Yu Lin,Xiaojun Jiang,Haiyan Hu,Kunyu Zhou,Qing Wang,Shifan Yu,Xilan Yang,Zhiqiang Wang,Fangkun Wu,Shihang Liu,Caixia Li,Mei Deng,Jian Ma,Guangeng Chen,Yuming Wei,Youliang Zheng,Yaxi Liu,*

a State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China,Wenjiang,Chengdu 611130,Sichuan,China

b Triticeae Research Institute,Sichuan Agricultural University,Wenjiang,Chengdu 611130,Sichuan,China

c School of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,Henan,China

d College of Resources,Sichuan Agricultural University,Wenjiang,Chengdu 611130,Sichuan,China

Keywords:Chinese Spring Grain number per spikelet High-density map KASP Quantitative trait locus

ABSTRACT Grain number per spikelet(GNS)is a key determinant of grain yield in wheat.A recombinant inbred line population comprising 300 lines was developed from a cross between a high GNS variety H461 and Chinese Spring from which the reference genome assembly of bread wheat was obtained.Both parents and the recombinant inbred lines were genotyped using the wheat 55K single nucleotide polymorphism(SNP) array.A high-density genetic map containing 21,197 SNPs was obtained.These markers covered each of the 21 chromosomes with a total linkage distance of 3792.71 cM.Locations of these markers in this linkage map were highly consistent with their physical locations in the genome assembly of Chinese Spring.The two parents and the whole RIL population were assessed for GNS in two consecutive years at two different locations.Based on multi-environment phenotype data and best liner unbiased prediction values,three quantitative trait loci(QTL)for GNS were identified.One of them located on chromosomes 2B and the other two on 2D.Phenotypic variation explained by these loci varied from 3.07%to 26.57%.One of these QTL,QGns.sicau-2D-2,was identified in each of all trials conducted.Based on the best linear unbiased prediction values,this locus explained 19.59%–26.57% of phenotypic variation.A KASP(Kompetitive Allele-Specific PCR) marker closely linked with this locus was generated and used to validate the effects of this locus in three different genetic backgrounds.The identified QTL and the KASP marker developed for it will be highly valuable in fine-mapping the locus and in exploiting it for markerassisted selection in wheat breeding programs.

1.Introduction

Wheat (Triticum aestivumL.) is one of the most widely cultivated crops in the world,and wheat grain is a major staple food for 20%of the global human population[1].The current annual rate of 0.9% increase in wheat productivity is considerably lower than the 2.4% needed to feed the growing world’s population by 2050[2].Hence,there is an urgent need to increase wheat productivity.Furthermore,owing to a continuous reduction in cultivated land and climate changes,breeding high-yielding wheat varieties must be one of the major strategies to increase the total food production.

Grain number per spikelet (GNS) is a key determinant of grain yield,for which the underlying genetic basis in wheat remains undefined.A higher GNS reportedly increases the yield of fieldgrown wheat[3].The gene Grain Number Increase 1(GNI1)located on chromosome 2A has been shown to be an important in regulating GNS[3].Additionally,quantitative trait loci(QTL)for GNS have been identified on 14 of the 21 chromosomes [4–7].Several of these QTL can be detected in multiple environments.Due to the low-densities of the linkage maps used,most of these QTL have been detected in exceedingly large genetic intervals.As a result,the reliability of marker-assisted selection for this trait is limited.

High-density genetic maps are important in genetic research,as they improve the resolution of QTL mapping in crops.With the advancement of sequencing technologies,several single nucleotide polymorphism(SNP)arrays have been successfully constructed for wheat research.They include the 9K [8],55K [12],90K [9],820K[10],and 660K arrays [11].The 55K SNP array including 53,063 tags was designed by the Chinese Academy of Agricultural Sciences,and synthesized by Affymetrix.This array has been widely used in recent research [12–14] which has provided abundant molecular markers for constructing high-density genetic maps for QTL mapping [12–15].Based on these high-density genetic maps,QTL have been detected within a short genetic interval[12–15].For example,with the use of this array and a recombinant inbred line (RIL) population comprising of 199 lines,a major and stable QTL for productive tiller number was identified and located in a 0.55-cM interval on chromosome 4B [12].Similarly,a major QTL for stripe rust resistance was identified in a 1.0-cM interval on chromosome 7BL using the wheat 55K SNP array [13].

