Genetic diversity and population structure analyses and genome-wide association studies of photoperiod sensitivity in cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genetic diversity and population structure analyses showed progressively narrowed diversity in US Upland cotton compared to land races. GWAS identified genomic regions and candidate genes for photoperiod sensitivity in cotton. Six hundred fifty-seven accessions that included elite cotton germplasm (DIV panel), lines of a public cotton breeding program (FB panel), and tropical landrace accessions (TLA panel) of Gossypium hirsutum L. were genotyped with cottonSNP63K array and phenotyped for photoperiod sensitivity under long day-length conditions. The genetic diversity analysis using 26,952 polymorphic SNPs indicated a progressively narrowed diversity from the landraces (0.230) to the DIV panel accessions (0.195) and FB panel (0.116). Structure analysis in the US germplasm identified seven subpopulations representing all four major regions of the US cotton belt. Three subpopulations were identified within the landrace accessions. The highest fixation index (FST) of 0.65 was found between landrace accessions of Guatemala and the Plains-type cultivars from Southwest cotton region while the lowest FST values were between the germplasms of Mid-South and Southeastern regions. Genome wide association studies (GWAS) of photoperiod response using 600 phenotyped accessions identified 14 marker trait associations spread across eight Upland cotton chromosomes. Six of these marker trait associations, on four chromosomes (A10, D04, D05, and D06), showed significant epistatic interactions. Targeted genomic analysis identified regions with 19 candidate genes including Transcription factor Vascular Plant One-Zinc Finger 1 (VOZ1) and Protein Photoperiod-Independent Early Flowering 1 (PIE1) genes. Genetic diversity and genome wide analyses of photoperiod sensitivity in diverse cotton germplasms will enable the use of genomic tools to systematically utilize the tropical germplasm and its beneficial alleles for broadening the genetic base in Upland cotton.

Identification and genomic characterization of major effect bacterial blight resistance locus (BB-13) in Upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Identification and genomic characterization of major resistance locus against cotton bacterial blight (CBB) using GWAS and linkage mapping to enable genomics-based development of durable CBB resistance and gene discovery in cotton. Cotton bacterial leaf blight (CBB), caused by Xanthomonas citri subsp. malvacearum (Xcm), has periodically been a damaging disease in the USA. Identification and deployment of genetic resistance in cotton cultivars is the most economical and efficient means of reducing crop losses due to CBB. In the current study, genome-wide association study (GWAS) of CBB resistance using an elite diversity panel of 380 accessions, genotyped with the cotton single nucleotide polymorphism (SNP) 63 K array, and phenotyped with race-18 of CBB, localized the CBB resistance to a 2.01-Mb region in the long arm of chromosome D02. Molecular genetic mapping using an F6 recombinant inbred line (RIL) population showed the CBB resistance in cultivar Arkot 8102 was controlled by a single locus (BB-13). The BB-13 locus was mapped within the 0.95-cM interval near the telomeric region in the long arm of chromosome D02. Flanking SNP markers, i04890Gh and i04907Gh of the BB-13 locus, identified from the combined linkage analysis and GWAS, targeted it to a 371-Kb genomic region. Candidate gene analysis identified thirty putative gene sequences in the targeted genomic region. Nine of these putative genes and two NBS-LRR genes adjacent to the targeted region were putatively involved in plant disease resistance and are possible candidate genes for BB-13 locus. Genetic mapping and genomic targeting of the BB13 locus in the current study will help in cloning the CBB-resistant gene and establishing the molecular genetic architecture of the BB-13 locus towards developing durable resistance to CBB in cotton.

Evaluation and genome-wide association study of resistance to bacterial blight race 18 in U.S. Upland cotton germplasm.

