Multi-environment BSA-seq using large F3 populations is able to achieve reliable QTL mapping with high power and resolution: An experimental demonstration in rice

Bulked-segregant analysis by deep sequencing(BSA-seq) is a widely used method for mapping QTL(quantitative trait loci) due to its simplicity, speed, cost-effectiveness, and efficiency. However, the ability of BSA-seq to detect QTL is often limited by inappropriate experimental designs, as evidenced by numerous practical studies. Most BSA-seq studies have utilized small to medium-sized populations, with F2populations being the most common choice. Nevertheless, theoretical studies have shown that using a large population with an appropriate pool size can significantly enhance the power and resolution of QTL detection in BSA-seq, with F_(3)populations offering notable advantages over F2populations. To provide an experimental demonstration, we tested the power of BSA-seq to identify QTL controlling days from sowing to heading(DTH) in a 7200-plant rice F_(3)population in two environments, with a pool size of approximately 500. Each experiment identified 34 QTL, an order of magnitude greater than reported in most BSA-seq experiments, of which 23 were detected in both experiments, with 17 of these located near41 previously reported QTL and eight cloned genes known to control DTH in rice. These results indicate that QTL mapping by BSA-seq in large F_(3)populations and multi-environment experiments can achieve high power, resolution, and reliability.

Combining QTL Mapping and Multi-Omics Identify Candidate Genes for Nutritional Quality Traits during Grain Filling Stage in Maize

The nutritional composition and overall quality of maize kernels are largely determined by the key chemical com-ponents:protein,oil,and starch.Nevertheless,the genetic basis underlying these nutritional quality traits during grainfilling remains poorly understood.In this study,the concentrations of protein,oil,and starch were studied in 204 recombinant inbred lines resulting from a cross between DH1M and T877 at four different stages post-pollination.All the traits exhibited considerable phenotypic variation.During the grain-filling stage,the levels of protein and starch content generally increased,whereas oil content decreased,with significant changes observed between 30 and 40 days after pollination.Quantitative trait locus(QTL)mapping was conducted and a total of 32 QTLs,comprising 14,12,and 6 QTLs for grain protein,oil,and starch content were detected,respectively.Few QTLs were consistently detectable across different time points.By integrating QTL analysis,glo-bal gene expression profiling,and comparative genomics,we identified 157,86,and 54 differentially expressed genes harboring nonsynonymous substitutions between the parental lines for grain protein,oil,and starch con-tent,respectively.Subsequent gene function annotation prioritized 15 candidate genes potentially involved in reg-ulating grain quality traits,including those encoding transcription factors(NAC,MADS-box,bZIP,and MYB),cell wall invertase,cellulose-synthase-like protein,cell division cycle protein,trehalase,auxin-responsive factor,and phloem protein 2-A13.Our study offers significant insights into the genetic architecture of maize kernel nutritional quality and identifies promising QTLs and candidate genes,which are crucial for the genetic enhance-ment of these traits in maize breeding programs.

Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.

SLAF marker based QTL mapping of fruit-related traits reveals a major-effect candidate locus ff2.1 for flesh firmness in melon

Flesh firmness(FF) is an important and complex trait for melon breeders and consumers. However, the genetic mechanism underlying FF is unclear. Here, a soft fruit melon(P5) and a hard fruit melon(P10) were crossed to generate F2, and the FF and fruit-related traits were recorded for two years. By performing quantitative trait locus(QTL) specificlocus amplified fragment(SLAF)(QTL-SLAF) sequencing and molecular marker-linkage analysis, 112 844 SLAF markers were identified, and 5 919 SNPs were used to construct a genetic linkage map with a total genetic distance of1 356.49 cM. Ten FF-and fruit-related QTLs were identified. Consistent QTLs were detected for fruit length(FL) and fruit diameter(FD) in both years, and QTLs for single fruit weight(SFW) were detected on two separate chromosomes in both years. For FF, the consistent major locus(ff2.1) was located in a 0.17-Mb candidate region on chromosome 2. Using 429 F2individuals derived from a cross between P5 and P10, we refined the ff2.1 locus to a 28.3-kb region harboring three functional genes. These results provide not only a new candidate QTL for melon FF breeding but also a theoretical foundation for research on the mechanism underlying melon gene function.

Identification of a candidate QTG for seed number per silique by integrating QTL mapping and RNA-seq in Brassica napus L.

