identified previously in a RIL population developed from a cross between Zhongmai 871 (ZM871) and a sister line Zhongmai 895 (ZM895), and the aim of this study was to perform fine mapping and validate the genetic effect of the QTL. It was delimited to an interval of approximately 2.0 Mb flanked by markers Kasp_5B29 and Kasp_5B31 (49.6–51.6 Mb) using 12 heterozygous recombinant plants obtained by selfing a residual BC1F6 line selected from the ZM871/ZM895//ZM871 population. A candidate gene was predicted following sequencing and differential expression analyses. Marker Kasp_5B_Tgw based on a SNP in TraesCS5B02G044800, the QTgw.caas-5B candidate, was developed and validated in a diversity panel of 166 cultivars. The precise mapping of QTgw.caas-5B laid a foundation for cloning of a predicted causal gene and provides a molecular marker for improving grain yield in wheat.


Introduction
Wheat is one of the most important food crops in the world, providing approximately 20% of the calories and 25% of the protein for humans (FAO 2017, http:// www. fao. org/ faost at/ en/). Although significant progress has already been made on yield improvement during the last 60 years, with a genetic gain of 0.7-1.0% annually (Fischer and Edmeades 2010; Gao et al. 2017), it is estimated that yield must still increase by more than 60% to meet predicted growth in the world population by 2050, despite the restricting effects of climate change and the declining area of available land due to urbanization and degradation (Langridge 2013). Improved yield potential is, therefore, still a major breeding objective. Identification and mining of genetic loci for grain yield will provide genetic resources and tools to improve yield potential.
Yield is a complex quantitative trait determined by thousand grain weight (TGW), grain number per spike and spike number per unit area, with TGW having the highest heritability among three components (Simmonds et al. 2014(Simmonds et al. , 2016Chen et al. 2020;Yang et al. 2020). TGW is determined by grain size and grain filling characteristics. Grain size can be divided into components grain length (GL), grain width (GW) and grain thickness (Kuchel et al. 2007;Simmons et al. 2014;Yang et al. 2020). Numerous genes for grain weight have been cloned in rice Li et al. 2019b), and many genes associated with grain weight in wheat were cloned by comparative genomics (Su et Zhang et al. 2012;Dong et al. 2014;Zhang et al. 2014;Jiang et al. 2015;Wang et al. 2015;Yue et al. 2015;Ma et al. 2016;Wang et al. 2016;Hanif et al. 2016;Hu et al. 2016;Simmonds et al. 2016;Sajjad et al. 2017;Yang et al. 2019;Cao et al. 2020). A large number of quantitative trait loci (QTL) for TGW, GL and GW have also been identified (Huang et al. 2003;Quarrie et al. 2005;Prashant et al. 2012;Cui et al. 2014;Wu et al. 2015;Sun et al. 2017;Cabral et al. 2018;Guan et al. 2018;Ma et al. 2018;Su et al. 2018;Zhai et al. 2018;Yang et al. 2020). However, few TGW genes have been isolated by map-based cloning due to the complexity of the wheat genome. With the current availability of genotyping arrays and release of wheat reference genome sequences (IWGSC 2018, https:// urgi. versa illes. inra. fr/ blast_ iwgsc/) fine mapping of major QTL for TGW has been achieved using near-isogenic lines (NILs) and residual heterozygosity (Brinton et al. 2017;Guan et al. 2019;Xu et al. 2019;Chen et al. 2020).
In a previous study (Yang et al. 2020) four QTL for TGW-related traits were identified on chromosomes 1AL, 2BS, 3AL and 5B. QTgw.caas-5B, detected across all of ten environments, explaining 5.7-17.1% of the phenotypic variances, was mapped to a 11.6 cM region between markers Kasp_5B5 and Kasp_5B12, extending from 45.3 to 394.2 Mb on chromosome 5B based on the Chinese Spring reference genome (IWGSC 2018, https:// urgi. versa illes. inra. fr/ blast_ iwgsc/). The present study was to precisely map QTgw.caas-5B by analysis of a residual heterozygous plant, predict one or more candidate genes and develop kompetitive allele-specific PCR (KASP) markers for accurate marker-assisted selection in breeding and research.