Bacterial blight (BB), caused by Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease to cotton production in many countries. In the U.S., Xcm race 18 is the most virulent and widespread race and can cause serious yield losses. Planting BB-resistant cotton cultivars is the most effective method of controlling this disease. In this study, 335 U.S. Upland cotton accessions were evaluated for resistance to race 18 using artificial inoculations by scratching cotyledons on an individual plant basis in a greenhouse. The analysis of variance detected significant genotypic variation in disease incidence, and 50 accessions were resistant including 38 lines with no symptoms on either cotyledons or true leaves. Many of the resistant lines were developed in the MAR (multi-adversity resistance) breeding program at Texas A&M University, whereas others were developed before race 18 was first reported in the U.S. in 1973, suggesting a broad base of resistance to race 18. A genome-wide association study (GWAS) based on 26,301 single nucleotide polymorphic (SNP) markers detected 11 quantitative trait loci (QTL) anchored by 79 SNPs, including three QTL on each of the three chromosomes A01, A05 and D02, and one QTL on each of D08 and D10. This study has identified a set of obsolete Upland germplasm with resistance to race 18 and specific chromosomal regions delineated by SNPs for resistance. The results will assist in breeding cotton for BB resistance and facilitate further genomic studies in fine mapping resistance genes to enhance the understanding of the genetic basis of BB resistance in cotton.

A genome-wide association study uncovers consistent quantitative trait loci for resistance to Verticillium wilt and Fusarium wilt race 4 in the US Upland cotton.

KEY MESSAGE: A high-resolution GWAS detected consistent QTL for resistance to Verticillium wilt and Fusarium wilt race 4 in 376 U.S. Upland cotton accessions based on six independent replicated greenhouse tests. Verticillium wilt (VW, caused by Verticillium dahliae Kleb.) and Fusarium wilt (FOV, caused by Fusarium oxysporum f.sp. vasinfectum Atk. Sny & Hans) are the most important soil-borne fungal diseases in cotton. To augment and refine resistance quantitative trait loci (QTL), we conducted a genome-wide association study (GWAS) using high-density genotyping with the CottonSNP63K array. Resistance of 376 US Upland cotton accessions to a defoliating VW and virulent FOV4 was evaluated in four and two independent replicated greenhouse tests, respectively. A total of 15 and 13 QTL for VW and FOV4 resistances were anchored by 30 (on five chromosomes) and 56 (on six chromosomes) significant single nucleotide polymorphic (SNPs) markers, respectively. QTL on c8, c10, c16, and c21 were consistent in two or more tests for VW resistance, while two QTL on c8 and c14 were consistent for FOV4 resistance in two tests. Two QTL clusters on c16 and c19 were observed for both VW and FOV4 resistance, suggesting that these genomic regions may harbor genes in response to both diseases. Using BLAST search against the sequenced TM-1 genome, 30 and 35 candidate genes were identified on four QTL for VW resistance and on three QTL for FOV4 resistance, respectively. These genomic regions were rich in NBS-LRR genes presented in clusters. The results create opportunities for further studies to determine the correlations of field resistance with these QTL, molecular examinations of VW and FOV4 resistances, marker-assisted selection (MAS) and eventual cloning of QTL for disease resistance in cotton.

High-density linkage map construction and QTL analyses for fiber quality, yield and morphological traits using CottonSNP63K array in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Improving fiber quality and yield are the primary research objectives in cotton breeding for enhancing the economic viability and sustainability of Upland cotton production. Identifying the quantitative trait loci (QTL) for fiber quality and yield traits using the high-density SNP-based genetic maps allows for bridging genomics with cotton breeding through marker assisted and genomic selection. In this study, a recombinant inbred line (RIL) population, derived from cross between two parental accessions, which represent broad allele diversity in Upland cotton, was used to construct high-density SNP-based linkage maps and to map the QTLs controlling important cotton traits. RESULTS: Molecular genetic mapping using RIL population produced a genetic map of 3129 SNPs, mapped at a density of 1.41 cM. Genetic maps of the individual chromosomes showed good collinearity with the sequence based physical map. A total of 106 QTLs were identified which included 59 QTLs for six fiber quality traits, 38 QTLs for four yield traits and 9 QTLs for two morphological traits. Sub-genome wide, 57 QTLs were mapped in A sub-genome and 49 were mapped in D sub-genome. More than 75% of the QTLs with favorable alleles were contributed by the parental accession NC05AZ06. Forty-six mapped QTLs each explained more than 10% of the phenotypic variation. Further, we identified 21 QTL clusters where 12 QTL clusters were mapped in the A sub-genome and 9 were mapped in the D sub-genome. Candidate gene analyses of the 11 stable QTL harboring genomic regions identified 19 putative genes which had functional role in cotton fiber development. CONCLUSION: We constructed a high-density genetic map of SNPs in Upland cotton. Collinearity between genetic and physical maps indicated no major structural changes in the genetic mapping populations. Most traits showed high broad-sense heritability. One hundred and six QTLs were identified for the fiber quality, yield and morphological traits. Majority of the QTLs with favorable alleles were contributed by improved parental accession. More than 70% of the mapped QTLs shared the similar map position with previously reported QTLs which suggest the genetic relatedness of Upland cotton germplasm. Identification of QTL clusters could explain the correlation among some fiber quality traits in cotton. Stable and major QTLs and QTL clusters of traits identified in the current study could be the targets for map-based cloning and marker assisted selection (MAS) in cotton breeding. The genomic region on D12 containing the major stable QTLs for micronaire, fiber strength and lint percentage could be potential targets for MAS and gene cloning of fiber quality traits in cotton.

