Map-based cloning of q LP_(A01.1),a favorable allele from the Gossypium tomentosum chromosome segment line

Cotton is an important natural fiber crop worldwide which plays a vital role in our daily life.High yield is a constant goal of cotton breeding,and lint percentage(LP)is one of the important components of cotton fiber yield.A stable QTL controlling LP,qLP_(A01.1),was identified on chromosome A01 from Gossypium hirsutum introgressed lines with G.tomentosum chromosome segments in a previous study.To fine-map qLP_(A01.1),an F2 population with 986individuals was established by crossing G.hirsutum cultivar CCRI35 with the chromosome segment substitution line HT_390.A high-resolution genetic map including 47 loci and spanning 56.98 cM was constructed in the QTL region,and qLP_(A01.1)was ultimately mapped into an interval corresponding to an~80 kb genome region of chromosome A01in the reference genome,which contained six annotated genes.Transcriptome data and sequence analysis revealed that S-acyltransferase protein 24(GoPAT24)might be the target gene of qLP_(A01.1).This result provides the basis for cotton fiber yield improvement via marker-assisted selection(MAS)and further studies on the mechanism of cotton fiber development.

Upregulation of the glycine-rich protein-encoding gene GhGRPL enhances plant tolerance to abiotic and biotic stressors by promoting secondary cell wall development

Abiotic and biotic stressors adversely affect plant survival,biomass generation,and crop yields.As the global availability of arable land declines and the impacts of global warming intensify,such stressors may have increasingly pronounced effects on agricultural productivity.Currently,researchers face the overarching challenge of comprehensively enhancing plant resilience to abiotic and biotic stressors.The secondary cell wall plays a crucial role in bolstering the stress resistance of plants.To increase plant resistance to stress through genetic manipulation of the secondary cell wall,we cloned a cell wall protein designated glycine-rich protein-like(GhGRPL)from cotton fibers,and found that it is specifically expressed during the period of secondary cell wall biosynthesis.Notably,this protein differs from its Arabidopsis homolog,AtGRP,since its glycine-rich domain is deficient in glycine residues.GhGRPL is involved in secondary cell wall deposition.Upregulation of GhGRPL enhances lignin accumulation and,consequently,the thickness of the secondary cell walls,thereby increasing the plant’s resistance to abiotic stressors,such as drought and salinity,and biotic threats,including Verticillium dahliae infection.Conversely,interference with GhGRPL expression in cotton reduces lignin accumulation and compromises that resistance.Taken together,our findings elucidate the role of GhGRPL in regulating secondary cell wall development through its influence on lignin deposition,which,in turn,reinforces cell wall robustness and impermeability.These findings highlight the promising near-future prospect of adopting GhGRPL as a viable,effective approach for enhancing plant resilience to abiotic and biotic stress factors.

Development of a target capture sequencing SNP genotyping platform for genetic analysis and genomic breeding in rapeseed

Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.

Identification and Characterization of ZF-HD Genes in Response to Abscisic Acid and Abiotic Stresses in Maize

The zinc finger homeodomain(ZF-HD)genes belong to the homeobox gene family,playing critical roles in flower development and stress response.Despite their importance,however,to date there has been no genome-wide identification and characterization of the ZF-HD genes that are probably involved in stress responses in maize.In this study,24 ZF-HD genes were identified,and their chromosomal locations,protein properties,duplication patterns,structures,conserved motifs and expression patterns were investigated.The results revealed that the ZF-HD genes are unevenly distributed on nine chromosomes and that most of these genes lack introns.Six and two ZF-HD genes have undergone segmental and tandem duplication,respectively,during genome expansion.These 24 ZF-HD transcription factors were classified into six major groups on the basis of protein molecular evolutionary relationship.The expression profiles of these genes in different tissues were evaluated,resulting in producing two distinct clusters.ZF-HD genes are preferentially expressed in reproductive tissues.Furthermore,expression profiles of the 24 ZF-HD genes in response to different kinds of stresses revealed that ten genes were simultaneously up-regulated under ABA,salt and PEG treatments;meanwhile four genes were simultaneously down-regulated.These findings will pave the way for deciphering the function and mechanism of ZF-HD genes on how to implicate in abiotic stress.

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.