In this study,an RIL population was developed from a cross between H461 and Chinese Spring,the two genotypes with contrasting GNS.The two parental lines and 300 RILs were phenotyped in four environments and genotyped using the 55K SNP array.A high-density map was constructed and used to identify GNSrelated QTL.Three additional RIL populations were used to validate the effects of the identified QTL in different genetic backgrounds.

2.Materials and methods

2.1.Plant materials and phenotype evaluation

A population between H461 and Chinese Spring was obtained for this study.H461 is a locally adapted line with large spikes and high GNS(Table S1)[16–18].Chinese Spring was selected from the landrace Cheng-du-guang-tou,and was used to construct the high-quality reference genome by the International Wheat Genome Sequencing Consortium[19,20].Following the initial crossing,the population was advanced to F8generation using the method of single-seed descent.The population (HCS) comprises 300 RILs.

Effects of the major QTL detected in the mapping population was validated in three additional RIL populations including H461 × CN16 (HCN;249 F13lines),H461 × CM107 (HCM;200 F8lines),and H461 × CY20 (HCY;200 F8lines).The GNS of the common parent H461 is significantly higher than those of the other four parents.The HCS population was evaluated in Chongzhou(30°32′N,103°38′E) in 2019 (CZ2019) and 2020 (CZ2020),and in Wenjiang (30°43′N,103°51′E) in 2019 (WJ2019) and 2020(WJ2020).GNS,anthesis date (AD),plant height (PH),spikelet number(SN),grain number per spike(GNPS),and thousand kernel weight(TKW)of the HCN,HCM,and HCY populations were evaluated in CZ2019 and WJ2019.The field arrangements and experimental designs in the four environments were performed as described in our previous studies [16,21].The main shoots of 10 plants of each line were randomly selected to determine GNS,using the spikelets in the center of the spike.AD,PH,SN,GNPS,and TKW were measured as reported in our previous studies [22].

The mean values calculated in each environment were used in further analyses.To minimize environmental effect,the best linear unbiased prediction(BLUP)for all traits in all tested environments was calculated using SAS 8.1 (SAS Institute Inc.,Cary,NC,USA) as reported previously [22,23].The broad-sense heritability of GNS was calculated as:H2=Vg/(Vg+Vge/n+Ve/nr);where,Vg,Vge,andVgare the estimates of variances of genotype,genotype × environment interaction,and random error variances,respectively,whilenandris the number of environments and replicates [24].The differences between the two parents H461 and Chinese Spring were evaluated using a Student’st-test with SPSS 20.0 (IBM Corp.,Armonk,NY,USA).

2.2.Genotyping

Genomic DNA was extracted from single plants for all parental lines and RILs of all the four populations using the CTAB method[25].DNA samples with an A260/280ratio of 1.70–2.10,an A260/230ratio>1.50,and a concentration of>25 ng μL-1were used for genotyping using the wheat 55 K SNP array containing 53,063 markers.Genotyping was conducted by China Golden Marker Biotechnology Co.,Ltd.(Beijing,China).

2.3.Genetic map construction and QTL detection

For linkage map construction,SNPs with a minor allele frequency of <0.3 or missing genotype information of >10% among the 300 RILs in the population HCS were rejected.The retained SNPs were binned based on their segregation patterns among the RILs with the ‘‘Distortion value” at 0.01 using the Bin function in IciMapping 4.2(http://www.isbreeding.net/).A single marker with the least amount of missing data in each bin (bin marker) was selected for linkage map construction.Bin markers were grouped and ordered with IciMapping 4.2 [26]using the Kosambi mapping function [27].Logarithm of odds (LOD) ≥7 was used based on a preliminary analysis of SNPs with LOD scores which ranged from 2 to 10.The flanking sequences of SNPs were blasted against IWGSC RefSeq v1.0 to determine their chromosomal and physical locations (http://www.wheatgenome.org/),and linkage groups were assigned to specific chromosomes accordingly.Where multiple linkage groups were formed for a single chromosome,the genotypic data were further analysed using the MAP function in IciMapping 4.2 [26].This analysis allowed the successful integration of the double linkage groups for chromosomes 2A,3B,4D,and 6D into a single linkage group each.The integrated genetic maps were drawn using MapChart 2.3.2 (http://www.biometris.nl/uk/Software/MapChart/).