Screening Germplasm and Quantification of Components Contributing to Thrips Resistance in Cotton.

Three hundred and ninety-one Gossypium hirsutum and 34 Gossypium barbadense accessions were screened for thrips resistance under field conditions at the Upper Coastal Plain Research Station in Rocky Mount, North Carolina in years 2014 and 2015. Visual damage ratings, thrips counts, and seedling dry weights were recorded at 2.5, 3.5, and 4.5 wk after planting, respectively. Population density and thrips arrival times varied between years. Data from the three separate damage scoring dates provided a better estimate of resistance or susceptibility to thrips than ratings from the individual dates over the season. Tobacco thrips [Frankliniella fusca (Hinds) (Thysanoptera: Thripidae)], followed by western flower thrips [Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)], were the dominant thrips species observed in the study. Five resistant G. barbadense accessions and five moderately resistant upland cotton accessions were identified from field evaluations. Greenhouse experiments were conducted in Fall 2015 and Spring 2016 to determine if plant height, growth rate, leaf pubescence, and leaf area were significantly different in resistant and susceptible groups of G. hirsutum and G. barbadense accessions identified from the field screenings. Leaf pubescence and relative growth rate were significantly higher in resistant accessions compared with susceptible accessions in absence of thrips. There was no difference for plant height and leaf area between resistant and susceptible groups. Results suggest thrips-resistant plants have a possible competitive advantage through faster growth and higher trichome density, which limits thrips movement.

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.

Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of Upland cotton (Gossypium hirsutum L.).

Leaf shape varies spectacularly among plants. Leaves are the primary source of photoassimilate in crop plants, and understanding the genetic basis of variation in leaf morphology is critical to improving agricultural productivity. Leaf shape played a unique role in cotton improvement, as breeders have selected for entire and lobed leaf morphs resulting from a single locus, okra (l-D1), which is responsible for the major leaf shapes in cotton. The l-D1 locus is not only of agricultural importance in cotton, but through pioneering chimeric and morphometric studies, it has contributed to fundamental knowledge about leaf development. Here we show that an HD-Zip transcription factor homologous to the LATE MERISTEM IDENTITY1 (LMI1) gene of Arabidopsis is the causal gene underlying the l-D1 locus. The classical okra leaf shape allele has a 133-bp tandem duplication in the promoter, correlated with elevated expression, whereas an 8-bp deletion in the third exon of the presumed wild-type normal allele causes a frame-shifted and truncated coding sequence. Our results indicate that subokra is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele and that normal is a derived mutant allele that came to predominate and define the leaf shape of cultivated cotton. Virus-induced gene silencing (VIGS) of the LMI1-like gene in an okra variety was sufficient to induce normal leaf formation. The developmental changes in leaves conferred by this gene are associated with a photosynthetic transcriptomic signature, substantiating its use by breeders to produce a superior cotton ideotype.

