Mining elite loci and candidate genes for root morphology-related traits at the seedling stage by genome-wide association studies in upland cotton(Gossypium hirsutum L.)

Root system architecture plays an essential role in water and nutrient acquisition in plants,and it is significantly involved in plant adaptations to various environmental stresses.In this study,a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage,including main root length(MRL),root fresh weight(RFW),total root length(TRL),root surface area(RSA),root volume(RV),and root average diameter(AvgD).The correlation analysis of the six root morphological traits revealed strong positive correlations of TRL with RSA,as well as RV with RSA and AvgD,whereas a significant negative correlation was found between TRL and AvgD.Subsequently,a genome-wide association study(GWAS)was performed using the root phenotypic and genotypic data reported previously for the 242 accessions using 56,010 single nucleotide polymorphisms(SNPs)from the CottonSNP80K array.A total of 41 quantitative trait loci(QTLs)were identified,including nine for MRL,six for RFW,nine for TRL,12 for RSA,12 for RV and two for AvgD.Among them,eight QTLs were repeatedly detected in two or more traits.Integrating these results with a transcriptome analysis,we identified 17 candidate genes with high transcript values of transcripts per million(TPM)≥30 in the roots.Furthermore,we functionally verified the candidate gene GH_D05G2106,which encodes a WPP domain protein 2in root development.A virus-induced gene silencing(VIGS)assay showed that knocking down GH_D05G2106significantly inhibited root development in cotton,indicating its positive role in root system architecture formation.Collectively,these results provide a theoretical basis and candidate genes for future studies on cotton root developmental biology and root-related cotton breeding.

Suppressing a mitochondrial calcium uniporter activates the calcium signaling pathway and promotes cell elongation in cotton

Mitochondrial calcium uniporter(MCU)is a conserved calcium ion(Ca^(2+))transporter in the mitochondrial inner membrane of eukaryotic cells.How MCU proteins regulate Ca^(2+)flow and modulate plant cell development remain largely unclear.Here,we identified the gene GhMCU4 encoding a MCU protein that negatively regulates plant development and fiber elongation in cotton(Gossypium hirsutum).GhMCU4expressed constitutively in various tissues with the higher transcripts in elongating fiber cells.Knockdown of GhMCU4 in cotton significantly elevated the plant height and root length.The calcium signaling pathway was significantly activated and calcium sensor genes,including Ca^(2+)dependent modulator of interactor of constitutively active ROP(GhCMI1),calmodulin like protein(GhCML46),calciumdependent protein kinases(GhCPKs),calcineurin B-like protein(GhCBLs),and CBL-interacting protein kinases(GhCIPKs),were dramatically upregulated in GhMCU4-silenced plants.Metabolic processes were preferentially enriched,and genes related to regulation of transcription were upregulated in GhMCU4-silenced plants.The contents of Ca^(2+)and H_(2)O_(2)were significantly increased in roots and leaves of GhMCU4-silenced plants.Fiber length and Ca^(2+)and H_(2)O_(2)contents in fibers were significantly increased in GhMCU4-silenced plants.This study indicated that GhMCU4 plays a negative role in regulating cell elongation in cotton,thus expanding understanding in the role of MCU proteins in plant growth and development.

The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene(named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene,GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis,also has potential breeding value by engineering GaPC to develop colored petals or fibers for multifunctional utilization of cotton.

Genome-wide analysis of the WRKY transcription factor gene family in Gossypium raimondii and the expression of orthologs in cultivated tetraploid cotton

WRKY proteins are members of a family of transcription factors in higher plants that function in plant responses to various physiological processes.We identified 120 candidate WRKY genes from Gossypium raimondii with corresponding expressed sequence tags in at least one of four cotton species,Gossypium hirsutum,Gossypium barbadense,Gossypium arboreum,and G.raimondii.These WRKY members were anchored on 13 chromosomes in G.raimondii with uneven distribution.Phylogenetic analysis showed that WRKY candidate genes can be classified into three groups,with 20 members in group I,88 in group II,and 12 in group III.The88 genes in group II were further classified into five subgroups,groups IIa–e,containing 7,16,37,15,and 13 members,respectively.We characterized diversity in amino acid residues in the WRKY domain and/or other zinc finger motif regions in the WRKY proteins.The expression patterns of WRKY genes revealed their important roles in diverse functions in cotton developmental stages of vegetative and reproductive growth and stress response.Structural and expression analyses show that WRKY proteins are a class of important regulators of growth and development and play key roles in response to stresses in cotton.