Seed number per silique(SNPS)is one of seed yield components in rapeseed,but its genetic mechanism remains elusive.Here a double haploid(DH)population derived from a hybrid between female 6Q006with 35–40 SNPS and male 6W26 with 10–15 SNPS was investigated for SNPS in the year 2017,2018,2019 and 2021,and genotyped with Brassica 60K Illumina Infinium SNP array.An overlapping major QTL(qSNPS.C09)explaining 51.50%of phenotypic variance on average was narrowed to a 0.90 Mb region from 44.87 Mb to 45.77 Mb on chromosome C09 by BSA-seq.Subsequently,two DEGs in this interval were detected between extreme individuals in DH and F_2populations by transcriptome sequencing at7 and 14 days after pollination siliques.Of which,BnaC09g45400D encoded an adenine phosphoribosyltransferase 5(APT5)has a 48-bp InDel variation in the promoter of two parents.Candidate gene association analysis showed that this InDel variation was associated with SNPS in a nature population of rapeseed,where 54 accessions carrying the same haplotype as parent 6Q006 had higher SNPS than103 accessions carrying the same haplotype as parent 6W26.Collectively,the findings are helpful for rapeseed molecular breeding of SNPS,and provide new insight into the genetic and molecular mechanism of SNPS in rapeseed.

Unconditional and Conditional QTL Mapping for Tiller Numbers at Various Stages with Single Segment Substitution Lines in Rice(Oryza sativa L.)

Tiller is one of the most important agronomic traits which influences quantity and quality of effective panicles and finally influences yield in rice. It is important to understand ＂static＂ and ＂dynamic＂ information of the QTLs for tillers in rice. This work was the first time to simultaneously map unconditional and conditional QTLs for tiller numbers at various stages by using single segment substitution lines in rice. Fourteen QTLs for tiller number, distributing on the corresponding substitution segments of chromosomes 1, 2, 3, 4, 6, 7 and 8 were detected. Both the number and the effect of the QTLs for tiller number were various at different stages, from 6 to 9 in the number and from 1.49 to 3.49 in the effect, respectively. Tiller number QTLs expressed in a time order, mainly detected at three stages of 0-7 d, 14-21 d and 35-42 d after transplanting with 6 positive, 9 random and 6 negative expressing QTLs, respectively. Each of the QTLs expressed one time at least during the whole duration of rice. The tiller number at a specific stage was determined by sum of QTL effects estimated by the unconditional method, while the increasing or decreasing number in a given time interval was controlled by the total of QTL effects estimated by the conditional method. These results demonstrated that it is highly effective and accurate for mapping of the QTLs by using single segment substitution lines and the conditional analysis methodology.

QTL mapping in A-genome diploid Asiatic cotton and their congruence analysis with AD-genome tetraploid cotton in genus Gossypium

Asiatic cotton （Gossypium arboreum L.） is an Old World cultivated cotton species. The sinense race was planted extensively in China. Due to the advances in spinning technology during the last century, the species was replaced by the New World allotetraploid cotton G. hirsutum L. Gossypium arboreum is still grown in India and Pakistan and also used as an elite in current cotton breeding programs. In addition, G. arboreum serves as a model for genomic research in Gossypium. In the present study, we generated an A-genome diploid cotton intraspecific genetic map including 264 SSR loci with three morphological markers mapped to 13 linkage groups. The map spans 2,508.71 cM with an average distance of 9.4 cM between adjacent loci. A population containing 176 F2：3 families was used to perform quantitative trait loci （QTL） mapping for 17 phenotypes using Multiple QTL Model （MQM） of MapQTL ver 5.0. Overall, 108 QTLs were detected on 13 chromosomes. Thirty-one QTLs for yield and its components were detected in the F2 population. Forty-one QTLs for yield and its components were detected in the F2：3 families with a total of 43 QTLs for fiber qualities. Two QTLs for seed cotton weight/plant and lint index and three QTLs for seed index were consistently detected both in F2 and F2：3. Most QTLs for fiber qualities and yields were located at the same interval or neighboring intervals. These results indicated that the negative correlation between fiber qualities and yield traits may result from either pleiotropic effect of one gene or linkage effects of multiple closely linked genes.