2.4.QTL mapping analysis

QTL for GNS were detected using IciMapping 4.2 with the inclusive composite interval mapping[28].To increase the authenticity and reliability of QTL detected,LOD scores were set at ≥3.0 and loci detected in only one environment were removed [29–31].QTL were named according to the rules established by the International Rules of Genetic Nomenclature (http://wheat.pw.usda.gov/ggpages/wgc/98/Intro.htm).‘‘Gns”and‘‘sicau”represent‘‘grain number per spikelet” and ‘‘Sichuan Agricultural University,”respectively.

2.5.Marker development and QTL validation

Tightly linked SNPs for GNS-related QTL were converted to kompetitive allele-specific PCR(KASP)markers.KASP primers were designed and analyzed using the method described previously by Suzuki et al.[16,32].The RILs were grouped into two classes based on their alleles present in parents and their lines:1)lines carrying homozygous alleles from H461(named as‘‘a(chǎn)a”)and 2)lines carrying homozygous alleles from non-H461 parents (named as ‘‘AA”).Student’st-test was used to calculate the differences in the GNS between the classes.

3.Results

3.1.Screening of SNP markers for genetic map construction

With the use of the wheat 55K SNP array,25,563 SNPs were polymorphic between H461 and Chinese Spring.Following the removal of SNPs with a minor allele frequency of <0.3 or missing genotype information of >10%,24,288 of the SNPs were retained and used for the linkage map construction.When the ‘‘Distortion Value” was set at 0.01,21,197 of the SNPs clustered in 3087 bins and the corresponding bin markers were mapped to the wheat genetic map.

3.2.Details of the high-density wheat genetic map

The 3087 bin markers formed 21 linkage groups,corresponding to each of the wheat chromosomes (Fig.S1;Table 1).The cosegregated/redundant markers were integrated into the genetic map.Thus,the genetic map contained 21,197 SNPs spanning 3792.71 cM (Table S2).Of them 7730 (36.47%),8650 (40.81%),and 4817 (22.72%) of the SNP markers were mapped to the A,B,and D subgenomes,respectively.The map lengths of the A,B,and D subgenomes were 1220.52,1079.82,and 1492.37 cM,respectively.The number of markers ranged from 273 (4D) to 2154 (2A) for the 21 chromosomes,with an average of 1009 per chromosome.Chromosome length ranged from 99.60 (Chromosome 1B) to 279.46 cM (Chromosome 7D).The order of markers on the genetic map constructed was highly consistent with the order on the physical map,indicating high accuracy and reliability of the constructed genetic map (Fig.1).

3.3.Phenotypic variation for GNS

The GNS of H461 was significantly(P<0.01)higher than that of Chinese Spring (Table 2).Transgressive segregation was observed in the mapping population (Table 2;Fig.S2).Based on the BLUP values,the broad-sense heritability of GNS was 0.80,indicating strong genetic effects.Strong interaction was detected between GNS and environments (P<0.01),with correlation coefficients ranging from 0.73 to 0.84 (Table 3).Similarly,based on the BLUP values,GNS obtained from the four experiments showed strong correlation (Table 3).

3.4.Identification of QTL conferring GNS

Three QTL were identified for GNS.One of them was located on chromosome 2B and the other two on 2D.Alleles for higher GNS at these three loci were all from the parent H461.Phenotypic variation explained (PVE) by these loci ranged from 3.07% to 26.57%(Table 4).Two QTL were detected across environments.A major and stable QTL,QGns.sicau-2D-2,was located on the long arm of chromosome 2D at 627.83–632.87 Mb (flanking between AX-109316972 and AX-110906716),and the PVE ranged from 19.59%to 26.57%.Another stable QTL,QGns.sicau-2B,was located on the long arm of chromosome 2B at 788.13–792.18 Mb (flanking between AX-108770043 and AX-108927717),and the PVE ranged from 3.32% to 9.36%.Additionally,QGns.sicau-2D-1was detected in two of the four environments,and the PVE ranged from 3.07%to 6.08%,according to the BLUP values.

