Recent progression and future perspectives in cotton genomic breeding

Upland cotton is an important global cash crop for its long seed fibers and high edible oil and protein content.Progress in cotton genomics promotes the advancement of cotton genetics,evolutionary studies,functional genetics,and breeding,and has ushered cotton research and breeding into a new era.Here,we summarize high-impact genomics studies for cotton from the last 10 years.The diploid Gossypium arboreum and allotetraploid Gossypium hirsutum are the main focus of most genetic and genomic studies.We next review recent progress in cotton molecular biology and genetics,which builds on cotton genome sequencing efforts,population studies,and functional genomics,to provide insights into the mechanisms shaping abiotic and biotic stress tolerance,plant architecture,seed oil content,and fiber development.We also suggest the application of novel technologies and strategies to facilitate genome-based crop breeding.Explosive growth in the amount of novel genomic data,identified genes,gene modules,and pathways is now enabling researchers to utilize multidisciplinary genomics-enabled breeding strategies to cultivate"super cotton",synergistically improving multiple traits.These strategies must rise to meet urgent demands for a sustainable cotton industry.

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.)is one of the most important fiber crops worldwide.In the last two decades,transgenesis and genome editing have played important roles in cotton improvement.However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation.Here,we developed a shoot apical meristem(SAM)cell-mediated transformation system(SAMT)that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton(Gossypium hirsutum),Sea island cotton(Gossypium barbadense),and Asiatic cotton(Gossypium arboreum).Through SAMT,we successfully introduced two foreign genes,GFP and RUBY,into SAM cells of some recalcitrant cotton genotypes.Within 2–3 months,transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants.The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP-and RUBY-positive plants,as well as in their progenies,indicating that the transgenes were stably integrated into the genome and transmitted to the next generation.Furthermore,using SAMT,we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing.In summary,the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.

Genome-wide analysis of BES1 genes in Gossypium revealed their evolutionary conserved roles in brassinosteroid signaling

Brassinosteroids(BRs), which are essential phytohormones for plant growth and development, are important for cotton fiber development. Additionally, BES1 transcription factors are critical for BR signal transduction. However, cotton BES1 family genes have not been comprehensively characterized. In this study, we identified 11 BES1 genes in G. arboreum, 11 in G.raimondii, 16 in G. barbadense, and 22 in G. hirsutum. The BES1 sequences were significantly conserved in the Arabidopsis thaliana, rice, and upland cotton genomes. A total of 94 BES1 genes from 10 different plant species were divided into three clades according to the neighbor-joining and minimum-evolution methods. Moreover, the exon/intron patterns and motif distributions were highly conserved among the A. thaliana and cotton BES1 genes. The collinearity among the orthologs from the At and Dt subgenomes was estimated. Segmental duplications in the At and Dt subgenomes were primarily responsible for the expansion of the cotton BES1 gene family. Of the GhBES1 genes, GhBES1.4_At/Dt exhibited BL-induced expression and was predominantly expressed in fibers. Furthermore, Col-0/mGhBES1.4_At plants produced curled leaves with long and bent petioles. These transgenic plants also exhibited decreased hypocotyl sensitivity to brassinazole and constitutive BR induced/repressed gene expression patterns. The constitutive BR responses of the plants overexpressing mGhBES1.4_At were similar to those of the bes1-D mutant.

Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum

Genetic transformation in some plant species,including cotton(Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing(VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different c DNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA(si RNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of si RNA in target sequence: the disruption energy of the target(DGdisruption), the differential stability of si RNA duplex ends(DSSE), and the internal stability at positions 9–14 of the si RNA antisense strand(AIS), which are calculated by Sfold program(http://sfold.wadsworth.org). We find that the si RNAs with low DGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, Gh PDS,Gh CLA1, Gh AOS1, and Gh CXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.

Fighting against fall armyworm by using multiple genes pyramiding and silencing(MGPS) technology

The pest Fall Armyworm(FAW),Spodoptera frugiperda belonging to the order Lepidoptera,is one of the most devastating insect pests resulting in severe economic losses to maize,rice,wheat,cotton and soybean in South America,Africa and Asia.It consists of two morphologically indistinguishable strains:the“corn strain”feeding mainly on maize,cotton and sorghum;the“rice strain”mostly feeding on rice and various grasses(Gouin et al.,2017).Adults of FAW have strong migration ability to fly 100 km per night.In 2018 it was detected in India and then spreads to Bangladesh,Thailand,Myanmar,and China in 2019(http://www.fao.org/fall-armyworm/en/).It is a polyphagous lepidopteran herbivore,attacks more than 350 plant species,and causes destructive damage to many economically important crops.The FAW causes up to 30%-60% yield losses.

Freeze substitution Hi-C,a convenient and cost-effective method for capturing the natural 3D chromatin conformation from frozen samples

Chromatin interactions functionally affect genome architecture and gene regulation,but to date,only fresh samples must be used in High-through chromosome conformation capture(Hi-C)to keep natural chromatin conformation intact.This requirement has impeded the advancement of 3 D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time.Here,we develop a freeze substitution Hi-C(FS-Hi-C)technique that overcomes the need for fresh samples.FS-Hi-C can be used with samples stored in liquid nitrogen(LN2):the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution.After confirming that the FS step preserves the natural chromosome conformation during sample thawing,we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum.Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains,we found that FS-HiC outperforms the in situ Hi-C in terms of library quality,reproducibility,and valid interactions.Thus,FS-HiC will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.