QTL Mapping for Cucumber Fruit Size and Shape with Populations from Long and Round Fruited Inbred Lines

To gain insight into the molecular and genetic basis of fruit size and shape in cucumber,we conducted Quantitative Trait Locus(QTL)mapping with F2 and BC1F1 populations derived froma cross between the Northern-China type inbred line CNS21 and the Southern-China type inbred line RNS7.Populations were evaluated during two developmental stages,ovary at anthesis and commercial fruit(immature fruit).A total of 13 major-effect QTLs(R2>10%)were detected for six traits,and one of them,fruit shape index 2.1,explained more than 50%of phenotypic variation.All QTLs distributed on chromosome(chr)1 and 2.We constructed a physical map containing almost all of the QTLs with their physical location from previous studies.For fruit size and shape,the highest number of QTLs were mapped on chr1 and chr6,and the fewest number were mapped on chr2 and chr7.At least one consensus region was presented on each chromosome.In addition,three candidate genes were predicted between the long-fruited and round-fruited inbred lines by comparing the sequences of the consensus region of chr3,where many QTLs for marketable fruit length had been detected.Our results provide a base for additional QTLs and molecular markers for fruit size and shape in cucumber breeding.

Conditional QTL Mapping of Sedimentation Volume on Seven Quality Traits in Common Wheat

To evaluate the possible genetic interrelationships between flour components and the sedimentation volume（SD）,a doubled haploid（DH） population comprising 168 lines were used to identify the conditional quantitative trait loci（QTLs） for SD in three environments.Ten additive QTLs and 15 pairs of epistatic QTLs were detected for SD through unconditional and conditional QTL mapping.Three major additive QTLs were detected for SD conditioned on the seven quality traits.Two additive QTLs were found to be independent of these traits.Three additive QTLs were suppressed by three of the seven traits because of non-detection in unconditional mapping.Three pairs of epistatic QTLs were completely affected by the seven traits because of detection in unconditional mapping but no-detection in conditional mapping.Twelve pairs of epistatic QTLs were detected in conditional mapping.Our results indicated that conditional mapping could contribute to a better understanding of the interdependence of different and closely correlated traits at the QTL molecular level,especially some minor QTLs were found.The conditional mapping approach provides new insights that will make it possible to avoid the disadvantages of different traits by breeding through molecular design.

QTL mapping of general combining abilities of four traits in maize using a high-density genetic map

General combining abilities （GCAs） are very important in utilization of heterosis in maize breeding. However, its genetic basis is unclear. In the present study, a set of 118 doubled haploid （DH） lines were induced from F1 generations produced from the cross between the inbred line Zheng 58 and the inbred line W499 belonging to the Reid subgroup. Using the MaizeSNP50 BeadChip, a high-density genetic map was constructed based on the DH population which included 1 147 bin markers with an average interval length of 2.00 cM. Meanwhile, the DH population was crossed with three testers including W16-5, HD568, and W556, which belong to the Sipingtou subgroup. The GCAs of the ear height （EH）, the kernel moisture content （KMC）, the kernel ratio （KR）, and the yield per plant （YPP） were estimated using these hybrids in three environments. Combining the high-density genetic map and the GCAs, a total of 14 QTLs were detected for the GCAs of the four traits. Especially, one pleiotropic QTL was identified on chromosome 1 between the SNP SYN16067 and the SNP PZE-101169244 which was simultaneously associated with the GCAs of the EH, the KR, and the YPP. These QTLs pave the way for further dissecting the genetic architecture underlying GCAs of the traits, and they may be used to enhance GCAs of inbred lines under the fixed heterotic pattern ReidxSipingtou in China through a marker-assisted selection approach.

Genetic Analysis and QTL Mapping of Mature Seed Culturability in Indica Rice

Genetic segregation analysis for mature seed culturability was conducted using recombinant inbred lines derived from a cross of indica rice, Yangdao 6 and Pei＇ai 64s. Three indices of seed culturability, the frequency of callus induction, the frequency of brown callus and the increase of callus weight were investigated. A combined genetic map constructed with simple sequence repeat （SSR）, sequence tag site （STS）, cleaved amplified polymorphic sequences （CAPS） and single nucleotide polymorphism （SNP） markers covered a total distance of 1 732.5 cM, averaging approximately 12 cM between two neighboring loci. Three QTLs on chromosomes 7, 7 and 10 were detected for the frequency of callus induction; three QTLs on chromosomes 6, 7 and 9 were detected for the frequency of brown callus; and two QTLs on chromosomes 5 and 7 were detected for the increase of callus weight. Common QTLs mapped at the interval flanking RM5481 and RM6835 on chromosome 7 were identified to be involved in the frequency of callus induction and the frequency of brown callus, explaining 7.29% and 12.52% of phenotypic variation, respectively. A total of 14 epistatic effects were detected for the three indices of mature seed culturability. ~

