Cotton germplasm improvement and progress in Pakistan

[Abstract] Cotton (Gossypium spp.) contributes significantly to the economy of cotton-producing countries. Pakistan is the fourth-largest producer of cotton after China, the USA and India. The average yield of cotton is about 570.99− 2 in Pakistan. Climate change and different biotic stresses are causing reduction in cotton production. Transgenic approaches have unique advantage to tackle all these problems. However, how to confer permanent resistance in cotton against insects through genetic modification, is still a big challenge to address. Development of transgenic cotton has been proven to be effective. But its effectiveness depends upon several factors, including heterogeneity, seed purity, diffusion of varieties, backcrossing and ethical concerns. Cotton biotechnology was initiated in Pakistan in 1992–1993 with a focus on acquiring cotton leaf curl virus (CLCuV)-resistant insect-resistant, and improving fiber quality. This review summarizes the use of molecular markers, QTLs, GWAS, and gene cloning for cotton germplasm improvement, particularly in Pakistan.
[Title] Cotton germplasm improvement and progress in Pakistan

[Authors] RAZZAQ Abdul, ZAFAR Muhammad Mubashar, ALI Arfan, HAFEEZ Abdul, BATOOL Wajeeha, SHI Yuzhen,  GONG Wankui & YUAN Youlu

Journal of Cotton Research 2021, 41

Identification of candidate genes controlling fiber quality traits in upland cotton through integration of meta-QTL, significant SNP and transcriptomic data

[Background] Meta-analysis of quantitative trait locus (QTL) is a computational technique to identify consensus QTL and refine QTL positions on the consensus map from multiple mapping studies. The combination of meta-QTL intervals, significant SNPs and transcriptome analysis has been widely used to identify candidate genes in various plants.

[Results] In our study, 884 QTLs associated with cotton fiber quality traits from 12 studies were used for meta-QTL analysis based on reference genome TM-1, as a result, 74 meta-QTLs were identified, including 19 meta-QTLs for fiber length; 18 meta-QTLs for fiber strength; 11 meta-QTLs for fiber uniformity; 11 meta-QTLs for fiber elongation; and 15 meta-QTLs for micronaire. Combined with 8 589 significant single nucleotide polymorphisms associated with fiber quality traits collected from 15 studies, 297 candidate genes were identified in the meta-QTL intervals, 20 of which showed high expression levels specifically in the developing fibers. According to the function annotations, some of the 20 key candidate genes are associated with the fiber development.

[Conclusion] This study provides not only stable QTLs used for marker-assisted selection, but also candidate genes to uncover the molecular mechanisms for cotton fiber development.

[Title] Identification of candidate genes controlling fiber quality traits in upland cotton through integration of meta-QTL, significant SNP and transcriptomic data
[Authors] XU Shudi, PAN Zhenyuan, YIN Feifan, YANG Qingyong, LIN Zhongxu, WEN Tianwang, ZHU Longfu, ZHANG Dawei and NIE Xinhui

Journal of Cotton Research 2020, 334

Insect resistance management in Bacillus thuringiensis cotton by MGPS (multiple genes pyramiding and silencing)

[Abstract] The introduction of Bacillus thuringiensis (Bt) cotton has reduced the burden of pests without harming the environment and human health. However, the efficacy of Bt cotton has decreased due to field-evolved resistance in insect pests over time. In this review, we have discussed various factors that facilitate the evolution of resistance in cotton pests. Currently, different strategies like pyramided cotton expressing two or more distinct Bt toxin genes, refuge strategy, releasing of sterile insects, and gene silencing by RNAi are being used to control insect pests. Pyramided cotton has shown resistance against different cotton pests. The multiple genes pyramiding and silencing (MGPS) approach has been proposed for the management of cotton pests. The genome information of cotton pests is necessary for the development of MGPS-based cotton. The expression cassettes against various essential genes involved in defense, detoxification, digestion, and development of cotton pests will successfully obtain favorable agronomic characters for crop protection and production. The MGPS involves the construction of transformable artificial chromosomes, that can express multiple distinct Bt toxins and RNAi to knockdown various essential target genes to control pests. The evolution of resistance in cotton pests will be delayed or blocked by the synergistic action of high dose of Bt toxins and RNAi as well as compliance of refuge requirement.

