Isolation, characterization and mapping of genes differentially expressed during fibre development between Gossypium hirsutum and G. barbadense by cDNA-SRAP

Gossypium hirsutum and G. barbadense are two cultivated tetraploid cotton species with differences in fibre quality. The fibre of G. barbadense is longer, stronger and finer than that of G. hirsutum. To isolate genes expressed differently between the two species during fibre development, cDNA-SRAP (sequence-related amplified polymorphism) was applied. This technique was used to analyse genes at different stages of fibre development in G. hirsutum cv. Emian22 and G. barbadense acc. 3-79, the parents of our interspecific mapping population. A total of 4096 SRAP primer combinations were used to screen polymorphism between the DNA of the parents, and 275 highly polymorphic primers were picked out to analyse DNA and RNA from leaves and fibres at different developmental stages of the parents. A total of 168 DNA fragments were isolated from gels and sequenced: 54, 30, 38 and 41 from fibres of 5, 10, 15 and 20 days post-anthesis, respectively, and five from multi stages. To genetically map these sequences, 104 sequence-specific primers were developed and were used to screened polymorphism between the mapping parents. Finally, six markers were mapped on six chromosomes of our backbone interspecific genetic map. This work can give us a primary knowledge of differences in mechanism of fibre development between G. hirsutum and G. barbadense.     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.     

Genetic dissection of lint yield and fiber quality traits of <Emphasis Type="Italic">G. hirsutum</Emphasis> in <Emphasis Type="Italic">G. barbadense</Emphasis> background

Gossypium hirsutum L. is a widely cultivated species characterized by its high yield and wide environmental adaptability, while Gossypium barbadense is well known for its superior fiber quality. In the present report, we, for the first time, developed G. hirsutum chromosome segment introgression lines (ILs) in a G. barbadense background (GhILs_Gb) and genetically dissected the inheritance of lint yield and fiber quality of G. hirsutum in G. barbadense background. The GhILs_Gb contains introgressed segments spanning 4121.20 cM, which represents 82.20% of the tetraploid cotton genome, with an average length of 18.65 cM. A total of 39 quantitative trait loci (QTLs) for six traits are identified in this IL population planted in Xinjiang. Four QTL clusters are detected. Of them, however, three clusters have deleterious effects on fiber length and strength and boll weight, and only one cluster on Chr. D9 can be used in marker-assisted selection (MAS) to increase lint percentage and decrease micronaire value in G. barbadense. QTL mapping showed that most of yield-related QTLs detected have positive effects and increase lint yield in G. barbadense, while most of fiber quality-related QTLs have deleterious effects except for micronaire. It suggested that G. hirsutum evolved to have a high lint yield. Several lines improved in lint percentage and boll size in G. barbadense by introgressed one fragment of G. hirsutum have been developed from the GhILs_Gb. The ILs developed, and the analyses presented here will enhance the understanding of the genetics of lint yield and fiber quality in G. hirsutum and facilitate further molecular breeding to improve lint yield in G. barbadense.     

Bidirectional cytoplasmic and nuclear introgression in the New World cottons Gossypium barbadense and G. Hirsutum (Malvaceae)

Patterns of interspecific cytoplasmic (plastid and mitochondrial) and nuclear introgression are typically asymmetrical: cytoplasmic gene flow or “capture” is frequently observed without evidence of nuclear introgression. In contrast, nuclear introgression without concomitant cytoplasmic introgression has rarely been demonstrated. Gossypium barbadense L. and G. hirsutum L. have large indigenous ranges in the New World semiarid tropics, with an extensive area of sympatry in the Caribbean and Central America. Numerous accessions of both species were surveyed for diagnostic cpDNA restriction sites. These data, in conjunction with previous information on nuclear markers, lead to several conclusions: 1) introgression between G. hirsutum and G. barbadense is bidirectional for both nuclear and cytoplasmic genes; 2) patterns of introgression between the two species are not symmetrical—in G. barbadense, introgression of G. hirsutum alleles is largely restricted to modem cultivars and is uncommon in areas of sympatry; in contrast, introgression of G. barbadense alleles into G. hirsutum is relatively common in areas of sympatry and is rare in modem cultivars; 3) nuclear introgression is geographically more widespread and more frequently detected than cytoplasmic introgression. Several mechanisms may underlie the differential patterns of cytoplasmic and nuclear gene flow observed, including differential fitness of infraspecific and interspecific cytonuclear combinations and selection against female function in interspecific backcrosses. Possible explanations for the observed asymmetrical patterns of introgression include differences in population sizes combined with phenological differences that bias interspecific pollen transfer.     

A Comparison of Leaf Anatomy in Field-grown Gossypium hirsutum and G. barbadense

Gossypium hirsutum L. (upland cotton) and G. barbadense L. (Pimacotton) are two of the most important fibre producing cottonspecies in cultivation. When grown side-by-side in the field,G.hirsutum has higher photosynthetic and transpiration rates (Luet al., 1997. Australian Journal of Plant Physiology24: 693–700).The present study was undertaken to determine if the differencesin physiology can be explained by leaf and canopy morphologyand anatomy. Scanning electron microscopy was used to comparethe leaf anatomy of field-grown upland (‘Delta’and ‘Pine Land 50’) and Pima (‘S6’)cotton. Compared to G. hirsutum, mature leaves of G. barbadenseare larger and thinner, with a thinner palisade layer. G. barbadenseleaves show significant cupping or curling which allows fora more even absorption of insolation over the course of theday and much more light penetration into the canopy. AlthoughG. barbadense leaves have a 70–78% higher stomatal densityon both the abaxial and the adaxial surfaces, its stomates areonly one third the size of those of G. hirsutum. This resultsin G. barbadense having only about 60% of the stomatal surfacearea per leaf surface area compared to G. hirsutum. These resultsare indicative of the anatomical and physiological differencesthat may limit the yield potential of G. barbadense in certaingrowing environments. Copyright 2000 Annals of Botany Company Cotton, leaf anatomy, leaf development, photosynthesis, Gossypium hirsutum, Gossypium barbadense, stomatal density     

