Mapping a leaf senescence gene els1 by BSR-Seq in common wheat

Miomio Li,Beiei Li,Gungho Guo,Yongxing Chen,Jingzhong Xie,Ping Lu,Qiuhong Wu,Deyun Zhng,Huizhi Zhng,Jin Yng,Pnpn Zhng,Yn Zhng,Zhiyong Liu,*

aCollege of Agronomy&Biotechnology,China Agricultural University,Beijing 100193,China

bState Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

cUniversity of Chinese Academy of Sciences,Beijing 100049,China

dCollege of Horticulture,China Agricultural University,Beijing 100193,China

1.Introduction

Leaf senescence,an orderly regulated and active process,is the last stage of leaf development.During this process,plant organs,and cells undergo a sequence of complex changes in cellular physiology and biochemistry,and nutrients are redistributed to the sink tissues,such as developing fruits and seeds[1,2].Crops with delayed leaf senescence or stay green maintain extended periods of photosynthetic competence and have higher seed weights and grain yields[3,4].However,premature senescence of functional leaves during grain-filling results in reduced yield and quality,on account of disrupted physiological function in leaves due to reducing transport of photosynthetic products from leaves to seeds and a shortened period for grain filling[5].

The onset and progression of senescence can be modulated by a variety of environmental signals including drought,nutrient limitation,light,and various plant hormones.Drought stress decreases crop yields by inducing abscisic acid(ABA)and premature leaf senescence in barley[6].A delay in leaf senescence results in delayed N remobilisation and a negative impact on protein deposition in the grain,thus reducing grain quality[7].H2S suppresses chlorophyll degradation of detached Arabidopsis leaves in dark-induced leaf senescence[8].Plant hormones play key roles in response to senescence.ABA can induce expression of several senescence-associated genes(SAG)in Arabidopsis thaliana[9].Exogenous ethylene enhances visible leaf yellowing and several ethylene biosynthesis genes are upregulated in senescing leaves in Arabidopsis[10].The Arabidopsis JA-insensitive mutant,coronatine insensitive 1(coi1),fails to display JA-dependent senescence[11].

Many endogenous factors involved in leaf senescence have been identified and characterized in Arabidopsis,rice,maize,sorghum,and cotton[12–16].Most of the characterized senescence-associated genesare transcription factors,including WRKY,NAC,MADS,MYB,bZIP,and bHLH family members,indicating that leaf senescence is regulated by complex transcriptional regulatory networks[2,12].For example,OsMYC2 acts as a positive regulator of leaf senescence by direct or indirect regulation of SAGs in rice[17];RPK1 plays an important role in ABA-dependent leaf senescence in Arabidopsis[18];miRNA was also found to be involved in early leaf senescence.Overexpression of miR164 represses EIN3-induced early-senescence phenotypes in the model plant Arabidopsis[19].Stay-green QTL were detected in chromosomes 3 and 4 by genotyping-by-sequencing(GBS)technology explained 8%–24%of the phenotypic variation in sorghum,which could be exploited to improve grain yield in molecular breeding programs[20].Five stay-green QTL were significantly correlated with yield in maize.QTL-linked markers can help accelerate development of delayed leaf senescence in maize varieties through molecular marker-assisted selection[21].

In comparison with studies in other species,research on leaf senescence in wheat is relatively backward.Wang et al.[22]reported a wheat stay-green mutant,tasg1,with delayed senescence.The wheat high grain protein content gene GPCB1 originating from wild emmer and encoding NAC transcription factor NAM-B1 is associated with early leaf senescence[23].Under favorable growing conditions,the yield contribution of the stay-green trait in wheat results from increased ear fertility and number of grains.In a more restrictive scenario,it favors an increase in grain mass by longer filling time at the end of the life cycle[24].

Wheat has a large genome(～17 Gb)that is about eight times larger than that of maize and 40 times larger than that of rice.The large genome size and presence of highly repetitive DNA sequences(80–90%)makes fine mapping of target genes,a necessary step for gene cloning,a formidable challenge[25].BSR-Seq,a method that combines bulked segregant analysis(BSA)and RNA-Seq has been used as a mapping strategy that offers the promise of rapid discovery of novel genes and genetic markers linked to target genes[26].The advantages of this approach are high-throughput and cost-effectiveness in analyzing large genomes,such as,hexaploid maize and wheat[26,27].Using BSR-Seq analysis the maize brown midrib 2(bm2)gene was mapped to a small region of chromosome 1 containing a putative methylenetetrahydrofolate reductase(MTHFR)gene[28].Wheat stripe rust resistance genes YrZH22 and YrMM58 were rapidly mapped on chromosomes 4BL[29]and 2AS[30],respectively,by combining BSR-Seq with comparative genomics analysis.Stripe rust resistance gene Yr15 was also rapidly mapped to a 0.77 cM chromosomal interval in chromosome 1BS in hexaploid wheat by applying BSR-Seq analysis[27].

