Genome resources for three modern cotton lines guide future breeding efforts.

Cotton (Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars ('UGA230', 'UA48' and 'CSX8308') and updating the 'TM-1' cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from 'Pima' cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.

Detecting Cotton Leaf Curl Virus Resistance Quantitative Trait Loci in Gossypium hirsutum and iCottonQTL a New R/Shiny App to Streamline Genetic Mapping.

Cotton leaf curl virus (CLCuV) causes devastating losses to fiber production in Central Asia. Viral spread across Asia in the last decade is causing concern that the virus will spread further before resistant varieties can be bred. Current development depends on screening each generation under disease pressure in a country where the disease is endemic. We utilized quantitative trait loci (QTL) mapping in four crosses with different sources of resistance to identify single nucleotide polymorphism (SNP) markers associated with the resistance trait to allow development of varieties without the need for field screening every generation. To assist in the analysis of multiple populations, a new publicly available R/Shiny App was developed to streamline genetic mapping using SNP arrays and to also provide an easy method to convert and deposit genetic data into the CottonGen database. Results identified several QTL from each cross, indicating possible multiple modes of resistance. Multiple sources of resistance would provide several genetic routes to combat the virus as it evolves over time. Kompetitive allele specific PCR (KASP) markers were developed and validated for a subset of QTL, which can be used in further development of CLCuV-resistant cotton lines.

Leveraging National Germplasm Collections to Determine Significantly Associated Categorical Traits in Crops: Upland and Pima Cotton as a Case Study.

Observable qualitative traits are relatively stable across environments and are commonly used to evaluate crop genetic diversity. Recently, molecular markers have largely superseded describing phenotypes in diversity surveys. However, qualitative descriptors are useful in cataloging germplasm collections and for describing new germplasm in patents, publications, and/or the Plant Variety Protection (PVP) system. This research focused on the comparative analysis of standardized cotton traits as represented within the National Cotton Germplasm Collection (NCGC). The cotton traits are named by 'descriptors' that have non-numerical sub-categories (descriptor states) reflecting the details of how each trait manifests or is absent in the plant. We statistically assessed selected accessions from three major groups of Gossypium as defined by the NCGC curator: (1) "Stoneville accessions (SA)," containing mainly Upland cotton (Gossypium hirsutum) cultivars; (2) "Texas accessions (TEX)," containing mainly G. hirsutum landraces; and (3) Gossypium barbadense (Gb), containing cultivars or landraces of Pima cotton (Gossypium barbadense). For 33 cotton descriptors we: (a) revealed distributions of character states for each descriptor within each group; (b) analyzed bivariate associations between paired descriptors; and (c) clustered accessions based on their descriptors. The fewest significant associations between descriptors occurred in the SA dataset, likely reflecting extensive breeding for cultivar development. In contrast, the TEX and Gb datasets showed a higher number of significant associations between descriptors, likely correlating with less impact from breeding efforts. Three significant bivariate associations were identified for all three groups, bract nectaries:boll nectaries, leaf hair:stem hair, and lint color:seed fuzz color. Unsupervised clustering analysis recapitulated the species labels for about 97% of the accessions. Unexpected clustering results indicated accessions that may benefit from potential further investigation. In the future, the significant associations between standardized descriptors can be used by curators to determine whether new exotic/unusual accessions most closely resemble Upland or Pima cotton. In addition, the study shows how existing descriptors for large germplasm datasets can be useful to inform downstream goals in breeding and research, such as identifying rare individuals with specific trait combinations and targeting breakdown of remaining trait associations through breeding, thus demonstrating the utility of the analytical methods employed in categorizing germplasm diversity within the collection.

Analysis of a tetraploid cotton line Mac7 transcriptome reveals mechanisms underlying resistance against the whitefly Bemisia tabaci.