Biotechnology ofα-linolenic acid in oilseed rape(Brassica napus)using FAD2 and FAD3 from chia(Salvia hispanica)

α-Linolenic acid(ALA,18:3Δ9,12,15)is an essential fatty acid for humans since it is the precursor for the biosynthesis of omega-3 long-chain polyunsaturated fatty acids(LC-PUFA).Modern people generally suffer from deficiency of ALA because most staple food oils are low or lack ALA content.Biotechnological enrichment of ALA in staple oil crops is a promising strategy.Chia(Salvia hispanica)has the highest ALA content in its seed oil among known oil crops.In this study,the FAD2 and FAD3 genes from chia were engineered into a staple oil crop,oilseed rape(Brassica napus),via Agrobaterium tumefaciens-mediated transformation of their LP4-2A fusion gene construct driven by the seed-specific promoter P_(NapA).In seeds of T0,T1,and T2 lines,the average ALA contents were 20.86,23.54,and 24.92%,respectively,which were 2.21,2.68,and 3.03 folds of the non-transformed controls(9.42,8.78,and 8.22%),respectively.The highest seed ALA levels of T0,T1,and T2 plants were 38.41,35.98,and 39.19%respectively,which were 4.10-4.77 folds of the respective controls.FA-pathway enzyme genes(BnACCD,BnFATA,BnSAD,BnSCD,BnDGAT1,BnDGAT2,and BnDGAT3)and positive regulatory genes(BnWRI1,BnLEC1,BnL1L,BnLEC2,BnABI3,BnbZIP67,and BnMYB96)were all significantly up-regulated.In contrast,BnTT1,BnTT2,BnTT8,BnTT16,BnTTG1,and BnTTG2,encoding negative oil accumulation regulators but positive secondary metabolism regulators,were all significantly down-regulated.This means the foreign ShFAD2-ShFAD3 fusion gene,directly and indirectly,remodeled both positive and negative loci of the whole FA-related network in transgenic B.napus seeds.

Detection of new candidate genes controlling seed weight by integrating gene coexpression analysis and QTL mapping in Brassica napus L.

Seed weight is a component of seed yield in rapeseed(Brassica napus L.).Although quantitative trait loci(QTL)for seed weight have been reported in rapeseed,only a few causal quantitative trait genes(QTGs)have been identified,resulting in a limitation in understanding of seed weight regulation.We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species.Among the 10 modules in this gene coexpression network,modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein,and photosynthesis.A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19(SCPL19)was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines.Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Genome-Wide Identification and Expression Analysis of the Phytocyanin Gene Family in Nicotiana tabacum

Phytocyanin(PC)is a class of plant-specific blue copper proteins involved in electron transport,plant growth,development,and stress resistance.However,PC proteins have not been systematically evaluated in tobacco plants.We determined the whole-genome sequences of the PC family in the tobacco cultivar‘K326.’The transcriptome data were used to analyze the expression of the NtPC family at different development stages and tissue-specific genes.Real-time fluorescence quantitative analysis was used to analyze the expression of the NtPC gene family under low temperature and methyl jasmonate stress.The tobacco NtPC family contained 110 members and was divided into four subfamilies:early nodulin-like protein(NtENODL),uclacyanin-like protein,stellacyanin1-like protein,and plantacyanin-like protein.According to phylogenetic and structural analyses,the NtPC family could be divided into eight structural types.Fifty-three NtPCs were randomly distributed on 22 of 24 tobacco chromosomes.Collinearity analysis revealed 33 pairs of genes belonging to the NtPC family.Gene ontology analysis showed that the PC genes are components of the plasma membrane and may participate in plasma membrane-related functions.The NtPC family contained numerous elements related to hormonal and abiotic stress responses and was specifically expressed in the tobacco prosperous,maturation,and budding periods.Tissue-specific expression analysis showed that some genes were tissue specific.The expression of NtENODL58 and other genes was significantly induced by low-temperature and methyl jasmonate stress.Thus,the NtPC gene family plays an important role in plant stress response.

SsdchA is a novel secretory cellobiohydrolase driving pathogenicity in Sclerotinia sclerotiorum

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7(GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar(PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover,the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.

A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.In this study,we demonstrate that Golden2-like 1a(BnGLK1a)plays an important role in regulating chloroplast development and photosynthetic efficiency.Overexpressing BnGLK1a resulted in significant increases in chlorophyll content,the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus,while knocking down BnGLK1a transcript levels through RNA interference(RNAi)had the opposite effects.A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2(BnPYL1-2)and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit(BnCSN5A),which play essential roles in regulating chloroplast development and photosynthesis.Consistent with this,BnGLK1a-RNAi lines of B.napus display hypersensitivity to the abscisic acid(ABA)response.Importantly,overexpression of BnGLK1a resulted in a 10%increase in thousand-seed weight,whereas seeds from BnGLK1a-RNAi lines were 16%lighter than wild type.We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.Our results not only provide insights into the mechanisms of BnGLK1a function,but also offer a potential approach for improving the productivity of Brassica species.