[Title]Insect resistance management in Bacillus thuringiensis cotton by MGPS (multiple genes pyramiding and silencing)

[Authors] ZAFAR Muhammad Mubashar, RAZZAQ Abdul, FAROOQ Muhammad Awais, REHMAN Abdul, FIRDOUS Hina, SHAKEEL Amir, MO Huijuan and REN Maozhi

Journal of Cotton Research 2020, 333

Genetic map construction and functional characterization of genes within the segregation distortion regions (SDRs) in the F2:3 populations derived from wild cotton species of the D genome

[Background] Segregation distortion (SD) is a common phenomenon among stable or segregating populations, and the principle behind it still puzzles many researchers. The F2:3 progenies developed from the wild cotton species of the D genomes were used to investigate the possible plant transcription factors within the segregation distortion regions (SDRs). A consensus map was developed between two maps from the four D genomes, map A derived from F2:3 progenies of Gossypium klotzschianum and G. davidsonii while Map B from G. thurberi and G. trilobumF2:3 generations. In each map, 188 individual plants were used.

[Results] The consensus linkage map had 1 492 markers across the 13 linkage groups with a map size of 1 467.445 cM and an average marker distance of 1.037 0 cM. Chromosome D502 had the highest percentage of SD with 58.6%, followed by Chromosome D507 with 47.9%. Six thousand and thirty-eight genes were mined within the SDRs on chromosome D502 and D507 of the consensus map. Within chromosome D502 and D507, 2 308 and 3 730 genes were mined, respectively, and were found to belong to 1 117 gourp out of which 622 groups were common across the two chromosomes. Moreover, genes within the top 9 groups related to plant resistance genes (R genes), whereas 188 genes encoding protein kinase domain (PF00069) comprised the largest group. Further analysis of the dominant gene group revealed that 287 miRNAs were found to target various genes, such as the gra-miR398, gra-miR5207, miR164a, miR164b, miR164c among others, which have been found to target top-ranked stress-responsive transcription factors such as NAC genes. Moreover, some of the stress- responsive cis-regulatory elements were also detected. Furthermore, RNA profiling of the genes from the dominant family showed that higher numbers of genes were highly upregulated under salt and osmotic stress conditions, and also they were highly expressed at different stages of fiber development.

[Conclusion] The results indicated the critical role of the SDRs in the evolution of the key regulatory genes in plants.

[Title]Genetic map construction and functional characterization of genes within the segregation distortion regions (SDRs) in the F2:3 populations derived from wild cotton species of the D genome

[Authors] KIRUNGU Joy Nyangasi, MAGWANGA Richard Odongo, SHIRAKU Margaret Linyerera, LU Pu, MEHARI Teame Gereziher, XU Yuanchao, HOU Yuqing, AGONG Stephen Gaya, ZHOU Yun, CAI Xiaoyan,  ZHOU Zhongli, WANG Kunbo & LIU Fang 

Journal of Cotton Research 2020, 332

Bt cotton seed purity in Burkina Faso: status and lessons learnt

[BackgroundSince the commercial release of Bt cotton in Burkina Faso in 2009, the issue of seed purity in producers’ fields has rarely been addressed in an unbiased and objective manner. The potential for contamination of conventional seed varieties with Bt traits and the consequent threat to the continuation of organic cotton production has been documented. However, studies are rare on the varietal purity of Bt cotton seeds, despite the implications for the effectiveness and sustainability of their use.

This paper compensates for the lack of research on the varietal purity of cotton seeds in Burkina Faso by reporting the results of Enzyme linked immunosorbent assay tests collected in 2015 on samples of both conventional and Bt varieties from 646 fields.

[Results] According to the conservative criteria used to declare the presence of a Bt gene in a given variety (more than 10% of seeds of conventional variety exhibit Bt traits, and at least 90% of seeds of Bt variety exhibit Bt traits), seed purity was very questionable for both types of variety. For the supposedly conventional variety, the Cry1Ac gene was observed in 63.6% of samples, the Cry2Ab gene was observed in 59.3% of samples, and both genes were detected in 52.2% of the seed samples. Only 29.3% of the seeds that were supposed to be of conventional type contained no Bt genes. Conversely, for the labeled Bt variety, the Cry1Ac gene was found in only 59.6% of samples, the Cry2Ab gene was found in 53.6% of the samples, and both genes were found in 40.4% of the samples. Finally, for the seeds that were supposed to contain both genes (Bollguard 2), both Cry1Ac and Cry2Ab genes were found in only 40.4% of the samples, only one of the genes was found in 32.4% of the samples, and 27.2% of the seeds in the samples contained neither.

