Fine mapping of two recessive genes TaFLA1 and TaSPL8 controlling flag leaf angle in bread wheat

Flag leaf angle is one of the key target traits in high yield wheat breeding.A smaller flag leaf angle reduces shading and enables plants to grow at a higher density,which increases yield.Here we identified a mutant,je0407,with an 84.34%-89.35%smaller flag leaf angle compared with the wild type.The mutant also had an abnormal lamina joint and no ligule or auricle.Genetic analysis indicated that the ligule was controlled by two recessive genes,which were mapped to chromosomes 2AS and 2DL.The mutant allele on chromosome 2AS was named Tafla1b,and it was fine mapped to a 1 Mb physical interval.The mutant allele on chr.2DL was identified as Taspl8b,a novel allele of TaSPL8 with a missense mutation in the second exon,which was used to develop a cleaved amplified polymorphic sequence marker.F3 and F4 lines derived from crosses between Jing411 and je0407 were genotyped to investigate interactions between the Tafla1b and Taspl8b alleles.Plants with the Tafla1b/Taspl8a genotype had 58.41%-82.76%smaller flag leaf angles,6.4%-24.9%shorter spikes,and a greater spikelet density(0.382 more spikelets per cm)compared with the wild type.Plants with the Tafla1a/Taspl8b genotype had 52.62%-82.24%smaller flag leaf angles and no differences in plant height or spikelet density compared with the wild type.Tafla1b/Taspl8b plants produced erect leaves with an abnormal lamina joint.The two alleles had dosage effects on ligule formation and flag leaf angle,but no significant effect on thousand-grain weight.The mutant alleles provide novel resources for improvement of wheat plant architecture.

Fine mapping and cloning of the sterility gene Bra2Ms in nonheading Chinese cabbage(Brassica rapa ssp.chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage(Brassica rapa ssp.chinensis).However,the molecular mechanisms underlying male sterility in B.rapa are still largely unclear.We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus,Bra2Ms,on the A2 chromosome.Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation.Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B.rapa.The Bra039753 gene encodes a plant homeodomain(PHD)-finger protein and is considered a potential candidate gene for Bra2Ms.Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage.Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants,which led to an amino acid insertion in the Bra039753 protein.In addition,the 369 bp fragment insertion was found to cosegregate with the male sterility trait.This study identified a novel locus related to male sterility in non-heading Chinese cabbage,and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Fine mapping of the powdery mildew resistance gene PmXQ-0508 in bread wheat

In a wheat breeding line XQ-0508 showing consistent resistance to powdery mildew disease,a recessive gene,designated PmXQ-0508,was identified and mapped to a distal region on chromosome arm 2BS.Of three resistance-associated genes in this region,one encoding a protein kinase was selected as the primary candidate for PmXQ-0508.Ten closely linked DNA markers developed in the study could be used for marker-assisted selection for powdery-mildew resistance in breeding programs.

Fine mapping and validation of a stable QTL for thousand-kernel weight in wheat(Triticum aestivum L.)

Thousand-kernel weight(TKW)is a measure of grain weight,a target of wheat breeding.The object of this study was to fine-map a stable quantitative trait loci(QTL)for TKW and identify its candidate gene in a recombinant inbred line(RIL)population derived from the cross of Kenong 9204(KN9204)and Jing411(J411).On a high-density genetic linkage map,24,26 and 25 QTL were associated with TKW,kernel length(KL),and kernel width(KW),respectively.A major and stable QTL,QTkw-2D,was mapped to an8.3 cM interval on chromosome arm 2DL.By saturation of polymorphic markers in its target region,QTkw-2D was confined to a 9.13 Mb physical interval using a secondary mapping population derived from a residually heterozygous line(F6:7).This interval was further narrowed to 2.52 Mb using QTkw-2D near-isogenic lines(NILs).NILs~(KN9204)had higher fresh and dry weights than NILsJ411at various grain-filling stages.The TKW and KW of NILs~(KN9204)were much higher than those of NILsJ411in field trials.By comparison of both DNA sequence and expression between KN9204 and J411,TraesCS2D02G460300.1(TraesKN2D01HG49350)was assigned as a candidate gene for QTkw-2D.This was confirmed by RNA sequencing(RNA-seq)of QTkw-2D NILs.These results provide the basis of map-based cloning of QTkw-2D,and DNA markers linked to the candidate gene may be used in marker-assisted selection.