3.5.Validation of the major and stable QTL,QGns.sicau-2D-2,in different genetic backgrounds and its effect on yield-related traits

To validate the major and stable QTLQGns.sicau-2D-2in different genetic backgrounds,the SNP marker tightly linked to this locus (AX-109316972) was converted into a KASP marker (KASPAX-109316972).This marker detected polymorphism between H461 and the other four parents (Chinese Spring,CN16,CM107,and CY20).In the populations HCN,HCM,and HCY,GNS in lines carrying the‘‘a(chǎn)a”allele was significantly(P<0.01)higher than that in the lines carrying the‘‘AA”allele(Fig.2).The differences ranged from 13.57% to 22.96%,with an average of 17.01% for the three populations (Fig.2).For yield-related traits,QGns.sicau-2D-2had a significant (P<0.01) and positive influence on GNPS,but it showed no effect on AD,PH,or SN in any of the three populations(Table 5).QGns.sicau-2D-2had a significant (P<0.01) and positive influence on TKW only in the HCM population.

4.Discussion

In the present study,an RIL population obtained from a cross between H461 and Chinese Spring was genotyped using the wheat 55K SNP array.H461 is an excellent line with high agronomic performance[16–18].In contrast,Chinese Spring is selected from the landrace Cheng-du-guang-tou and is widely used by the international community for constructing the reference genome for allohexaploid wheat [19,20].Owing to the large difference in the genetic background between them and the redundant number of markers in the wheat 55K SNP array,a large number of polymorphic markers were found between the two parental lines H461 and Chinese Spring.Such a large number of polymorphic markers and the large number of lines (300) in the RIL population under this study significantly improved the power of detecting recombination events,thus the construction of a highly accurate and highdensity genetic map.The map constructed contained 21,197 markers spanning 3792.71 cM across the 21 chromosomes (Table 1;Fig.S1).A comparison of the genetic and physical maps revealed that the orders of the markers on the genetic map was highly consistent with their orders on the physical map(Fig.1),likely because that Chinese Spring is one of the parents for the RIL population.Furthermore,most of the recombination events occurred in the distal portions of the chromosomal arms,whereas only a few occurred around the centromere.This finding was consistent with those of previous studies[11,33,34].In previous studies,the wheat 55K SNP array was used to construct high-density genetic maps for RIL populations[12–14].For example,using a RIL population with 186 lines,a high-density genetic map was constructed with 8225 SNP markers spanning 3593.37 cM [13].Based on this genetic map,a major and stable QTL for stripe rust resistance was identified in a 1.0-cM interval (corresponding to a physical interval of 2.0 Mb between AX-108819274 and AX-110470708) on chromosome arm 7BL.[13].Additionally,Liu et al.[12] constructed a high-density map containing 12,109 SNP markers spanning over 3021.04 cM in a RIL population with 199 lines.Using this genetic map,a novel major QTL for productive tiller(QPtn.sau-4B)number was identified and placed in a 0.55-cM interval,corresponding to a 3.3-Mb physical length[12].Clearly,the wheat 55K SNP array satisfies the purpose of constructing high-density genetic maps that can be successfully used to locate QTL associated with complex traits to small intervals.However,compared with the maps used in these studies,the genetic map constructed and used here based on the H461/Chinese Spring population showed an even higher density,suggesting that shorter QTL intervals could be detected upon further analysis.

GNS is a key determinant of grain yield in wheat.Results from previous studies showed that increased GNS was associated with increase in the expression ofGNI1without any adverse effects on the number of spikes,number of spikelets per spike,nor grain size,thereby increasing yield by increasing grain number [3].Thus,improving GNS might be a promising breeding strategy to enhancegrain yield in wheat.H461 has excellent agronomic performance.Among the main yield component,this variety has high GNS,high grain filling rate,high thousand kernel weight,and large spike size(Table S1)[16–18].In the present study,three QTL conferring GNS were identified.They were located on chromosomes 2B and,2D and 2D,respectively,and alleles increasing GNS for the three loci were all from H461.Among these QTL,QGns.sicau-2D-2is a major and stable QTL located in the interval between 222.96 and 229.64 cM on chromosome 2D.By comparing with findings from previous studies,a major gene (GNI-A1)on chromosome 2A was identified and cloned using a population of recombinant inbred substitution lines derived from a cross between durum wheat cultivar Langdon and the line DIC-2A [3].GNI-D1located at 490,117,188–490,118,827 bp on chromosome 2D is an allele ofGNI-A1.QGns.sicau-2D-2is located between 627.83 and 632.87 Mb on chromosome 2D,indicating it is a novel GNS locus.Similarly,QGns.sicau-2Bis located at 788.13–792.18 Mb on chromosome 2B,which is quite distant fromGNI-B1(at 573,974,813–573,975,706 bp).The different locations suggested thatQGns.sicau-2Bis likely a novel locus.Furthermore,the major QTLQGns.sicau-2D-2showed an average effect of 17.01% and the allele from H461 increased GNS in each of the three different genetic backgrounds (Fig.2;Table 5),indicating a great potential for use in wheat yield improvement.