Two factors are responsible for the severe lack of seed purity. First, conventional varieties are being contaminated with Bt traits because of a failure to revise the seed production scheme in Burkina Faso to prevent cross-pollination. Second, the original Bt seeds provided to Burkina Faso lacked varietal purity.

The organic sector plays a very minor role in the cotton sector of Burkina Faso (production of organic cotton totaled 453 t in 2018/2019, out of national cotton production of 183 000 t). Nevertheless, the lack of purity in conventional seed varieties is a threat to efforts to expand certified organic cotton production. The poor presence of Bt proteins in supposed Bt varieties undermines their effectiveness in controlling pests and increases the likelihood of the development of resistance among pest populations.

[Conclusion] Our results show the extent of purity loss when inadequate attention is paid to the preservation of seed purity. Pure conventional seeds could vanish in Burkina Faso, while Bt seeds do not carry the combination of the expected Bt traits. Any country wishing to embark on the use of Bt cotton, or to resume its use, as in the case of Burkina Faso, must first adjust its national seed production scheme to ensure that procedures to preserve varietal purity are enforced. The preservation of varietal purity is necessary to enable the launch or the continuation of identity-cotton production. In addition, the preservation of varietal purity is necessary to ensure the sustainable effectiveness of Bt cotton. In order to ensure that procedures to preserve varietal purity are observed, seed purity must be tested regularly, and test results must be published.

[Title] Bt cotton seed purity in Burkina Faso: status and lessons learnt

[Authors] BOURGOU Larbouga, KARGOUGOU Ester, SAWADOGO Mahamadou and FOK Michel

Journal of Cotton Research 2020, 330

Apprehending the potential of BABY BOOM transcription factors to mitigate cotton regeneration and transformation

[Abstract] Since the advent of transgenic technology, the incorporation of gene(s) encoding traits of economic importance in cotton is being practiced worldwide. However, factors like recalcitrant nature of cotton cultivars, in vitro regeneration via tissue culture (especially via somatic embryogenesis), genotype dependency, long and toilsome protocols impede the pace of development of transgenic cotton. Besides that, types and age of explants, media composition, plant growth regulators and other environmental factors affect in vitro cotton regeneration significantly. The studies of genetic control of in vitro regeneration in plants have elucidated the role of certain transcription factor genes that are induced and expressed during somatic embryogenesis. Among these transcription factors, BABY BOOM (BBM) plays a very important role in signal transduction pathway, leading to cell differentiation and somatic embryos formation. The role of BBM has been established in plant cell proliferation, growth and development even without exogenous growth regulators. This review intends to provide an informative summary of regeneration and transformation problems in cotton and the latest developments in utilization of BBM transcription factors in cotton. We believe that the use of BBM will not only ease cotton genetic improvement but will also accelerate cotton breeding programmes.

[Title] Apprehending the potential of BABY BOOM transcription factors to mitigate cotton regeneration and transformation

[Authors] YAVUZ Caner, TILLABOEVA Shakhnozakhan & BAKHSH Allah

Journal of Cotton Research 2020, 329

Phenylpropanoid metabolism and pigmentation show divergent patterns between brown color and green color cottons as revealed by metabolic and gene expression analyses

[Background] Naturally-colored cotton has become increasingly popular because of their natural properties of coloration, UV protection, flame retardant, antibacterial activity and mildew resistance. But poor fiber quality and limited color choices are two key issues that have restricted the cultivation of naturally-colored cotton. To identify the possible pathways participating in fiber pigmentation in naturally-colored cottons, five colored cotton accessions in three different color types (with green, brown and white fiber) were chosen for a comprehensive analysis of phenylpropanoid metabolism during fiber development.

[Result] The expression levels of flavonoid biosynthesis pathway genes in brown cotton fibers were significantly higher than those in white and green cotton fibers. Total flavonoids and proanthocyanidin were higher in brown cotton fibers relative to those in white and green cotton fibers, which suggested that the flavonoid biosynthesis pathway might not participate in the pigmentation of green cotton fibers. Further expression analysis indicated that the genes encoding enzymes for the synthesis of caffeic acid derivatives, lignin and lignan were activated in the developing fibers of the green cotton at 10 and 15 days post-anthesis.

[Conclusion] Our results strengthen the understanding of phenylpropanoid metabolism and pigmentation in green and brown cotton fibers, and may improve the breeding of naturally-colored cottons.