Whitefly inflicts both direct and indirect losses to cotton crop. Whitefly resistant cotton germplasm is a high priority and considered among the best possible solutions to mitigate this issue. In this study, we evaluated cotton leaf curl disease (CLCuD) resistant cotton line Mac7 under whitefly stress. Furthermore, we utilized the already available transcriptome data of Mac7 concerning whitefly stress to elucidate associated mechanisms and identify functionally important genes in cotton. In transcriptomic data analysis, differentially expressed genes (DEGs) were found involved in complex relay pathways, activated on whitefly exposure. The response implicates signalling through resistance genes (R-genes), MAPK, ROS, VQs or RLKs, transcription factors, which leads to the activation of defence responses including, Ca2+messengers, phytohormonal cross-talk, gossypol, flavonoids, PhasiRNA and susceptibility genes (S-genes). The qRT-PCR assay of 10 functionally important genes also showed their involvement in differential responses at 24 and 48 h post whitefly infestation. Briefly, our study helps in understanding the resistant nature of Mac7 under whitefly stress.

Effects of Interspecific Chromosome Substitution in Upland Cotton on Cottonseed Macronutrients.

Nutrients, including macronutrients such as Ca, P, K, and Mg, are essential for crop production and seed quality, and for human and animal nutrition and health. Macronutrient deficiencies in soil lead to poor crop nutritional qualities and a low level of macronutrients in cottonseed meal-based products, leading to malnutrition. Therefore, the discovery of novel germplasm with a high level of macronutrients or significant variability in the macronutrient content of crop seeds is critical. To our knowledge, there is no information available on the effects of chromosome or chromosome arm substitution on cottonseed macronutrient content. The objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and content of the cottonseed macronutrients Ca, K, Mg, N, P, and S in chromosome substitution lines (CS). Nine chromosome substitution lines were grown in two-field experiments at two locations in 2013 in South Carolina, USA, and in 2014 in Mississippi, USA. The controls used were TM-1, the recurrent parent of the CS line, and the cultivar AM UA48. The results showed major variability in macronutrients among CS lines and between CS lines and controls. For example, in South Carolina, the mean values showed that five CS lines (CS-T02, CS-T04, CS-T08sh, CS-B02, and CS-B04) had higher Ca level in seed than controls. Ca levels in these CS lines varied from 1.88 to 2.63 g kg-1 compared with 1.81 and 1.72 g kg-1 for TM-1 and AMUA48, respectively, with CS-T04 having the highest Ca concentration. CS-M08sh exhibited the highest K concentration (14.50 g kg-1), an increase of 29% and 49% over TM-1 and AM UA48, respectively. Other CS lines had higher Mg, P, and S than the controls. A similar trend was found at the MS location. This research demonstrated that chromosome substitution resulted in higher seed macronutrients in some CS lines, and these CS lines with a higher content of macronutrients can be used as a genetic tool towards the identification of desired seed nutrition traits. Also, the CS lines with higher desired macronutrients can be used as parents to breed for improved nutritional quality in Upland cotton, Gossypium hirsutum L., through improvement by the interspecific introgression of desired seed nutrient traits such as Ca, K, P, S, and N. The positive and significant (p ≤ 0.0001) correlation of P with Ca, P with Mg, S with P, and S with N will aid in understanding the relationships between nutrients to improve the fertilizer management program and maintain higher cottonseed nutrient content.

Effects of Interspecific Chromosome Substitution in Upland Cotton on Cottonseed Micronutrients.