Mapping and genomic targeting of the major leaf shape gene (L) in Upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: A major leaf shape locus (L) was mapped with molecular markers and genomically targeted to a small region in the D-genome of cotton. By using expression analysis and candidate gene mapping, two LMI1 -like genes are identified as possible candidates for leaf shape trait in cotton. Leaf shape in cotton is an important trait that influences yield, flowering rates, disease resistance, lint trash, and the efficacy of foliar chemical application. The leaves of okra leaf cotton display a significantly enhanced lobing pattern, as well as ectopic outgrowths along the lobe margins when compared with normal leaf cotton. These phenotypes are the hallmark characteristics of mutations in various known modifiers of leaf shape that culminate in the mis/over-expression of Class I KNOX genes. To better understand the molecular and genetic processes underlying leaf shape in cotton, a normal leaf accession (PI607650) was crossed to an okra leaf breeding line (NC05AZ21). An F2 population of 236 individuals confirmed the incompletely dominant single gene nature of the okra leaf shape trait in Gossypium hirsutum L. Molecular mapping with simple sequence repeat markers localized the leaf shape gene to 5.4 cM interval in the distal region of the short arm of chromosome 15. Orthologous mapping of the closely linked markers with the sequenced diploid D-genome (Gossypium raimondii) tentatively resolved the leaf shape locus to a small genomic region. RT-PCR-based expression analysis and candidate gene mapping indicated that the okra leaf shape gene (L (o) ) in cotton might be an upstream regulator of Class I KNOX genes. The linked molecular markers and delineated genomic region in the sequenced diploid D-genome will assist in the future high-resolution mapping and map-based cloning of the leaf shape gene in cotton.

Genetic diversity and population structure in the US Upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genetic diversity and population structure in the US Upland cotton was established and core sets of allelic richness were identified for developing association mapping populations in cotton. Elite plant breeding programs could likely benefit from the unexploited standing genetic variation of obsolete cultivars without the yield drag typically associated with wild accessions. A set of 381 accessions comprising 378 Upland (Gossypium hirsutum L.) and 3 G. barbadense L. accessions of the United States cotton belt were genotyped using 120 genome-wide SSR markers to establish the genetic diversity and population structure in tetraploid cotton. These accessions represent more than 100 years of Upland cotton breeding in the United States. Genetic diversity analysis identified a total of 546 alleles across 141 marker loci. Twenty-two percent of the alleles in Upland accessions were unique, specific to a single accession. Population structure analysis revealed extensive admixture and identified five subgroups corresponding to Southeastern, Midsouth, Southwest, and Western zones of cotton growing areas in the United States, with the three accessions of G. barbadense forming a separate cluster. Phylogenetic analysis supported the subgroups identified by STRUCTURE. Average genetic distance between G. hirsutum accessions was 0.195 indicating low levels of genetic diversity in Upland cotton germplasm pool. The results from both population structure and phylogenetic analysis were in agreement with pedigree information, although there were a few exceptions. Further, core sets of different sizes representing different levels of allelic richness in Upland cotton were identified. Establishment of genetic diversity, population structure, and identification of core sets from this study could be useful for genetic and genomic analysis and systematic utilization of the standing genetic variation in Upland cotton.

Molecular genetic description of the cryptic wheat-Aegilops geniculata introgression carrying rust resistance genes Lr57 and Yr40 using wheat ESTs and synteny with rice.

The cryptic wheat-alien translocation T5DL.5DS-5MgS(0.95), with leaf rust and stripe rust resistance genes Lr57 and Yr40 transferred from Aegilops geniculata (UgMg) into common wheat, was further analyzed. Molecular genetic analysis using physically mapped ESTs showed that the alien segment in T5DL.5DS-5MgS(0.95) represented only a fraction of the wheat deletion bin 5DS2-0.78-1.00 and was less than 3.3 cM in length in the diploid wheat genetic map. Comparative genomic analysis indicated a high level of colinearity between the distal region of the long arm of chromosome 12 of rice and the genomic region spanning the Lr57 and Yr40 genes in wheat. The alien segment with genes Lr57 and Yr40 corresponds to fewer than four overlapping BAC or PAC clones of the syntenic rice chromosome arm 12L. The wheat-alien translocation breakpoint in T5DL.5DS-5MgS(0.95) was further localized to a single BAC clone of the syntenic rice genomic sequence. The small size of the terminal wheat-alien translocation, as established precisely with respect to Chinese Spring deletion bins and the syntenic rice genomic sequence, further confirmed the escaping nature of cryptic wheat-alien translocations in introgressive breeding. The molecular genetic resources and information developed in the present study will facilitate further fine-scale physical mapping and map-based cloning of the Lr57 and Yr40 genes.

The major threshability genes soft glume (sog) and tenacious glume (Tg), of diploid and polyploid wheat, trace their origin to independent mutations at non-orthologous loci.