Fine Mapping of AST Gene in Arabidopsis

The ast ( anthocyanin spotted testa) mutant, which was induced by carbon ion radiation, was a single recessive gene mutant of Arabidopsis thaliana (L.) Heynh. with spotted pigment in seed coats, and involved in the anthocyanin biosynthesis. To clone the AST gene by map-based cloning strategy, a series of molecular markers were designed according to the SNPs (single nucleotide polymophisms) and insertion/deletion polymophisms in the Arabidopsis database. With these molecular markers, the fine-structure mapping of the AST gene was finished, the AST locus was located in BAC clone T13M11. It was suggested that the AST candidate gene was T13M11. 8 in the T13M11 This gene was 1432 bp long with 6 exons and 5 introns. The putative protein of T13M11. 8 gene was similar to dihydroflavonol 4-reductase (DFR), which was an important enzyme in the anthocyanin biosynthesis pathway.

Fine mapping and marker-assisted selection (MAS) of a low glutelin content gene in rice

Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. Following an SDS-PAGE screen of rice germplasm from Taihu Valley of China, Japonica selection W3660 is identified to be a novel mutant characterized with low glutelin content. For fine mapping the mutant gene for low glutelin content, F2 and F3 populations were derived from a cross between W3660 and Jingrennuo. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The dis- tances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. By semi-quantitative RT-PCR analysis, the transcripts of GluB4/GluB5 genes located within the region do not change. However, GluB5 gene located proximal to SSR2-001/SSR2-004 was specifically reduced. SSR profiles of seven Japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker- assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively. This provides a standard starting point for the breeding of low glutelin content rice varieties in China.

Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice(Oryza sativa)

A thermo-insensitive pale green leaf mutant （pgl2） was isolated from T-DNA inserted transgenic lines of rice （Oryza sativa L. subsp, japonica cv. Nipponbare）. Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2gene, an F2 population was constructed by crossing the mutant with Longtefu （Oryza sativa L. subsp, indica）. The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll （Chl） content, while the ratio of Chl a / Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.

Fine Mapping of C（Chromogen for Anthocyanin） Gene in Rice

Seven residual heterozygous lines （RHLs） displaying different genotypic compositions in the genomic region covering probable locations of C （Chromogen for anthocyanin） gene on the short arm of rice chromosome 6 were selected from the progenies of the indica cross Zhenshan 97B/Milyang 46. Seeds were harvested from each of the seven plants, and the resultant F2：3 populations were used for fine mapping of C gene. It was shown in the populations that the apiculus coloration matched to basal leaf sheath coloration in each plant. By relating the coloration performances of the populations with the genotypic compositions of the RHLs, the C locus was located between rice SSR markers RM314 and RM253. By using a total of 1279 F2：3 individuals from two populations showing coloration segregation, the C locus was then located between RM111 and RM253, with genetic distances of 0.7 cM to RM111 and 0.4 cM to RM253. Twenty-two recombinants found in the two populations were assayed with seven more markers located between RM111 and RM253, including six SSR markers and one marker for the C gene candidate, OsCl. The C locus was delimited to a 59.3-kb region in which OsC1 was located.

Phenotype of Rice Floury Endosperm Mutant flo7 and Fine Mapping of Mutated Gene

The major storage substance in rice endosperm is starch, which accounts for 80% of dry matter weight. In this study, rice mutant flo7, selected from the progeny of Nipponbare＇s tissue culture, displayed floury and opaque endosperm. Compared with its corresponding wild type （WT） Nipponbare, the mutant rio7 produced longer, narrower, thinner and lighter grains. The levels of glucose, fructose and sucrose in the mutant flo7 endosperm were higher than those in the WT endosperm, whereas the protein content was not affected. With respect to both amylose content and gel consistency, the mutant flo7 was lower than WT, but its alkali value was higher. Scanning electron microscopic examinations showed that the endosperm of the mutant flo7 contained irregular, loosely packed and compound starch granules. Genetic analysis indicated that the mutant phenotype was determined by a single recessive nuclear gene The flo7 locus was mapped to a region on the long arm of chromosome 12, within a 95.1 kb interval defined by the markers C2-11 and C5-15. There are 13 open reading frames in the mapping interval. Transcription profiling of the developing grains showed that a number of genes involved in starch synthesis were affected differently in the mutant flo7.