Plant materials
Zhongmai 895 (hereafter ZM895), with a current production area around 0.7 million ha annually is a leading cultivar in the Yellow and Huai River Valleys Winter Wheat Region of China. ZM895 and Zhongmai 871 (hereafter ZM871), developed by pedigree selection and bulked as fixed lines at F 5 , are sister lines that can be traced back to a single F 2 plant of cross Zhoumai 16/Liken 4. The detailed information of ZM895 and ZM871 was described in a previous study (Yang et al. 2020). One residual heterozygous line (L2925) within the marker interval of QTgw.caas-5B was selected from BC 1 F 6 generation of the ZM871/ZM895//ZM871 population (Fig. 1a, b). A heterozygous recombinant plant (HRL2925) from L2925 was self-pollinated, generating 12 heterozygous recombinant plants (designated RL1 to RL12) and 119 homozygous plants with 57 having 5B+ alleles (ZM895 genotype) and 62 having 5B− (ZM871 genotype), in which two groups of homozygous plants were used for a preliminary evaluation of the phenotypic effects of QTgw.caas-5B on TGW, GL and GW (Fig. 1c, d). Genetic backgrounds of HRL2925 were evaluated by genotyping two 5B+ and two 5B− homozygous lines generated from two kinds of homozygous plants, respectively, using the wheat 50 K SNP array developed in collaboration with the Capital-Bio, Beijing, China (https:// www. capit albio tech. com/). Twenty 5B+ and 20 5B− homozygous lines were used to measure TGW, GL and GW at different grain developmental stages and for RNA-sequencing. In addition, 52-77 NILs from each of RL1 to RL12 were identified using QTgw.caas-5B-flanking markers to narrow the region of candidate genes ( Fig. 1d; Table 2). A diverse panel of 166 cultivars (Li et al. 2019a) was used to validate the effects of QTgw.caas-5B.

Field trials and trait measurement
The progeny from HRL2925 were sown in ten 3. Wanshen SC-G seed detector (Hangzhou Wanshen Detection Technology Co., Ltd) was used to record TGW, GL and GW. Thirty random spikes were harvested from each plot of all 20 homozygous individuals in the contrasting 5B+ and 5B− groups to measure TGW, GL and GW. The same parameters were measured on the 119 homozygous plants from HRL2925 were measured on grains from 6-10 spikes of each plant and 52-77 homozygous progenies from each of RL1-RL12. For the diversity panel, TGW was determined by weighing 500 grains, and GL and GW were measured on 20 random grains from each plot to obtain mean length and width values, respectively (Li et al. 2019a).

Grain sampling
Twenty 5B+ and 20 5B− homozygous lines grown at Xinxiang in 2018-2019 were used for the study. Main stems at anthesis were marked with red tags at 09:00-11:00 am every day. Ten grains from outer florets of five spikelets in the middle regions of tagged spikes were sampled at 09:00-11:00 am at 4, 8, 12, 16, 20, 25 and 30 days post-anthesis (DPA). At each time point 10 spikes per plot were sampled, including 200 grains (20 spikes × 10 grains each spike) for each group. GL and GW were measured using image analysis software (Image-Pro Plus 6.0, http:// www. media cy. com/) after scanning the grain samples placed on a scanner panel with grain creases placed downwards. Following measurement, the grains were dried in an oven at 135 °C for 15 min and then at 65 °C until a constant weight. The TGW of the dried grain samples harvested at various DPA was determined, three biological replications were performed for each time point.

RNA and DNA extraction and RNA-sequencing
Two grains sampled from the outer florets of spikelets in the middle of tagged spikes of main stems at 4, 8, 12, 16, 20 Statistical comparisons of TGW between 5B+ and 5B− genotypes among self-pollinated progenies of each RL are shown at the right. *, **, ***and ns, significant at P < 0.05, P < 0.01, P < 0.001 and non-significant, respectively and 25 DPA were snap-frozen in liquid nitrogen and stored at −80 °C. For 20 homozygous lines with 5B+ and 20 with 5B− selected from 119 self-pollinated homozygous plants of HRL2925, 40 grains were sampled (20 spikes × 2 grains each spike) with three biological replications at each time point.
Total RNA was isolated using the TRIzol protocol (Invitrogen, Carlsbad, CA, USA). After quality testing, a single RNA library for each sample was constructed, and the library preparations were sequenced on an Illumina Hiseq platform with 250-300 bp paired-end reads at Novegene Bioinformatics Technology in Beijing (http:// www. novog ene. com/). FeatureCounts v1.5.0-p3 was used to count the number of reads mapped to each gene. Then FPKM of each gene was calculated based on the length of the gene and reads count mapped. Differential expression analysis of 5B+ and 5B− genotypes were performed with three biological replications at each stage using the DESeq2 R package (1.10.1), which provides the statistical routines for determining differential expression in digital gene expression data using a model based on a negative binomial distribution. The resulting P-value was adjusted using the Benjamini and Hochberg's approach for controlling false discovery rate. Genes with an adjusted P < 0.05 determined by DESeq2 were considered as differentially expressed. Genomic DNA was extracted from young leaves of experimental lines using the CTAB method.
Array-based SNP markers or SNPs from RNA-sequencing and whole-genome resequencing upstream of the physical location of the Kasp_5B2 locus and between Kasp_5B2 and Kasp_5B6 were converted to KASP markers for fine mapping of QTgw.caas-5B. Allele-specific and common reverse primers for each KASP marker were designed using Poly-Marker (Ramirez-Gonzalez et al. 2015, http:// www. polym arker. info/).