Micronutrients are essential for plant growth and development, and important for human health nutrition and livestock feed. Therefore, the discovery of novel germplasm with significant variability or higher micronutrients content in crop seeds is critical. Currently, there is no information available on the effects of chromosome or chromosome arm substitution in cotton on cottonseed micronutrients. Thus, the objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and levels of micronutrients B, Fe, Cu, Zn, Mn, and Ni in cottonseed from chromosome substitution (CS) cotton lines. Our hypothesis was that interspecific chromosome substitution in cotton can affect cottonseed micronutrients content, resulting in significant differences and variabilities of these nutrients among CS lines and between CS lines and the controls. Nine CS lines were grown in two-field experiments at two locations (in 2013 in South Carolina, USA; and in 2014 in Mississippi, USA). TM-1 (the recurrent parent of the CS line) and AM UA48 (cultivar) were used as control. The results showed significant variability among CS lines compared to the controls AM UA48 and TM-1. For example, in South Carolina (SC), B concentration in cottonseed ranged from 10.35 mg kg-1 in CS-M02 to 13.67 mg kg-1 in CS-T04. The concentration of Cu ranged from 4.81 mg kg-1 in CS-B08sh to 7.65 mg kg-1 in CS-T02, and CS-T02 was higher than both controls. The concentration of Fe ranged from 36.09 mg kg-1 to 56.69 mg kg-1 (an increase up to 57%), and six CS lines (CS-B02, CS-B08sh, CS-M02, CS-M04, CS-T02, and CS-T04) had higher concentration than both controls in 2013. In 2014 at the Mississippi location (MS), similar observation was found with CS lines for micronutrients content. The CS lines with higher concentrations of these micronutrients can be used as a genetic tool toward QTL identification for desired seed traits because these lines are genetically similar with TM-1, except the substituted chromosome or chromosome segment pairs from the alien species. Chromosome substitution provides an effective means for upland cotton improvement by targeted interspecific introgression, yielding CS lines that facilitate trait discovery, such as seed micronutritional qualities, due to increased isogenicity and markedly reduced complexity from epistatic interactions with non-target alien chromosomes. The positive correlation between B, Cu, and Fe at both locations, between Ni and Mn, between Zn and Cu, and between Zn and Ni at both locations signify the importance of a good agricultural and fertilizer management of these nutrients to maintain higher cottonseed nutrient content.

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum).

Cultivated cotton (Gossypium hirsutum) is the most important fibre crop in the world. Cotton leaf curl disease (CLCuD) is the major limiting factor and a threat to textile industry in India and Pakistan. All the local cotton cultivars exhibit moderate to no resistance against CLCuD. In this study, we evaluated an exotic cotton accession Mac7 as a resistance source to CLCuD by challenging it with viruliferous whiteflies and performing qPCR to evaluate the presence/absence and relative titre of CLCuD-associated geminiviruses/betasatellites. The results indicated that replication of pathogenicity determinant betasatellite is significantly attenuated in Mac7 and probably responsible for resistance phenotype. Afterwards, to decipher the genetic basis of CLCuD resistance in Mac7, we performed RNA sequencing on CLCuD-infested Mac7 and validated RNA-Seq data with qPCR on 24 independent genes. We performed co-expression network and pathway analysis for regulation of geminivirus/betasatellite-interacting genes. We identified nine novel modules with 52 hubs of highly connected genes in network topology within the co-expression network. Analysis of these hubs indicated the differential regulation of auxin stimulus and cellular localization pathways in response to CLCuD. We also analysed the differential regulation of geminivirus/betasatellite-interacting genes in Mac7. We further performed the functional validation of selected candidate genes via virus-induced gene silencing (VIGS). Finally, we evaluated the genomic context of resistance responsive genes and found that these genes are not specific to A or D sub-genomes of G. hirsutum. These results have important implications in understanding CLCuD resistance mechanism and developing a durable resistance in cultivated cotton.

Whole genome sequencing of Asia II 1 species of whitefly reveals that genes involved in virus transmission and insecticide resistance have genetic variances between Asia II 1 and MEAM1 species.

BACKGROUND: Whiteflies (Bemisia tabaci) are phloem sap-sucking pests that because of their broad host range and ability to transmit viruses damage crop plants worldwide. B. tabaci are now known to be a complex of cryptic species that differ from each other in many characteristics such as mode of interaction with viruses, invasiveness, and resistance to insecticides. Asia II 1 is an indigenous species found on the Indian sub-continent and south-east Asia while the species named as Middle East Asia Minor 1 (MEAM1), likely originated from the Middle-East and has spread worldwide in recent decades. The purpose of this study is to find genomic differences between these two species. RESULTS: Sequencing of the nuclear genome of Asia II 1 with Illumina HiSeq and MiSeq generated 198.90 million reads that covers 88% of the reference genome. The sequence comparison with MEAM1 identified 2,327,972 SNPs and 202,479 INDELs. In Total, 1294 genes were detected with high impact variants. The functional analysis revealed that some of the genes are involved in virus transmission including 4 genes in Tomato yellow leaf curl virus (TYLCV) transmission, 96 in Tomato crinivirus (ToCV) transmission, and 14 genes in insecticide resistance. CONCLUSIONS: These genetic differences between Asia II 1 and MEAM1 may underlie the major biological differences between the two species such as virus transmission, insecticide resistance, and range of host plants. The present study provides new genomic data and information resources for Asia II 1 that will not only contribute to the species delimitation of whitefly, but also help in conceiving future research studies to develop more targeted management strategies against whitefly.