Fine mapping and genetic analysis of resistance genes,Rsc18,against soybean mosaic virus

Soybean mosaic virus(SMV) affects seed quality and production of soybean(Glycine max(L.) Merr.) worldwide.SC18 is one of the dominant SMV strains in South China,and accession Zhonghuang 24 displayed resistance to SC18.The F_(1),F_(2) and 168 F_(11) recombinant inbred lines(RILs) population derived from a hybridization between Zhonghuang 24(resistant,R) and Huaxia 3(susceptible,S) were used in this study.According to the segregation ratios of the F_(2) generation(3 R:1 S) and the recombinant inbred lines(RILs) population(1 R:1 S),one dominant locus may regulate the resistance to SC18 in Zhonghuang 24.By using composite interval mapping(CIM),Rsc18 was mapped to a 415.357-kb region on chromosome 13.Three candidate genes,including one NBS-LRR type gene and two serine/threonine protein type genes,were identified according to the genetic annotations,which may be related to the resistance to SC18.The q RT-PCR demonstrated that these genes were up-regulated in the R genotype compared to the control.In conclusion,the findings of this research enhanced the understanding about the R genes at the Rsc18 locus.Moreover,our results will provide insights for designing molecular markers to improve marker-assisted selection and developing new varieties with resistance to SC18.

Fine Mapping QTLs Affecting Milk Production Traits on BTA6 in Chinese Holstein with SNP Markers

Our previous studies demonstrated that the region around markers BMS470 and BMS1242 on BTA6 showed a linkage to 305-d milk yield and composition traits in the Chinese Holstein population. We herein focused on such narrow region to fine map milk production QTLs with 15 SNPs across 25 Mb with each SNP in 1 Mb within most regions in a Chinese Holstein population with daughter design. 1 449 Holstein cows and 11 sires were genotyped for such SNPs by using TaqMan probe and RFLP assays. Multipoint linkage analysis across family revealed a QTL affecting milk yield between PPARGC1A C4075T and SLC34A2 T1713C. Meanwhile, within family analysis found three milk yield QTLs （two in CR T60984131G-CEP135 C501T and one in PDLIM5 A106C-OPN T3907, a fat yield QTLin UGDH T1670C-CR T60984131G region, and two protein yield QTLs in TBC1D1 G501C-UGDH T1670C and PPARGC1A C4075T-SLC34A2 T1713C, respectively. Associations between aforementioned significant SNP markers and milk production traits were further implemented. We found significant associations of PPARGC1A C4075T, SLC34A2 T1713C with milk yield （P0.05, P0.01, P0.01）, UGDH T1670C, and CR T60984131G with fat yield （P0.01, P0.01）, and PPARGC1A C4075T, SLC34A2 T1713C, UGDH T1670C and OPN T3907 with protein yield （P0.01, P0.01, P0.01, P0.01）. Our findings implied that QTLs affecting milk production traits on BTA6 were pleictropism or multigenic effect and PPARGC1A and OPN may be the causal mutations behind milk production QTLs on BTA6 in the Chinese Holstein population.

Identification and Fine Mapping of Heading Date Related Mutant Gene in Rice

A rice heading-date-related mutant was isolated from a ^60Co-y-ray-induced mutation pool of Zhejing 22, a conventional japonica cultivar in Zhejiang Province, China. The mutant was characterized by a delayed heading date of almost 20 d longer than the wild type plant. Genetic analysis revealed that the mutation was controlled by a single nuclear-encoded recessive gene that was designed as HD（t） （heading date tentatively）. To isolate the HD（t） gene, a map-based cloning approach was employed using 479 F2 mutant individual plants derived from the cross between the hd（t） mutant （japonica） x Zhenshan 97 （indica）. Finally, the HD（t） gene was mapped to an approximate 53 kb region between the insertion and deletion （InDel） markers of 10-61W and 10-66W on chromosome 10. According to the genome sequence of Nipponbare, the target region contains 11 annotated genes. It is helpful for future cloning of HD（t） gene based on this fine mapping results.

Phenotypic characterization and fine mapping of mps1,a premature leaf senescence mutant in rice (Oryza sativa L.)