Statistical analysis
For the statistical analyses in progeny tests, grain development and diversity panel, phenotypic differences between the allelic pairs were determined by Student's t tests with SAS 9.2 software (SAS Institute Inc, Cary, NC, USA). BLUE (best linear unbiased estimation) value of the phenotypic data for each line/cultivar in each environment was used for the analyses.

Generation of fine mapping population using residual heterozygous lines
A heterozygous line L2925 screened from a BC 1 F 6 population of the ZM871/ZM895//ZM871 had homozygous background across the mapping interval spanning QTgw. caas-5B (Fig. 1a, b; Yang et al. 2020). One recombinant plant (HRL2925) from L2925 was self-pollinated and generated 12 heterozygous recombinant plants (Fig. 1c, d). The heterozygous interval was flanked by SNP markers AX-110372788 and AX-95631395 from 24.5 to 53.7 Mb in the HRL2925 based on the wheat 50 K SNP array. The genetic similarity between homozygous progenies for 5B+ and 5B− was more than 99% according to 50 K SNP array data, indicating that the segregating progenies from the HRL2925 were suitable for fine mapping (Table S2). Within each family of selfed progenies from 12 recombinant plants, homozygous non-recombinant plants, namely 5B+ NILs and 5B− NILs, were genotyped with markers according to heterozygous interval and phenotypes evaluated for fine mapping.

Phenotypic validation of NILs for QTgw.caas-5B
After a progeny test, a significant difference in TGW was detected between the genotypes 5B+ with 57 plants and 5B− with 62 plants from selfing progenies of HRL2925 (Fig. 2). In order to improve the accuracy of phenotypic evaluation, 20 homozygous lines with 5B+ and 20 with 5B− generated from two kinds of homozygous plants were evaluated at Xinxiang and Anyang (Henan province) to verify the effects of QTgw.caas-5B. Student's t tests indicated significantly (P < 0.05) higher TGW and GL in 5B+ lines than their contrasting 5B− lines (Fig. 3). These demonstrated that the ZM895 allele at QTgw.caas-5B had a positive effect on TGW.
To further analyze the genetic effect of QTgw.caas-5B, the dynamic change of grain weight and size at different developmental stages between the above 5B+ and 5B− homozygous lines were investigated. Twenty lines with 5B+ and 20 with 5B− were used to determine the differences on grain morphometric parameters. Student's t tests indicated significantly (P < 0.05) higher GL in 5B+ lines than those in 5B− from the 12 DPA, while 5B+ lines also had significantly (P < 0.05) higher TGW than those of 5B− from the 25 DPA (Fig. 4, Table 1). No significant differences were observed in GW between the 5B+ and 5B− genotypes at all the developmental stages. This suggests that the increased TGW is attributed to the increased grain length in the 5B+ genotypes.

Prediction of candidate genes for QTgw.caas-5B
Based on resequencing data for the parents, SNPs or InDels were found in the coding or intron regions of TraesCS5B02G044500, TraesCS5B02G044600, TraesCS5B02G044700, TraesCS5B02G044900, TraesCS5B02G045500, TraesCS5B02G045800, TraesCS5B02G045900 and TraesCS5B02G046000, whereas the other nine high-confidence genes lacked sequence polymorphisms between two parents. SNPs are synonymous mutation in TraesCS5B02G044500, TraesCS5B02G044600, TraesCS5B02G044700, TraesCS5B02G045900 and TraesCS5B02G046000. They are not likely to cause delirious  with 5B− at different grain development stages. *and ns, significant at P < 0.05 and non-significant, respectively effects on the proteins. Whereas, SNPs are missense mutation in TraesCS5B02G044900, TraesCS5B02G045500 and TraesCS5B02G045800.
The results of RNA-seq indicated that only TraesCS5B02G044800 among the 17 high-confidence genes in the 49.6-51.6 Mb region on chromosome 5B showed higher expression level in the 5B− genotype, whereas the transcript was not detected in the genotype 5B+ (Fig. 5, Table 3). The other 16 high-confidence genes, including TraesCS5B02G044900, TraesCS5B02G045500 and TraesCS5B02G045800 which had missense mutations, showed no differential expression levels between homozygous 5B+ and 5B− genotypes (Table S3). A SNP was located at 824 bp upstream of the initiation codon ATG in the promoter region of TraesCS5B02G044800 by resequencing the parents. Thus, TraesCS5B02G044800 was considered a candidate gene for QTgw.caas-5B.