Insights into the Evolution of the New World Diploid Cottons (Gossypium, Subgenus Houzingenia) Based on Genome Sequencing.

We employed phylogenomic methods to study molecular evolutionary processes and phylogeny in the geographically widely dispersed New World diploid cottons (Gossypium, subg. Houzingenia). Whole genome resequencing data (average of 33× genomic coverage) were generated to reassess the phylogenetic history of the subgenus and provide a temporal framework for its diversification. Phylogenetic analyses indicate that the subgenus likely originated following transoceanic dispersal from Africa about 6.6 Ma, but that nearly all of the biodiversity evolved following rapid diversification in the mid-Pleistocene (0.5-2.0 Ma), with multiple long-distance dispersals required to account for range expansion to Arizona, the Galapagos Islands, and Peru. Comparative analyses of cpDNAversus nuclear data indicate that this history was accompanied by several clear cases of interspecific introgression. Repetitive DNAs contribute roughly half of the total 880 Mb genome, but most transposable element families are relatively old and stable among species. In the genic fraction, pairwise synonymous mutation rates average 1% per Myr, with nonsynonymous changes being about seven times less frequent. Over 1.1 million indels were detected and phylogenetically polarized, revealing a 2-fold bias toward deletions over small insertions. We suggest that this genome down-sizing bias counteracts genome size growth by TE amplification and insertions, and helps explain the relatively small genomes that are restricted to this subgenus. Compared with the rate of nucleotide substitution, the rate of indel occurrence is much lower averaging about 17 nucleotide substitutions per indel event.

Genes regulating gland development in the cotton plant.

In seeds and other parts of cultivated, tetraploid cotton (Gossypium hirsutum L.), multicellular groups of cells lysigenously form dark glands containing toxic terpenoids such as gossypol that defend the plant against pests and pathogens. Using RNA-seq analysis of embryos from near-isogenic glanded (Gl2 Gl2 Gl3 Gl3 ) versus glandless (gl2 gl2 gl3 gl3 ) plants, we identified 33 genes that expressed exclusively or at higher levels in embryos just prior to gland formation in glanded plants. Virus-induced gene silencing against three gene pairs led to significant reductions in the number of glands in the leaves, and significantly lower levels of gossypol and related terpenoids. These genes encode transcription factors and have been designated the 'Cotton Gland Formation' (CGF) genes. No sequence differences were found between glanded and glandless cotton for CGF1 and CGF2 gene pairs. The glandless cotton has a transposon insertion within the coding sequence of the GoPGF (synonym CGF3) gene of the A subgenome and extensive mutations in the promoter of D subgenome homeolog. Overexpression of GoPGF (synonym CGF3) led to a dramatic increase in gossypol and related terpenoids in cultured cells, whereas CRISPR/Cas9 knockout of GoPGF (synonym CGF3) genes resulted in glandless phenotype. Taken collectively, the results show that the GoPGF (synonym CGF3) gene plays a critical role in the formation of glands in the cotton plant. Seed-specific silencing of CGF genes, either individually or in combination, could eliminate glands, thus gossypol, from the cottonseed to render it safe as food or feed for monogastrics.

Transcriptomics reveals multiple resistance mechanisms against cotton leaf curl disease in a naturally immune cotton species, Gossypium arboreum.