[Title] Phenylpropanoid metabolism and pigmentation show divergent patterns between brown color and green color cottons as revealed by metabolic and gene expression analyses

[Authors] LI Zhonghua, SU Qian, XU Mingqi, YOU Jiaqi, KHAN Anam Qadir, LI Junyi, ZHANG Xianlong, TU Lili & YOU Chunyuan

Journal of Cotton Research 2020, 327

A genome-wide identification of the BLH gene family reveals BLH1 involved in cotton fiber development

[Background] Cotton is the world’s largest and most important source of renewable natural fiber. BEL1-like homeodomain (BLH) genes are ubiquitous in plants and have been reported to contribute to plant development. However, there is no comprehensive characterization of this gene family in cotton. In this study, 32, 16, and 18 BLH genes were identified from the G. hirsutumG. arboreum, and G. raimondii genome, respectively. In addition, we also studied the phylogenetic relationships, chromosomal location, gene structure, and gene expression patterns of the BLH genes.

[Result] The results indicated that these BLH proteins were divided into seven distinct groups by phylogenetic analysis. Among them, 25 members were assigned to 15 chromosomes. Furthermore, gene structure, chromosomal location, conserved motifs, and expression level of BLH genes were investigated in G. hirsutum. Expression profiles analysis showed that four genes (GhBLH1_3GhBLH1_4GhBLH1_5, and GhBLH1_6) from BLH1 subfamily were highly expressed during the fiber cell elongation period. The expression levels of these genes were significantly induced by gibberellic acid and brassinosteroid, but not auxin. Exogenous application of gibberellic acid significantly enhanced GhBLH1_3GhBLH1_4, and GhBLH1_5transcripts. Expression levels of GhBLH1_3 and GhBLH1_4 genes were significantly increased under brassinosteroid treatment.

[Conclusion] The BLH gene family plays a very important role in many biological processes during plant growth and development. This study deepens our understanding of the role of the GhBLH1gene involved in fiber development and will help us in breeding better cotton varieties in the future.

[Title] A genome-wide identification of the BLH gene family reveals BLH1 involved in cotton fiber development

[Authors] LIU Cuixia, LI Zhifang, DOU Lingling, Yi YUAN, ZOU Changsong, SHANG Haihong, CUI Langjun & XIAO  Guanghui

Journal of Cotton Research 2020, 326

Global identification of genes associated with xylan biosynthesis in cotton fiber

[Background] Mature cotton fiber secondary cell wall comprises largely of cellulose (> 90%) and small amounts of xylan and lignin. Little is known about the cotton fiber xylan biosynthesis by far.

[Result] To comprehensively survey xylan biosynthetic genes in cotton fiber, we identified five IRX9, five IRX10, one IRX14, six IRX15, two FRA8, one PARVUS, eight GUX, four GXM, two RWA, two AXY9, 13 TBL genes by using phylogenetic analysis coupled with expression profile analysis and co-expression analyses. In addition, we also identified two GT61 members, two GT47 members, and two DUF579 family members whose homologs in Arabidopsis were not functionally characterized. These 55 genes were regarded as the most probable genes to be involved in fiber xylan biosynthesis. Further complementation analysis indicated that one IRX10 like and two FRA8 related genes were able to partially recover the irregular xylem phenotype conferred by the xylan deficiency in their respective Arabidopsis mutant. We conclude that these genes are functional orthologs of respective genes that are implicated in GX biosynthesis.

[Conclusion] The list of 55 cotton genes presented here provides not only a solid basis to uncover the biosynthesis of xylan in cotton fiber, but also a genetic resource potentially useful for future studies aiming at fiber improvement via biotechnological approaches.

[Title] Global identification of genes associated with xylan biosynthesis in cotton fiber

[Authors] CHEN Feng, GUO Yanjun, CHEN Li, GAN Xinli, LIU Min, LI Juan & XU Wenliang

Journal of Cotton Research 2020, 325

Development and application of perfect SSR markers in cotton

[Background] This study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.

[Result] Using bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.

[Conclusion] A set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

[Title] Development and application of perfect SSR markers in cotton

[Authors] WU Yuzhen, HUANG Longyu, ZHOU Dayun, FU Xiaoqiong, LI Chao, WEI Shoujun, PENG Jun & KUANG Meng

Journal of Cotton Research 2020, 321