QTL mapping of quality traits in peanut using whole-genome resequencing

Oil and protein content and fatty acid composition are quality traits in peanut.Elucidating the genetic mechanisms underlying these traits may help researchers to obtain improved cultivars by molecular breeding.Whole-genome resequencing of a recombinant inbred population of 318 lines was performed to construct a high-density linkage map and identify QTL for peanut quality.The map,containing 4561 bin markers,covered 2032 c M with a mean marker density of 0.45 c M.A total of 110 QTL for oil and protein content,and fatty acid composition were mapped on the 18 peanut chromosomes.The QTL q A05.1 was detected in four environments and showed a major phenotypic effect on the contents of oil,protein,and six fatty acids.The genomic region spanned by q A05.1,corresponding to a physical interval of approximately 1.5 Mb,contains two SNPs polymorphic between the parents that could cause missense mutations.The two SNP sites were employed as KASP markers and validated using lines with extremely high and low oil contents.These sites may be useful in the marker-assisted breeding of peanut cultivars with high oil contents.

First identification of two co-existing genome-wide significant sex quantitative trait loci(QTL) in red tilapia using integrative QTL mapping

Red tilapia(Oreochromis spp.)is one of the most popular fish in China due to its bright red appearance,fast growth rate,and strong adaptability.Understanding the sex determination mechanisms is of vital importance for the selection of all-male lines to increase aquacultural production of red tilapia.In this research,the genetic architecture for sex from four mapping populations(n=1090)of red tilapia was analyzed by quantitative trait loci(QTL)-seq,linkage-based QTL mapping,and linkage disequilibrium(LD)-based genome-wide association studies.Two genome-wide significant QTL intervals associated with sex were identified on ChrLG1(22.4-23.9 Mb)and ChrLG23(32.0-35.9 Mb),respectively.The QTL on ChrLG1 was detected in family 1(FAM1),FAM2,and FAM4,and the other QTL on ChrLG23 was detected in FAM3 and FAM4.Four microsatellite markers located within the QTL were successfully developed for marker-assisted selection.Interestingly,three(Ipp,sox14,and amh)of the 12 candidate genes located near or on the two QTL intervals were abundantly expressed in males,while the remaining genes were more highly expressed in females.Seven genes(scly,ube3a,Ipp,gpr17,oca2,cog4,and atp10a)were significantly differentially expressed between the male and female groups.Furthermore,LD block analysis suggested that a cluster of genes on ChrLG23 may participate in regulating sex development in red tilapia.Our study provides important information on the genetic architecture of sex in red tilapia and should facilitate further exploration of sex determination mechanisms in this species.

QTL Mapping for Fiber Quality Traits Based on a Dense Genetic Linkage Map with SSR,TRAP,SRAP and AFLP Markers in Cultivated Tetraploid Cotton

Cotton is one of the most important economic crops in the world,and it provides natural fiber for the textile industry.With the advancement of the textile technology and increased consumption demands on cotton fiber,both cotton yield and quality should be enhanced.However,cotton yield

QTL mapping of agronomic and economic traits for four F_(2)populations of upland cotton

Background:Upland cotton(Gossypium hirsutum)accounts for more than 90%of the annual world cotton output because of its high yield potential.However,yield and fiber quality traits often show negative correlations.We constructed four F_(2)populations of upland cotton,using two normal lines(4133B and SGK9708)with high yield potential but moderate fiber quality and two introgression lines(Suyuan04–3 and J02–247)with superior fiber quality,and used them to investigate the genetic basis underlying complex traits such as yield and fiber quality in upland cotton.We also phenotyped eight agronomic and economic traits and mapped quantitative trait loci(QTLs).Results:Extensive phenotype variations and transgressive segregation were found across the segregation populations.We constructed four genetic maps of 585.97 centiMorgan(cM),752.45 cM,752.45 cM,and 1163.66 cM,one for each of the four F_(2)populations.Fifty QTLs were identified across the four populations(7 for plant height,27 for fiber quality and 16 for yield).The same QTLs were identified in different populations,including qBW4 and qBW2,which were linked to a common simple sequence repeat(SSR)marker,NAU1255.A QTL cluster containing eight QTLs for six different traits was characterized on linkage group 9 of the 4133B×Suyuan04–3 population.Conclusions:These findings will provide insights into the genetic basis of simultaneous improvement of yield and fiber quality in upland cotton breeding.