In this study we performed a genetic analysis of early leaf senescence wheat line M114 using a segregating F3family and its F3:4progenies as well as a newly developed F2mapping population.BSR-Seq strategy was used to develop a linkage map of early leaf senescence gene els1.

2.Materials and methods

2.1.Plant materials and mapping population

An early leaf senescence segregating F3family was identified in a breeding population from cross ZK 331/Xiangmai 99171//2*Luomai 30.The three parental lines had normal phenotype,and their F3:4progenies were subjected to phenotypic analysis.From the F4progenies,a homozygous early leaf senescence line M114 was selected to make cross with a normal line W301.The F1plants,F2segregating population of the cross M114/W301 were evaluated for early leaf senescence/normal phenotypes and used for genetic mapping.All the plants were grown at the field of Gaoyi Experimental Station in Shijiazhuang,Hebei province.

2.2.BSR-Seq analysis

Leaf tissue samples were collected from 35 early leaf senescent plants and 40 normal F4plants during the midflowering stage.These samples were frozen in liquid nitrogen and stored at?80 °C until used for total RNA extraction.Total RNA was extracted from tissues,then quantity and quality assessed.Two sequencing libraries were constructed using an Illumina RNA-Seq sample preparation kit and sequenced on an Illumina HiSeq4000 pair end sequencing platform.Raw RNA-Seq reads were trimmed to remove lowquality nucleotides using software Trimmomatic v0.32[31].Trimmed reads were aligned to the Chinese Spring reference sequence published by International Wheat Genome Sequencing Consortium(IWGSC)(http://www.wheatgenome.org/)using software STAR v2.4.0j[32].SNP and InDel calling was analyzed using software GATK v3.2-2 module“Haplotype Caller”[33].

2.3.DNA extraction

Genomic DNA was extracted by the CTAB method.The quality and quantity of the DNA were verified using 1.0%agarose gels and a Nanodrop 2000 spectrophotomer.Eight homozygous early leaf senescent and fifteen homozygous normal leaf plants from the F3:4progenies were used to construct two DNA bulks for SNP and markers validation.

2.4.Development and verification of molecular markers

After aligning trimmed reads and SNP-calling SNPs associated with early leaf senescence were identified by BSR-Seq analysis and selected for molecular marker development and validation.Specific PCR primers were designed from flanking sequences of selected SNPs using Primer 6.0 software.

Wheat microsatellite markers(Xgwm,Xwmc,Xbarc,Xcfa,and Xcfd series)mapped on chromosome 2B were chosen for polymorphism detection.Information on these markers is reported on the Grain Genes database(http://wheat.pw.usda.gov/).

2.5.Statistical analysis and genetic linkage map construction

Chi-squared(χ2)tests were performed to determine the goodness-of-fit of observed and expected segregation ratios using SPSS 20.0.Linkage analysis of polymorphic molecular markers and the gene was conducted using Mapmaker 3.0 software and LOD score threshold of 3.0.The genetic map was constructed with the software Mapdraw V2.1[34].

3.Results

3.1.Phenotypic and genetic analysis of early leaf senescence

In our breeding population,we identified an F3family that segregated in a 3 normal:1 early leaf senescent ratio and segregation at a single locus was confirmed by progeny tests(Table 1).Early leaf senescence in line homozygous F3:4line M114 began on bottom leaves at the tillering stage(Fig.1-A,B)and became very evident by the booting stage with lower leaves gradually drying from bottom leaves.In contrast,a representative normal line F3:4(W301)remained green and healthy.The F1plants of the cross M114/W301 were normal indicating recessive inheritance of the early senescence phenotype.The F2population of M114/W301 segregated in a 3:1 ratio(Table 1).These results suggest that early leaf senescence of M114 is conferred by a single recessive gene,provisionally designated els1.