Threshability is an important crop domestication trait. The wild wheat progenitors have tough glumes enveloping the floret that make spikes difficult to thresh, whereas cultivated wheats have soft glumes and are free-threshing. In hexaploid wheat, the glume tenacity gene Tg along with the major domestication locus Q control threshability. The Q gene was isolated recently and found to be a member of the AP2 class of transcription factors. However, only a few studies have reported on the tough glume trait. Here, we report comparative mapping of the soft glume (sog) gene of diploid Triticum monococcum L. and tenacious glume (Tg) gene of hexaploid T. aestivum L. using chromosome-specific SSR and RFLP markers. The sog gene was flanked by Xgwm71 and Xbcd120 in a 6.8 cM interval on chromosome 2A(m)S of T. monococcum whereas Tg was targeted to a 8.1 cM interval flanked by Xwmc503 and Xfba88 on chromosome 2DS of T. aestivum. Deletion bin mapping of the flanking markers assigned sog close to the centromere on 2AS, whereas Tg was mapped to the most distal region on 2DS. Both 2AS and 2DS maps were colinear ruling out the role of chromosome rearrangements for their non-syntenic positions. Therefore, sog and Tg are not true orthologues suggesting the possibility of a diverse origin.

Genomic targeting and mapping of tiller inhibition gene (tin3) of wheat using ESTs and synteny with rice.

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3AmL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat-rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene.

Characterization and mapping of cryptic alien introgression from Aegilops geniculata with new leaf rust and stripe rust resistance genes Lr57 and Yr40 in wheat.

Leaf rust and stripe rust are important foliar diseases of wheat worldwide. Leaf rust and stripe rust resistant introgression lines were developed by induced homoeologous chromosome pairing between wheat chromosome 5D and 5M(g) of Aegilops geniculata (U(g)M(g)). Characterization of rust resistant BC(2)F(5) and BC(3)F(6) homozygous progenies using genomic in situ hybridization with Aegilops comosa (M) DNA as probe identified three different types of introgressions; two cytologically visible and one invisible (termed cryptic alien introgression). All three types of introgression lines showed similar and complete resistance to the most prevalent pathotypes of leaf rust and stripe rust in Kansas (USA) and Punjab (India). Diagnostic polymorphisms between the alien segment and recipient parent were identified using physically mapped RFLP probes. Molecular mapping revealed that cryptic alien introgression conferring resistance to leaf rust and stripe rust comprised less than 5% of the 5DS arm and was designated T5DL.5DS-5M(g)S(0.95). Genetic mapping with an F(2)population of Wichita x T5DL.5DS-5M(g)S(0.95) demonstrated the monogenic and dominant inheritance of resistance to both diseases. Two diagnostic RFLP markers, previously mapped on chromosome arm 5DS, co-segregated with the rust resistance in the F(2) population. The unique map location of the resistant introgression on chromosome T5DL.5DS-5M(g)S(0.95) suggested that the leaf rust and stripe rust resistance genes were new and were designated Lr57 and Yr40. This is the first documentation of a successful transfer and characterization of cryptic alien introgression from Ae. geniculata conferring resistance to both leaf rust and stripe rust in wheat.

Identification and mapping of a tiller inhibition gene (tin3) in wheat.

Tillering is one of the most important agronomic traits in cereal crops because tiller number per plant determines the number of spikes or panicles per plant, a key component of grain yield and/or biomass. In order to characterize the underlying genetic variation for tillering, we have isolated mutants that are compromised in tillering ability using ethyl methanesulphonate (EMS)-based mutagenesis in diploid wheat (Triticum monococcum subsp. monococcum). The tillering mutant, tiller inhibition (tin3) produces only one main culm compared to the wild type with many tillers. The monoculm phenotype of tin3 is due to a single recessive mutation. Genetic and molecular mapping in an F(2) population of diploid wheat located the tin3 gene on the long arm of chromosome 3A(m). One codominant RFLP marker Xpsr1205 cosegregated with tin3 in the F(2) population. Physical mapping of PSR1205 in a set of Chinese Spring deletion lines of group-3 chromosomes placed the tin3 gene in the distal 10% of the long arm of chromosome 3A, which is a recombination-rich region in wheat. The implications of the mapping of tin3 on chromosome arm 3A(m)L are discussed with respect to putative orthologs of tin3 in the 3L colinear regions across various cereal genomes and other tillering traits in grasses.