Validation of KASP markers flanking QTgw.caas-5B in a germplasm panel
The Kasp_5B_Tgw based on the SNP with 'C' in ZM871 and 'A' in ZM895 alleles in the promoter region of TraesCS5B02G044800 for QTgw.caas-5B was used to genotype the diversity panel of 166 cultivars, among which 48 cultivars had the ZM895 genotype, and 118 had the ZM871 genotype (Tables 4 and S3). The ZM895 genotype showed significantly (P < 0.05) higher TGW and GL than the ZM871 genotype in all six environments as well as BLUE value. Differences in TGW and GL between the two genotypes ranged from 2.1 to 2.9 g (5.5 to 5.9%) and 0.30 to 0.40 mm (2.0 to 3.0%), respectively (Table 4). Other three KASP markers based on SNPs in TraesCS5B02G044900, TraesCS5B02G045500 and TraesCS5B02G045800 were run on the same diversity panel, but no significant differences in TGW were observed between two genotypes. These results provided further evidence for a significant effect of QTgw. caas-5B on TGW, and TraesCS5B02G044800 is probably a candidate gene for QTgw.caas-5B.

Residual heterozygous recombinant lines are useful stocks for fine mapping
Many QTL for grain-related traits have been identified in different genetic backgrounds (Huang et al. 2003;Quarrie et al. 2005;Prashant et al. 2012;Cui et al. 2014;Wu et al. 2015;Ma et al. 2018;Su et al. 2018;Zhai et al. 2018;Xu et al. 2019). Most were located in large chromosome intervals due to limited numbers of markers or lack of recombination events within the targeted QTL regions. Following release of the Chinese Spring reference genome sequence (IWGSC 2018, https:// urgi. versa illes. inra. fr/ blast_ iwgsc/) and development of new sequencing technologies densely populated genetic maps can easily be constructed, and genetic information for a specific map interval can be searched. Consequently, many researchers have employed fine mapping approaches to validate QTL or narrow genomic intervals within targeted QTL regions (Brinton et al. 2017;Guan et al. 2019;Chen et al. 2020). In this study, progenies of 12 heterozygous recombinant plants were genotyped and phenotyped to fine map QTgw.caas-5B to an approximately 2.0 Mb physical interval containing 17 high-confidence annotated genes.

Candidate genes for QTL controlling TGW
The approximate 2.0 Mb interval of QTgw.caas-5B was flanked by Kasp_5B29 and Kasp_5B31 (49.6-51.6 Mb). To identify candidate genes for TGW expression levels of the annotated were determined. TraesCS5B02G044800 showed as significantly lower expression level in the 5B+ genotype than in the 5B− genotype at all grain developmental Table 1 Mean thousand grain weight (TGW), grain length (GL) and grain width (GW) of 20 homozygous lines with 5B+ and 20 with 5B− from progenies of L2925 at different grain development stages a DPA days post-anthesis b Data are means ± SD c Phenotypic differences between contrasting genotypes followed by different letters are significant at P < 0.05

GL contributes to TGW at QTgw.caas-5B locus
Various studies suggest that the early stage of grain length development is important in determining final grain weight in wheat (Hasan et al. 2011;Guo et al. 2015;Simmonds et al. 2016;Brinton et al. 2017Brinton et al. , 2018. This study initially detected a clear difference in GL between 5B+ and 5B− genotypes at 12 DPA, whereas a corresponding difference in TGW between 5B+ and 5B− genotypes was first observed at 25 DPA. This supported the contention that GL is a main factor contributing to grain weight (Brinton et al. 2017).

Applications in wheat breeding
Major stable QTL for yield-related traits with tightly linked markers is very important for molecular breeding. In this study, a QTL for TGW on chromosome 5B showed stable effects on TGW and GL cross environments. Its presence in 48 accessions among a panel of 166 indicated that it had been a selected target for grain weight (or even yield) in past breeding programs, and thus Kasp_5B_Tgw represents a future target for marker-assisted selection to enhance grain size and weight. Moreover, the current results provide a basis for map-based cloning of the gene underlying the QTL.   0B 0B 5B− (ZM871) 1.02 ± 0.10 b A c 1.11 ± 0.06A 1.18 ± 0.23A 0.56 ± 0.18A 0.81 ± 0.14A 0.82 ± 0.34A Table 4 Mean thousand grain weight (TGW), grain length (GL) and grain width (GW) of genotypes 5B+ and 5B− in the germplasm panel of 166 wheat cultivars grown in six environments a Genotypes identified using marker Kasp_5BTgw b Number of cultivars with corresponding genotype c E4-E9, Anyang 2012-2013, Suixi 2012-2013, Anyang 2013, Suixi 2013, Suixi 2014and Shijiazhuang 2014, respectively d BLUE Best linear unbiased estimation e Data are shown as means ± SD f Phenotypic differences between two genotype 5B+ and 5B−. Asterisks indicate significant differences determined by Student's t tests. *, ** and ***, significant at P < 0.05, P < 0.01 and P < 0.001, respectively g GL and GW are mean lengths and widths of 20 grains