Over-Expression of Bn MAPK1 in Brassica napus Enhances Tolerance to Drought Stress

Mitogen-activated protein kinases （MAPKs） are a family of Ser/Thr protein kinases widely conserved in all eukaryotes and involved in responses to biotic and abiotic stresses. In this study, two over-expressing BnMAPK1 oilseed rape lines, ov3 and ov11, were used to study the drought-resistant mechanism of BnMAPK1 under natural drought and simulation drought through spraying 10%PEG 8000 in seedlings. Zhongyou 821 （WT） was used as control. Compared with wild type, transgenic seedlings had higher leaf water content, higher root activity, slightly higher peroxidase （POD） and superoxide dismutase （SOD） activity, higher proline content and lower malondialdehyde （MDA） content. The expression of drought-resistant related genes, including P5CSB, PLC, LEA4 and SCE1, have been up-regulated in some degree and the expressed time of transgenic lines were earlier than that of wild type. These results suggested that over-expression of BnMAPK1 can enhance the resistance to drought in oilseed rape （Brassica napus）.

ISSR-Based Molecular Characterization of an Elite Germplasm Collection of Sweet Potato(Ipomoea batatas L.) in China

To determine the genetic diversity and population structure of sweet potato accessions cultivated in China, and to establish the genetic relationships among their germplasm types, a representative collection of 240 accessions was analyzed using inter-simple sequence repeat （ISSR） markers. The mean genetic similarity coefifcient, Nei’s gene diversity, and shared allele distance of tested sweet potato accessions were 0.7302, 0.3167 and 0.2698, respectively. The 240 accessions could be divided into six subgroups and ifve subpopulations based on neighbor-joining （NJ） clustering and STRUCTURE results, and obvious genetic relationships among the tested sweet potato accessions were identiifed. The marker-based NJ clustering and population structure showed no distinct assignment pattern corresponding to lfesh color or geographical ecotype of the tested sweet potato germplasm. Analysis of molecular variance （AMOVA） revealed small but signiifcant difference between white and orange-lfeshed sweet potato accessions. Small but signiifcant difference were also observed among sweet potato accessions from the Southern summer-autumn sweet potato region, the Yellow River Basin spring and summer sweet potato region and the Yangtze River Basin summer sweet potato region. This study demonstrates that genetic diversity in the tested sweet potato germplasm collection in China is lower than that in some reported sweet potato germplasm collections from other regions. Pedigree investigations suggest that more diverse Chinese sweet potato varieties should be formed by broadening the selection scope of breeding parents and incorporating the introduced varieties into future breeding programs.

Two types of cinnamoyl-Co A reductase function divergently in accumulation of lignins,flavonoids and glucosinolates and enhance lodging resistance in Brassica napus

Brassica crops,which are of worldwide importance,provide various oil,vegetable and ornamental products,as well as feedstocks for animal husbandry and biofuel industry.Cinnamoyl-Co A reductase(CCR)is the entry point to the lignin pathway and a crucial locus in manipulation of associated traits,but CCRassociated metabolism and traits in Brassica crops have remained largely unstudied except in Arabidopsis thaliana.We report the identification of 16 CCR genes from Brassica napus and its parental species B.rapa and B.oleracea.The Bn CCR1 and Bn CCR2 subfamilies displayed divergent organ-specificity and participation in the yellow-seed trait.Their functions were dissected via overexpression of representative paralogs in B.napus.Bn CCR1 was expressed preferentially in G-and H-lignin biosynthesis and vascular development,while Bn CCR2 was expressed in S-lignin biosynthesis and interfascicular fiber development.Bn CCR1 showed stronger effects on lignification-related development,lodging resistance,phenylpropanoid flux control,and seed coat pigmentation,whereas Bn CCR2 showed a stronger effect on sinapate biosynthesis.Bn CCR1 upregulation delayed bolting and flowering time,while Bn CCR2 upregulation weakened the leaf vascular system in consequence of suppressed G-lignin accumulation.Bn CCR1 and Bn CCR2 were closely but almost oppositely linked with glucosinolate metabolism via inter-pathway crosstalk.We conclude that Bn CCR1 and Bn CCR2 subfamilies offer great but differing potential for manipulating traits associated with phenylpropanoids and glucosinolates.This study reveals the CCR1–CCR2 divergence in Brassicaceae and offers a resource for rapeseed breeding for lodging resistance,yellowseed traits,and glucosinolate traits.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

Genome-wide association study identifies novel loci and candidate genes for drought stress tolerance in rapeseed