Table 1 Details of markers in the constructed map.

Table 2 Phenotypic variation and heritability of grain number per spikelet for the parents and RIL population in different environments.

Table 3 Correlation coefficient of grain number per spikelet among different environments in RIL population.

Table 4 Quantitative trait loci for grain number per spikelet identified in different environments and using BLUP values.

Table 5 Effects of QGns.sicau-2D-2 on yield-related traits in the three validation populations based on BLUP values.

Based on the high-quality Chinese Spring reference genome[19],QGns.sicau-2D-2locates in a 5.05-Mb interval containing 72 genes.Clearly,fine mapping for this locus warrants further study.We believe that reducing the interval of this locus for markerdevelopment,fine mapping,and scanning candidate genes should be easier by comparing with populations that lack a Chinese Spring background.This is because that this variety is one of the parents for the mapping population used here and the high-quality reference genome of wheat is based on the use of this genotype.

In summary,a high-density genetic map was constructed using an RIL population with the wheat 55 K SNP array.The genetic map contained 21,197 SNPs spanning 3792.71 cM over 21 chromosomes.Three QTL for GNS were identified,and they explained 3.07%–26.57% of the phenotypic variation.Two of the QTL were identified in all environments as well as using the BLUP values.By comparing with results from previous studies,we found thatone of these loci,QGns.sicau-2D-2,was novel.Based on the developed KASP marker,the effects of this major QTL were validated in three additional genetic backgrounds.This assessment showed that this locus had an average effect of 17.01%.The identified QTL and the developed KASP marker will be valuable in fine mapping loci conferring GNS and for marker-assisted selection in wheat breeding programs.

Fig.2.Effects of QGns.sicau-2D-2 in the three validation populations.(A)HCN population;(B)HCM population;(C)HCY population.aa,lines carrying the homozygous alleles from H461;AA,lines carrying the homozygous alleles from the non-H461 parents;CZ2019,Chongzhou trials conducted in 2019;WJ2019,Wenjiang trials conducted in 2019;BLUP,best linear unbiased prediction.**,Significant at P <0.01.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

CRediT authorship contribution statement

Yu Lin:Methodology,Formal analysis,Validation,Visualization,Writing-original draft,Writing-review&editing.Xiaojun Jiang:Methodology,Formal analysis,Validation,Visualization,Writing -original draft,Writing -review &editing.Haiyan Hu:Formal analysis,Investigation,Resources.Kunyu Zhou:Formal analysis,Investigation,Resources.Qing Wang:Formal analysis,Investigation,Resources.Shifan Yu:Formal analysis,Investigation,Resources.Xilan Yang:Formal analysis,Investigation,Resources.Zhiqiang Wang:Formal analysis,Investigation.Fangkun Wu:Formal analysis,Investigation.Shihang Liu:Formal analysis,Investigation.Caixia Li:Formal analysis,Investigation.Mei Deng:Writing -review &editing.Jian Ma:Writing -review &editing.Guangdeng Chen:Writing -review &editing.Yuming Wei:Resources,Conceptualization,Supervision,Writing -review &editing.Youliang Zheng:Resources,Supervision,Writing-review&editing.Yaxi Liu:Conceptualization,Data curation,Funding acquisition,Project administration,Resources,Writing -original draft,Writing -review &editing.

Acknowledgments

This study was supported by the National Natural Science Foundation of China(31771794),the National Key Research and Development Program of China (2016YFD0101004 and 2017YFD0100900),and the International Science &Technology Cooperation Program of the Bureau of Science and Technology of Chengdu China (2015DFA306002015-GH03-00008-HZ).We thank the anonymous reviewers for critically reviewing the manuscript and for offering valuable suggestions.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.12.006.