Leaves play a key role in photosynthesis in rice plants. The premature senescence of such plants directly reduces the accumulation of photosynthetic products and also affects yield and grain quality significantly and negatively. A novel premature senescence mutant, mps1（mid-late stage premature senescence 1）, was identified from a mutant library consisting of ethyl methane sulfonate（EMS） induced descendants of Jinhui 10, an elite indica restorer line of rice. The mutant allele, mps1, caused no phenotypic differences from the wild type（WT）, Jinhui 10, but drove the leaves to turn yellow when mutant plants grew to the tillering stage, and accelerated leaf senescence from the filling stage to final maturation. We characterized the agronomic traits, content of photosynthetic pigments and photosynthetic efficiency of mps1 and WT, and fine-mapped MPS1. The results showed that the MPS1-drove premature phenotype appeared initially on the leaf tips at the late tillering stage and extended to the middle of leaves during the maturing stage. Compared to the WT, significant differences were observed among traits of the number of grains per panicle（–31.7%） and effective number of grains per panicle（–38.5%） of mps1 individuals. Chlorophyll contents among the first leaf from the top（Top 1st）, the second leaf from the top（Top 2nd） and the third leaf from the top（Top 3rd） of mps1 were significantly lower than those of WT（P〈0.05）, and the levels of photosynthetic efficiency from Top 1st to the forth leaf from the top（Top 4th） of mps1 were significantly lower than those of WT（P〈0.01）. Results from the genetic analysis indicated that the premature senescence of mps1 is controlled by a recessive nuclear gene, and this locus, MPS1 is located in a 37.4-kb physical interval between the markers Indel145 and Indel149 on chromosome 6. Genomic annotation suggested eight open reading frames（ORFs） within this physical region. All of these results will provide informative references for the further researches involving functional analyses and molecular mechanism exploring of MPS1 in rice.

Fine Mapping and Candidate Gene Prediction of the Quantitative Trait Locus qPL8 for Panicle Length in Rice

Rice panicle is the sink organ where assimilation product accumulates,and its morphology determines the rice yield.Panicle length has been suggested as a yield-related trait,but the genetic factor for its control is still limited.In this study,we carried out fine-mapping of qPL8,a QTL identified for panicle length in our previous work.Near isogenic line(NIL)with qPL8 exhibited elongated panicle without obvious effect on other panicle elements.With five key recombinants from NIL population,the locus was finally narrowed down to a 278-kb region,where 44 genes are annotated.By comparing the genomic sequence of two parents,17 genes were identified with SNPs or InDels variations in the coding region.Expression analysis showed that eight genes were up-regulated in the NIL with qPL8.Considering both the coding variation and expression status,several candidate genes for the locus were identified,and OsMADS37 was raised as the most possible candidate.Interestingly,an expression QTL(eQTL)also resides in the locus,leading to a cluster of gene expression variation in the region.This study will facilitate the application of qPL8 locus in rice breeding for yield potential.

Fine mapping of an adult-plant resistance gene to powdery mildew in soybean cultivar Zhonghuang 24

Powdery mildew(PM),caused by the fungus Microsphaera diffusa,causes severe yield losses in soybean[Glycine max(L.)Merr.]under suitable environmental conditions.Identifying resistance genes and developing resistant cultivars may prevent soybean PM damage.In this study,analysis of F_(1),F_(2),and F8:11 recombinant inbred line(RIL)populations derived from the cross between Zhonghuang 24(ZH24)and Huaxia 3(HX3)indicated that adult-plant resistance(APR)to powdery mildew in the soybean cultivar(cv.)ZH24 was controlled by a single dominant locus.A high-density genetic linkage map of the RIL population was used for fine mapping.The APR locus in ZH24 was mapped to a 281-kb genomic region on chromosome 16.Using 283 susceptible plants of another F2 population,the candidate region was finemapped to a 32.8-kb genomic interval flanked by the markers InDel14 and Gm16_428.The interval harbored five genes,including four disease resistance(R)-like genes,according to the Williams 82.a2.v1 reference genome.Quantitative real-time PCR assays of candidate genes revealed that the expression levels of Glyma.16g214300 and Glyma.16g214500 were changed by M.diffusa infection and might be involved in disease defense.Rmd_B13 showed all-stage resistance(ASR)to PM in soybean cv.B13.An allelism test in the F2 segregating population from the cross of ZH24 × B13 suggested that the APR locus Rmd_ZH24 and the ASR locus Rmd_B13 may be allelic or tightly linked.These results provide a reference marker-assisted selection in breeding programs.