Genetic dissection of tetraploid cotton resistant to Verticillium wilt using interspecific chromosome segment introgression lines

Verticillium wilt(caused by the pathogen Verticillium dahliae) is of high concern for cotton producers and consumers. The major strategy for controlling this disease is the development of resistant cotton(Gossypium spp.) cultivars. We used interspecific chromosome segment introgression lines(CSILs) to identify quantitative trait loci(QTL) associated with resistance to Verticillium wilt in cotton grown in greenhouse and inoculated with three defoliating V. dahliae isolates. A total of 42 QTL, including 23 with resistance-increasing and 19 with resistancedecreasing, influenced host resistance against the three isolates. These QTL were identified and mapped on 18 chromosomes(chromosomes A1, A3, A4, A5, A7, A8, A9, A12, A13, D1, D2,D3, D4, D5, D7, D8, D11, and D12), with LOD values ranging from 3.00 to 9.29. Among the positive QTL with resistance-increasing effect, 21 conferred resistance to only one V. dahliae isolate, suggesting that resistance to V. dahliae conferred by most QTL is pathogen isolate-specific. The At subgenome of cotton had greater effect on resistance to Verticillium wilt than the Dt subgenome. We conclude that pyramiding different resistant QTL could be used to breed cotton cultivars with broad-spectrum resistance to Verticillium wilt.

Association analysis of fiber quality traits and exploration of elite alleles in Upland cotton cultivars/accessions(Gossypium hirsutum L.)

Exploring the elite al eles and germplasm acces-sions related to fiber quality traits wil accelerate the breeding of cotton for fiber quality improvement. In this study, 99 Gossypium hirsutum L. accessions with diverse origins were used to perform association analysis of fiber quality traits using 97 polymorphic microsatel ite marker primer pairs. A total of 107 significant marker-trait associations were detected for three fiber quality traits under three different environments, with 70 detected in two or three environments and 37 detected in only one environment. Among the 70 significant marker-trait associations, 52.86% were reported previously, implying that these are stable loci for target traits. Furthermore, we detected a large number of elite al eles associated simulta-neously with two or three traits. These elite al eles were mainly from accessions col ected in China, introduced to China from the United States, or rare al eles with a frequency of less than＆amp;nbsp;5%. No one cultivar contained more than half of the elite al eles, but 10 accessions were col ected from China and the two introduced from the United States did contain more than half of these al eles. Therefore, there is great potential for mining elite al eles from germplasm accessions for use in fiber quality improvement in modern cotton breeding.

Variation induced by DNA rearrangement in a transgenic Bt+CpTI cotton strain

In the development, of transgenic Bt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T4 strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2-1/3 as large as that of the wild transgenic Bt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n=52; however, there are 4-8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCRamplification revealed that Bt and CpTl genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant pheno-type might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred.

Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in tbliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agro- bacterium tumefaciens-mediated transformation （ATMT） system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant translbnnants per 1 × 10^4 conidia. V. dahliae mutants lacking either VdATG8 or VdATGI2 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild- type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in E dahliae.

Identification of RAPD marker linked with fertility-restoring gene of cytoplasmic male sterile lines in upland cotton

Bulked segregant analysis was employed to construct two mixed DNA pools to screen the RAPd marker linked with the fertilityrestoring gene（\%Rf\-i）\% of upland cotton. A total of 425 arbitrary 10mer oligonucleotide primers were screened on two DNA pools, bulked male fertile and sterile DNAs isolated from BC\-3 segregating population of （06132R×Simian No. 3）. Three primers produced repeatable polymorphisms between the paired bulks and their parents. DNA was extracted and amplified with these three primers for 92 plants of （Zhong 12A1×06132R）F\-2. Based on the male fertility scoring and RAPD amplification, it is found that one RAPD marker fragment designated OPV15 300 was linked with the fertilityrestoring gene （R f \-1） with a recombination value of 13.0±2.57%.