Rapeseed(Brassica napus)is one of the most important oil crops worldwide;however,drought seriously curtails its growth and productivity.Identifying drought-tolerant germplasm is an efficient strategy for addressing water shortages.Here,we phenotyped a panel of 264 B.napus accessions at full-bloom stage using water loss ratio(WLR)as drought-tolerant index.It identified 8 low-WLR and 6 high-WLR accessions,regarded as droughttolerant and drought-sensitive,respectively.Comparing with drought-sensitive accessions at the seedling stage,drought-tolerant accessions had shown better performance in maintaining fresh and dry weights,and performed the higher expression of drought-induced marker genes under drought stress.Subsequently,a total of 139 SNPs(single nucleotide polymorphisms)were identified associated with the WLR using a genome-wide association study(GWAS)among 264 B.napus accessions,with the largest number SNPs at chromosome A10,and 13 SNPs significantly were associated with the WLR(-log_(10)(p-value)>6).Furthermore,4 putative candidate genes(BnaC09.RPS6,BnaC09.MATE,BnaA10.PPD5 and BnaC09.Histone)were screened involving in drought tolerance in B.napus.Together,our results highlight the WLR's importance in drought tolerance and establish the foundation for improving WLR-associated drought tolerance in rapeseed.

Sphingolipid inhibitor response gene GhMYB86 controls fiber elongation by regulating microtubule arrangement

Although the cell membrane and cytoskeleton play essential roles in cellular morphogenesis,the interaction between the membrane and cytoskeleton is poorly understood.Cotton fibers are extremely elongated single cells,which makes them an ideal model for studying cell development.Here,we used the sphingolipid biosynthesis inhibitor,fumonisin B1(FB1),and found that it effectively suppressed the myeloblastosis(MYB)transcription factor GhMYB86,thereby negatively affecting fiber elongation.A direct target of GhMYB86 is GhTUB7,which encodes the tubulin protein,the major component of the microtubule cytoskeleton.Interestingly,both the overexpression of GhMYB86 and GhTUB7 caused an ectopic microtubule arrangement at the fiber tips,and then leading to shortened fibers.Moreover,we found that GhMBE2 interacted with GhMYB86 and that FB1 and reactive oxygen species induced its transport into the nucleus,thereby enhancing the promotion of GhTUB7 by GhMYB86.Overall,we established a GhMBE2-GhMYB86-GhTUB7 regulation module for fiber elongation and revealed that membrane sphingolipids affect fiber elongation by altering microtubule arrangement.

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice(Oryza sativa L.)

The leaf blade consists of color and shape traits.Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis.A narrow and upper-albino leaf mutant(nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate.Under field conditions,the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development.The narrow-leaf trait is caused by a decreased number of small veins.In contrast to the wild type,the growth period was extended by approximately 8 d and agronomic traits,such as effective panicle number,percentage seed set and 1000-grain weight,declined significantly in the nul1 mutant.Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene.The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637.The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence.These results provide a foundation for cloning and function analysis of Nul1.

Phyto-Toxicity of Chromium in Maize： Oxidative Damage, Osmolyte Accumulation, Anti-Oxidative Defense and Chromium Uptake

Agricultural production systems are immensely exposed to different environmental stresses in which heavy metal stress receives serious concerns. This study was conducted to explore the deleterious effects of different chromium （Cr） stress levels, i.e., O, 30, 60, 90, 120, and 150 μmol L^-1, on two maize genotypes, Wandan 13 and Runnong 35. Both genotypes were evaluated by measuring their growth and yield characteristics, Cr accumulation in different plant tissues, alterations in osmolyte accumulation, generation of reactive oxygen species （ROS）, and anti-oxidative enzyme activity to scavenge ROS. The results showed that Cr stress decreased the leaf area, cob formation, 100-grain weight, shoot fresh biomass, and yield formation, while Cr accumulation in different maize tissues was found in the order of roots 〉 leaves 〉 stem ~ seeds in both genotypes. The increased Cr toxicity resulted in higher free proline, soluble sugars and total phenolic contents, and lower soluble protein contents. However, enhanced lipid peroxidation was noticed in the forms of malondialdehyde, hydrogen peroxide （H2O2） and thiobarbituric acid reactive substance accumulation, and electrolyte leakage. The hyperactivity of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, especially glutathione peroxidase and glutathione reductase indicated that these anti-oxidative enzymes had a central role in protecting maize from Cr toxicity, especially for Wandan 13. Moreover, higher uptake and less translocation of Cr contents into the grains of Wandan 13 implied its importance as a potential candidate against soil Cr pollution.