Fine mapping of a novel wax crystal-sparse leaf3 gene in rice

Cuticular wax plays an important role in protecting plants against water loss and pathogen infection and in the adaptations to environmental stresses. The genetic mechanism of the biosynthesis and accumulation of epicuticular wax in rice remains largely unknown. Here, we show a spontaneous mutant displaying wax crystal-sparse leaves and decreased content of epicuticular wax that was derived from the cytoplasmic male sterility （CMS） restorer line Zhenhui 714. Compared with the wild type Zhenhui 714, the mutant exhibited hydrophilic features on leaf surface and more sensitivity to drought stress. The mutation also caused lower grain number per panicle and thousand grain weight, leading to the decline of yield. Genetic analysis indicates that the mutation is controlled by a single recessive gene, named wax crystal-sparse leaf3 （wsl3）. Using segregation populations derived from crosses of mutant/Zhendao 88 and mutant/Wuyujing 3, respectively, the wsl3 gene was fine-mapped to a 110-kb region between markers c3-16 and c3-22 on chromosome 3. According to the rice reference genome and gene analysis, we conclude that a novel gene/mechanism involved in regulation of rice cuticular wax formation.

Fine mapping and candidate gene analysis of qHD1b,a QTL that promotes flowering in common wild rice(Oryza rufipogon)by up-regulating Ehd1

Heading date(flowering time)determines the adaptability of cultivars to different environments.We report the fine mapping and candidate gene analysis of qHD1b,a quantitative trait locus(QTL)responsible for early flowering that was derived from common wild rice(O.rufipogon)under both short-day and longday conditions.The introgression line IL7391,which carried segments from common wild rice in a Zhonghui 8015(ZH8015)background,exhibited early heading compared to the background and was crossed with ZH8015 to generate BC_(5)F_(2:3) families for QTL analysis.This enabled the identification of two heading-date QTL,named qHD1b and qHD7,of which the first was selected for further research.High-resolution linkage analysis was performed in BC_(5)F_(4:5) and BC_(5)F_(6) populations,and the location of qHD1b was confined to a 112.7-kb interval containing 17 predicted genes.Five of these genes contained polymorphisms in the promoter or coding regions and were thus considered as candidates.Expression analysis revealed a positive association between LOC_Os01g11940 expression and early heading.This locus was annotated as OsFTL1,which encodes an ortholog of Arabidopsis Flowering Locus T and was the most likely candidate gene for qHD1b.Our study revealed that qHD1b acts as a floral activator that promotes flowering by up-regulating Ehd1,Hd3a,RFT1,OsMADS14,and OsMADS15 under both shortday and long-day conditions.Field experiments showed that qHD1b affected several yield-related agronomic traits including 1000-grain weight and grain length.qHD1b could be useful for marker-assisted selection and breeding of early-maturing cultivars.

Characterization and Fine Mapping of Non-panicle Mutant (nop) in Rice

A mutant of panicle differentiation in rice called non-panicle （nop） was discovered in the progeny of a cross between 93-11 and Nipponbare. The mutant exhibits normal plant morphology but has apparently few tillers. The most striking change in nop is that its panicle differentiation is blocked, with masses of fluffy bract nodes generate from the positions where rachis branches normally develop in wild-type plants. Genetic analysis suggests that nop is controlled by a single recessive gene, which is temporarily named Nop（t）. Based on its mutant phenotype, Nop（t） represents a key gene controlling the initiation of inflorescence differentiation, By using simple sequence repeat markers and sequence tagged site markers, Nop（t） gene was fine mapped in a 102-kb interval on the long arm of chromosome 6. These results will facilitate the positional cloning and functional studies of the gene.

An Entropy-based Index for Fine-scale Mapping of Disease Genes

By comparing the entropy and the conditional entropy in a marker, an entropy-based index has been presented for fine-scale linkage disequilibrium gene mapping using high-density marker maps for human disease genes. The index can quantify the level of linkage disequilibrium （LD） between the marker and the disease susceptibility locus （DSL） of genes. The advantage of using the index is attributed to the fact that it does not depend on marker allele frequencies across loci. Moreover, it is parallel to Hardy-Weinberg disequilibrium （HWD） measure for DSL fine mapping. Through various simulations, the fine mapping perform- ances of the proposed entropy-based index was extensively investigated under various genetic parameters. The results show that the index presented is both robust and powerful for DSL mapping in genes.

An Entropy-based Index for Fine-scale Mapping of QTL

By comparing the entropy and conditional entropy in a marker, an entropy-based index for fine-scale linkage-disequilibrium gene mapping is presented using high-density marker maps in extreme samples for quantitative trait. The entropy-based index is the function of LD between the marker and the traitlocus and does not depend on marker allele frequencies across the loci. It is parallel to Hardy-Weinberg disequilibrium （HWD） measure for QTL fine mapping, but its power of fine mapping QTL is higher than that of HWD measure. Through simulations, the fine mapping performance of this entropy-based index is investigated extensively under various genetic parameters. The results show that the indices presented here are both robust and powerful.