3.2.BSR-Seq analysis

Applying BSR-Seq,the homozygous early leaf senescence and homozygous normal bulks produced 53,781,214 and 56,288,977 raw read pairs,respectively.＜1%of the raw read pairs were filtered after quality control.Trimmed reads were aligned to the Chinese Spring reference sequences.In total,76.74%and 85.30%of the filtered read pairs were uniquely mapped in the homozygous early leaf senescent and homozygous normal bulks,respectively.Subsequent SNP calling identified 151,928 high-quality variants(SNPs and InDels)between the two bulks.Eighty SNPs were associated with the Els1 locus at a cut off of allele frequency difference(AFD)＞0.8 and Fisher's exact test P-value ＜1e?10.The highest frequency SNPs(34/80)associated with the locus was identified on chromosome 2B(Fig.2).

Fig.2–Distribution of candidate SNPs associated with the els1 on wheat chromosomes.

3.3.Candidate SNPs validation and genetic mapping of els1

The Els1-associated SNPs on chromosome 2B were validated for polymorphisms between the M114 and W301 parents and between homozygous normal and homozygous early leaf senescent plant DNA bulks.Four SNP markers,WGGB302,WGGB303,WGGB304,and WGGB305(Table 2)showed clear polymorphism between the parental lines and DNA bulks and shown to be linked to Els1 locus after genotyping the segregating population(Fig.3).

Seventy six publically available SSR primer pairs on chromosome 2B were screened for polymorphism between the parental lines as well as homozygous normal and homozygous early leaf senescent plant DNA bulks.Only Xbarc91 was polymorphic between the parental as well as the bulk DNAs(Fig.4,Table 2).New SSR markers were developed using Chinese Spring 2B reference sequences near to the SNP markers linked to Els1.Two SSR markers,WGGB306 and WGGB307(Table 2)were polymorphic between the parental lines and DNA bulks(Fig.4,Table 2).

Four SNP markers,WGGB302,WGGB303,WGGB304,and WGGB305,and three SSR markers Xbarc91,WGGB306,and WGGB307 linked with Els1 were then genotyped in the 45 F3:4families and 127 homozygous early leaf senescence F2plants of the cross M114/W301.WGGB305 and WGGB307 were located 0.3 cM and 6.7 cM distal to Els1,respectively.WGGB303,WGGB304 and WGGB306 were located 1.2 cM proximal and Xbarc91 a further 0.6 cM from Els1.WGGB302 co-segregated with Els1(Fig.5).

3.4.Gene annotation

Genetic mapping results showed that Els1 was mapped within a 1.5 cM genetic interval between markers WGGB303 and WGGB305,corresponding to a 9.2 Mb physical genomic region in Chinese Spring chromosome 2BS.Sixty-nine putative genes were annotated in this physical genomic region(Table S1).Among them,two cytochrome P450,one NB-ARC-domain protein,one leucine-rich receptor-like protein kinase family protein,one transducin/WD40 repeat-like superfamily protein and one WRKY transcription factor were identified.

4.Discussion

Stay-green is a physiological mechanism that delays leaf senescence has attracted the attention of plant breeders as a means of increasing grain yield[35].Early leaf senescence limits crop productivity by restricting growth,which is important for plant development and especially for crop yield.Stay-green has been studied in several crops and leaf senescence-associated genes have been identified and cloned in a number of plant species,including Arabidopsis thaliana[2],barley[6],maize[14],and rice[17].Several stay-green QTL identified in sorghum,maize and rice were correlated with grain yield[20,21,36].There are few reports on early leaf senescence in wheat.In the present study,we confirmed an early leaf senescence phenotype in F4line M114.Leaf senescence was expressed from the tillering stage and lower leaves showed dry necrotic symptoms during reproductive phase.Genetic analysis revealed that the senescent phenotype was controlled by a recessive gene,which we named els1.Since all of the three parental lines had normal phenotype,the early leaf senescence trait identified in the F3segregating family should be a hidden deleterious locus in one of the original parents.However,identification of Els1 gene could benefit our understanding of the leaf growth and developmental process in wheat and provide a functional gene for molecular manipulation in producing wheat varieties with stay green phenotype.

Hybrid necrosis in wheat was also described by progressive chlorosis and necrosis of plant leaf and sheath tissues,which results in gradual premature death of leaves,tillers or plants,or loss of productivity in certain F1wheat hybrids[37].The degree of necrosis in F1hybrids was classified into three level,weak-hybrids produce normal seeds,moderate-hybrids produce premature seeds,and severe or strong-hybrids produce no seed[38].Hybrid necrosis is genetically controlled by two complementary dominant genes Ne1 and Ne2 located onchromosomes 5BL and 2BS,respectively[39].It has reported Ne2m has a very close relationship with leaf rust resistance gene Lr13 and they maybe the same gene[40].In this study,early leaf senescence phenotype in F4line M114 began on bottom leaves at the tillering stage and became very evident by the booting stage with lower leaves gradually drying from bottom leaves.This feature is somewhat similar with moderate hybrid necrosis(Ne2m).The mapping region of Els1 gene is close to Ne2/Lr13 locus in chromosome 2BS[40].However,further works need to be conducted to prove weather Els1 and Ne2/Lr13 are the same gene.