Prepulse inhibition (PPI) of tactile startle response in recombinant congenic strains of mice:QTL mapping and comparison with acoustic PPI

Prepulse inhibition （PPI） of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems. We analyzed the tactile startle response （TSR） and PPI of TSR （tPPI）, using light as a prepulse stimulus, in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them. Parental strains were significantly different for TSR, but were comparable for tPPI. Among the congenic strains, variation for TSR was significant in both genetic backgrounds, but that of tPPI was significant only for the C57BL/6J background. Provisional mapping for loci modulating TSR and tPPI was carried out. Using mapping data from our previous study on acoustic startle responses （ASR） and PPI of ASR （aPPI）, no common markers for aPPI and tPPI were identified. However, some markers were significantly associated with both ASR and TSR, at least in one genetic background. These results indicate cross-modal genetic regulation for the startle response but not for PPI, in these mouse strains.

Discovering Candidate Chromosomal Regions Linked to Kernel Size-Related Traits via QTL Mapping and Bulked Sample Analysis in Maize

Kernel size-related traits,including kernel length,kernel width,and kernel thickness,are critical components in determining yield and kernel quality in maize(Zea mays L.).Dissecting the phenotypic characteristics of these traits,and discovering the candidate chromosomal regions for these traits,are of potential importance for maize yield and quality improvement.In this study,a total of 139 F2:3 family lines derived from EHel and B73,a distinct line with extremely low ear height(EHel),was used for phenotyping and QTL mapping of three kernel sizerelated traits,including 10-kernel length(KL),10-kernel width(KWid),and 10-kernel thickness(KT).The results showed that only one QTL for KWid,i.e.,qKWid9 on Chr9,with a phenotypic variation explained(PVE)of 13.4%was detected between SNPs of AX-86298371 and AX-86298372,while no QTLs were detected for KL and KT across all 10 chromosomes.Four bulked groups of family lines,i.e.,Groups I to IV,were constructed with F2:3 family lines according to the phenotypic comparisons of KWid between EHel and B73.Among these four groups,Group I possessed a significantly lower KWid than EHel(P=0.0455),Group II was similar to EHel(P=0.34),while both Group III and Group IV were statistically higher than EHel(P<0.05).Besides,except Group IV exhibited a similar KWid to B73(P=0.11),KWid of Groups I to III were statistically lower than B73(P<0.00).By comparing the bulked genotypes of the four groups to EHel and B73,a stable chromosomal region on Chr9 between SNPs of AX-86298372 to AX-86263154,entirely covered by qKWid9,was identified to link KWid with the positive allele of increasing phenotypic effect to KWid from B73,similar to that of qKWid9.A large amount of enzyme activity and macromolecule binding-related genes were annotated within this chromosomal region,suggesting qKWid9 as a potential QTL for KWid in maize.

Constructing Molecular Marker Linkage Maps of Chromosome 14Sh and 22Sh and QTL Mapping for Major Traits by Use of Substitution Lines of Gossypium hirsutum L.

CSB14Sh,which is isogenic for its recurrent parent TM-1 except for chromosome 14 short arm,was crossed with TM-1,and the F2 population was produced.A total of 3800 SSR primer pairs covering the whole genome were used to screen polymorphism among two parents,TM-1 and CSB14Sh,

Genetic analysis and QTL mapping of growth period traits and plant height traits in soybean recombinant inbred lines from Dongnong 47 × PI 317334-B

High and stable yield is the main goal of soybean genetic improvement.In this study,association analysis was used to detect the quantitative trait loci(QTL)for the plant height,and soybean growth period using 182 SSR markers in the RIL population of 136 F_(4:8) lines,which developed from a cross between photoperiod-insensitive cultivar‘Dongnong 47’and photoperiod-sensitive variety PI317334–B.The results showed that 33 QTLs related to soybean growth period and plant height traits were detected by compound interval mapping,and were located on 12 linkage groups including N,C1,C2,J,D1a,B2,E,G,A2,O,L,I,with the contribution rate of 7.85–33.84%.These QTL loci and linkage markers related to soybean photoperiod sensitivity,would be helpful to identify key genes that control soybean photoperiod sensitivity,and provide an important basis for the breeding of new photoperiod-insensitive soybean varieties based on molecular design breeding.