Cotton leaf curl disease (CLCuD), caused by cotton leaf curl viruses (CLCuVs), is among the most devastating diseases in cotton. While the widely cultivated cotton species Gossypium hirsutum is generally susceptible, the diploid species G. arboreum is a natural source for resistance against CLCuD. However, the influence of CLCuD on the G. arboreum transcriptome and the interaction of CLCuD with G. arboreum remains to be elucidated. Here we have used an RNA-Seq based study to analyze differential gene expression in G. arboreum under CLCuD infestation. G. arboreum plants were infested by graft inoculation using a CLCuD infected scion of G. hirsutum. CLCuD infested asymptomatic and symptomatic plants were analyzed with RNA-seq using an Illumina HiSeq. 2500. Data analysis revealed 1062 differentially expressed genes (DEGs) in G. arboreum. We selected 17 genes for qPCR to validate RNA-Seq data. We identified several genes involved in disease resistance and pathogen defense. Furthermore, a weighted gene co-expression network was constructed from the RNA-Seq dataset that indicated 50 hub genes, most of which are involved in transport processes and might have a role in the defense response of G. arboreum against CLCuD. This fundamental study will improve the understanding of virus-host interaction and identification of important genes involved in G. arboreum tolerance against CLCuD.

Sub genome anchored physical frameworks of the allotetraploid Upland cotton (Gossypium hirsutum L.) genome, and an approach toward reference-grade assemblies of polyploids.

Like those of many agricultural crops, the cultivated cotton is an allotetraploid and has a large genome (~2.5 gigabase pairs). The two sub genomes, A and D, are highly similar but unequally sized and repeat-rich, which pose significant challenges for accurate genome reconstruction using standard approaches. Here we report the development of BAC libraries, sub genome specific physical maps, and a new-generation sequencing approach that will lead to a reference-grade genome assembly for Upland cotton. Three BAC libraries were constructed, fingerprinted, and integrated with BAC-end sequences (BES) to produce a de novo whole-genome physical map. The BAC map was partitioned by sub genomes through alignment to the diploid progenitor D-genome reference sequence with densely spaced BES anchor points and computational filtering. The physical maps were validated with FISH and genetic mapping of SNP markers derived from BES. Two pairs of homeologous chromosomes, A11/D11 and A12/D12, were used to assess multiplex sequencing approaches for completeness and scalability. The results represent the first sub genome anchored physical maps of Upland cotton, and a new-generation approach to the whole-genome sequencing, which will lead to the reference-grade assembly of allopolyploid cotton and serve as a general strategy for sequencing other polyploid species.

Multiple begomoviruses found associated with cotton leaf curl disease in Pakistan in early 1990 are back in cultivated cotton.

The first epidemic of cotton leaf curl disease (CLCuD) in early 1990's in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990's but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus, Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus, several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuBVeh. The implications of these findings in efforts to control CLCuD are discussed.

Diversity analysis of cotton (Gossypium hirsutum L.) germplasm using the CottonSNP63K Array.

BACKGROUND: Cotton germplasm resources contain beneficial alleles that can be exploited to develop germplasm adapted to emerging environmental and climate conditions. Accessions and lines have traditionally been characterized based on phenotypes, but phenotypic profiles are limited by the cost, time, and space required to make visual observations and measurements. With advances in molecular genetic methods, genotypic profiles are increasingly able to identify differences among accessions due to the larger number of genetic markers that can be measured. A combination of both methods would greatly enhance our ability to characterize germplasm resources. Recent efforts have culminated in the identification of sufficient SNP markers to establish high-throughput genotyping systems, such as the CottonSNP63K array, which enables a researcher to efficiently analyze large numbers of SNP markers and obtain highly repeatable results. In the current investigation, we have utilized the SNP array for analyzing genetic diversity primarily among cotton cultivars, making comparisons to SSR-based phylogenetic analyses, and identifying loci associated with seed nutritional traits. RESULTS: The SNP markers distinctly separated G. hirsutum from other Gossypium species and distinguished the wild from cultivated types of G. hirsutum. The markers also efficiently discerned differences among cultivars, which was the primary goal when designing the CottonSNP63K array. Population structure within the genus compared favorably with previous results obtained using SSR markers, and an association study identified loci linked to factors that affect cottonseed protein content. CONCLUSIONS: Our results provide a large genome-wide variation data set for primarily cultivated cotton. Thousands of SNPs in representative cotton genotypes provide an opportunity to finely discriminate among cultivated cotton from around the world. The SNPs will be relevant as dense markers of genome variation for association mapping approaches aimed at correlating molecular polymorphisms with variation in phenotypic traits, as well as for molecular breeding approaches in cotton.

RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants.

The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread.

Frequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan.

Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production on the Indian subcontinent, and is caused by monopartite begomoviruses accompanied by a specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Since the breakdown of resistance against CLCuD in 2001/2002, only one virus, the "Burewala" strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bur), and a recombinant form of CLCuMB have consistently been identified in cotton across the major cotton growing areas of Pakistan. Unusually a bipartite isolate of the begomovirus Tomato leaf curl virus was identified in CLCuD-affected cotton recently. In the study described here we isolated the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) from CLCuD-affected cotton. To assess the frequency and geographic occurrence of ToLCNDV in cotton, CLCuD-symptomatic cotton plants were collected from across the Punjab and Sindh provinces between 2013 and 2015. Analysis of the plants by diagnostic PCR showed the presence of CLCuKoV-Bur in all 31 plants examined and ToLCNDV in 20 of the samples. Additionally, a quantitative real-time PCR analysis of the levels of the two viruses in co-infected plants suggests that coinfection of ToLCNDV with the CLCuKoV-Bur/CLCuMB complex leads to an increase in the levels of CLCuMB, which encodes the major pathogenicity (symptom) determinant of the complex. The significance of these results are discussed.

RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly.

Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA) against both aquaporin (AQP) and a sucrase gene, alpha glucosidase (AGLU). These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR) was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci) and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Identification and genomic location of a reniform nematode (Rotylenchulus reniformis) resistance locus (Ren ari) introgressed from Gossypium aridum into upland cotton (G. hirsutum).

In this association mapping study, a tri-species hybrid, [Gossypium arboreum x (G. hirsutum x G. aridum)(2)], was crossed with MD51ne (G. hirsutum) and progeny from the cross were used to identify and map SSR markers associated with reniform nematode (Rotylenchulus reniformis) resistance. Seventy-six progeny (the 50 most resistant and 26 most susceptible) plants were genotyped with 104 markers. Twenty-five markers were associated with a resistance locus that we designated Ren(ari) and two markers, BNL3279_132 and BNL2662_090, mapped within 1 cM of Ren(ari). Because the SSR fragments associated with resistance were found in G. aridum and the bridging line G 371, G. aridum is the likely source of this resistance. The resistance is simply inherited, possibly controlled by a single dominant gene. The markers identified in this project are a valuable resource to breeders and geneticists in the quest to produce cotton cultivars with a high level of resistance to reniform nematode.

Microsatellites reveal genetic diversity in Rotylenchulus reniformis populations.

Rotylenchulus reniformis is the predominant parasitic nematode of cotton in the Mid South area of the United States. Although variable levels of infection and morphological differences have been reported for this nematode, genetic variability has been more elusive. We developed microsatellite-enriched libraries for R. reniformis, produced 1152 clones, assembled 694 contigs, detected 783 simple sequence repeats (SSR) and designed 192 SSR-markers. The markers were tested on six R. reniformis cultures from four states, Texas, Louisiana, Mississippi and Georgia, in the USA. Based on performance we selected 156 SSR markers for R. reniformis from which 88 were polymorphic across the six reniform nematode populations, showing as the most frequent motif the dinucleotide AG. The polymorphic information content of the markers ranged from 0.00 to 0.82, and the percentage of multiallelic loci of the isolates was between 40.9 and 45.1%. An interesting finding in this study was the genetic variability detected among the three Mississippi isolates, for which 22 SSR markers were polymorphic. We also tested the level of infection of these isolates on six cotton genotypes, where significant differences were found between the Texas and Georgia isolates. Coincidentally, 62 polymorphic markers were able to distinguish these two populations. Further studies will be necessary to establish possible connections, if any, between markers and level of pathogenicity of the nematode. The SSR markers developed here will be useful in the assessment of the genetic diversity of this nematode, could assist in management practices for control of reniform nematode, be used in breeding programs for crop resistance, and help in detecting the origin and spread of this nematode in the United States.