Table 2–SNP and SSR markers linked to Els1 locus developed from BSR-Seq and comparative genomics analysis.

Fig.3–Sanger sequencing profiles of four SNP markers tightly linked to Els1 locus in homozygous normal,heterozygous normal,and homozygous early leaf senescent plants.

In the present research,we took advantage of an available F3segregating population for early leaf senescence from a breeding program and 80 SNPs were associated with Els1 by BSR-Seq analysis.The high percentage of the SNPs(42.5%)on chromosome 2B enabled us to rapidly map Els1 on chromosome 2BS.Four SNP markers,WGGB302,WGGB303,WGGB304,and WGGB305,were tightly linked to Els1 and the gene was placed in a 1.5 cM genetic interval between WGGB303 and WGGB305,corresponding to a 9.2 Mb physical sequence.Marker WGGB302 co-segregating with Els1 could serve as a starting point for map-based cloning.

Only one polymorphic SSR marker,Xbarc91,was polymorphic among 76 publicly available SSR primer pairs on chromosome 2BS.The low percentage of polymorphic SSR markers detected in the study was at least in part due to the mapping population being F3:4family that originated from a single F2plant,Families segregating for a specific trait in breeding program canbeused for genetic analysis and mapping purposes.However,the narrow genetic background in a single heterozygous plant from an advanced generation hybrid can present difficulties for obtaining polymorphic markers linked to the trait.BSR-Seq offers one way of detecting SNPs or InDels for mapping in the target genomic.

Fig.4–PCR amplification patterns of three polymorphic SSR markers tightly linked to Els1.The arrows show polymorphic DNA fragments between normal and early leaf senescent lines.Lanes 1 and 2 are normal line W301 and early leaf senescence line M114.Lanes 3–6 represent homozygous normal F3families.Lanes 7–10 represent homozygous early leaf senescence F3 families.Lanes 11–14 represent heterozygous F3families.M,DNA ladder.

Fig.5–Genetic linkage map of Els1 on wheat chromosome 2BS.

Gene annotation of the corresponding 9.2 Mb genomic region in Chinese Spring revealed two cytochrome P450, one NB-ARC domain protein, one leucine-rich receptor-like protein kinase,one transduction/WD40 repeat-like superfamily protein and one WRKY transcription factor in the mapping interval(Table S1).Cytochrome P450 monooxygenase CYP89A9 as being responsible for nonfluorescent dioxobilin-type chlorophyll catabolites accumulation is involved in the formation of major chlorophyll catabolites during leaf senescence[41].In Arabidopsis,ORE9,a 693 amino acid polypeptide containing 18 leucine-rich repeats functions in limiting leaf longevity by removing,through ubiquitin-dependent proteolysis,target proteins that are required to delay programmed leaf senescence[42].Protein kinases and autophagy-related gene also were reported to play an important role in leaf senescence[43].In Arabidopsis,WD40 protein played important roles in development and also during stress signalling[44].The plant specific WRKY transcription factor family,especially WRKY4,WRKY6,WRKY11,and WRKY53 have been suggested to play an important role in leaf senescence[45].In particular,NB-ARC domain protein usually triggers R-gene mediated pathogen recognition with programing cell death that is a typical feature of necrosis,leaf rust resistance and leaf senescence[46].The coincidence of hybrid necrosis gene Ne2,leaf rust resistance gene Lr13 and early senescence gene Els1 in this genomic region may provide interesting information to understand the relationship between cell death and leaf development in wheat.

5.Conclusions

We identified early leaf senescent F4line M114 in wheat a breeding program.Genetic analysis indicated that the early leaf senescence is controlled by a single recessive allele designated els1.By applying BSR-Seq analysis,seven polymorphic markers tightly linked to els1 were developed.Ultimately,els1was mapped in a 1.5 cM genetic interval flanked by markers WGGB303 and WGGB305.The co-segregating marker WGGB302 provides a starting point for map-based cloning of Els1.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.01.004.

Acknowledgments

This work was financially supported by the National Key Research and Development Program of China(2017YFD0101004)and Science and Technology Service Network Initiative of Chinese Academy of Sciences(KFJ-STSZDTP-024).

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