Postinfection Development of Rotylenchulus reniformis on Resistant Gossypium barbadense Accessions

The reniform nematode (Rotylenchulus reniformis) causes significant cotton (Gossypium hirsutum) losses in the southeastern United States. The research objective was to describe the effects of two resistant G. barbadense lines (cultivar TX 110 and accession GB 713) on development and fecundity of reniform nematode. Nematode development and fecundity were evaluated on the resistant lines and susceptible G. hirsutum cultivar Deltapine 16 in three repeated growth chamber experiments. Nematode development on roots early and late in the infection cycle was measured at set intervals from 1 to 25 d after inoculation (DAI) and genotypes were compared based on the number of nematodes in four developmental stages (vermiform, swelling, reniform, and gravid). At 15, 20, and 25 DAI, egg production by individual females parasitizing each genotype was measured. Unique reniform nematode developmental patterns were noted on each of the cotton genotypes. During the early stages of infection, infection and development occurred 1 d faster on susceptible cotton than on the resistant genotypes. Later, progression to the reniform and gravid stages of development occurred first on the susceptible genotype, followed by G. barbadense cultivar TX 110, and finally G. barbadense accession GB 713. Egg production by individual nematodes infecting the three genotypes was similar. This study corroborates delayed development previously reported on G. barbadense cultivar TX 110 and is the first report of delayed infection and development associated with G. barbadense accession GB 713. The different developmental patterns in the resistant genotypes suggest that unique or additional loci may confer resistance in these two lines.     

Genome-wide identification and characterization of HSP70 gene family in four species of cotton

Heat shock proteins (HSPs) are important elements of the cellular group of molecular chaperones. Specifically, HSP70 proteins protect cells from being damaged when plants are exposed to environmental stresses. These proteins are catalysts that manage the correct folding of other proteins, and they play a key role in the development of tolerance against biotic and abiotic stresses. In the present study, 113 HSP70 genes were retrieved from the available genome assemblies of four cotton species, including Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. The HSP70 genes were clustered into 11 subfamilies based on phylogeny. One hundred and nine (109) gene duplications were found across these four species. Localization of genes revealed that several HSP70 genes reside in the cytoplasm. Synonymous and non-synonymous substitution rates revealed that functional segregation of HSP70 genes in cotton is due to purifying selection. Furthermore, HSP70 genes in cotton are expressed constitutively during developmental stages. These findings are valuable to understand the complex mechanism of HSP70 gene regulation that occurs in signaling pathways in response to plant stress.     

miR482 Regulation of NBS-LRR Defense Genes during Fungal Pathogen Infection in Cotton

In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the D_t (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci (gra-miR482d, f-i) in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.     

Resistant Germplasm in Gossypium Species and Related Plants to Rotylenchulus reniformis

Gossypium hirsutum, G. herbaceum, G. arboreum, G. barbadense, wild Gossypium spp., Hibiscus spp, and other Malvaceae were tested in the greenhouse to identify germplasm resistant to Rotylenchulus reniformis (Rr). Host resistance was based on Rr egg production per gram of root compared with known G. hirsutum susceptible ''Deltapine 16'' as check. G. longicalyx and Sida rhombifolia were nonhosts. High levels of resistance were found in G. stocksii, G. somalense, and G. barbadense ''Texas 110.'' Other cotton lines with potential value in breeding for Rr resistance were G. herbaceum P.I. 408775; G. arboreum P.I. 41895, P.I, 417891, CB 3839; and G. hirsutum 893. All these supported less than 20% of the egg production on the check. Seventy-three percent of the Hibiscus spp. tested were resistant. Female development and egg production reflected host resistance; healthy females and large egg masses were observed on susceptible plants, and degenerated females and small egg masses on resistant plants. Females penetrating nonhost G. longicalyx never matured to kidney shape.     

SSR marker-assisted improvement of fiber qualities in Gossypium hirsutum using G. barbadense introgression lines

Key message

This study demonstrates the first practical use of CSILs for the transfer of fiber quality QTLs into Upland cotton cultivars using SSR markers without detrimentally affecting desirable agronomic characteristics.

Abstract

Gossypium hirsutum is characterized by its high lint production and medium fiber quality compared to extra-long staple cotton G. barbadense. Transferring valuable traits or genes from G. barbadense into G. hirsutum is a promising but challenging approach through a traditional interspecific introgression strategy. We developed one set of chromosome segment introgression lines (CSILs), where TM-1, the genetic standard in G. hirsutum, was used as the recipient parent and the long staple cotton G. barbadense cv. Hai7124 was used as the donor parent by molecular marker-assisted selection (MAS). Among them, four CSILs, IL040-A4-1, IL080-D6-1, IL088-A7-3 and IL019-A2-6, found to be associated with superior fiber qualities including fiber length, strength and fineness QTL in Xinjiang were selected and backcrossed, and transferred these QTLs into three commercial Upland cotton cultivars such as Xinluzao (XLZ) 26, 41 and 42 grown in Xinjiang. By backcrossing and self-pollinating twice, five improved lines (3262-4, 3389-2, 3326-3, 3380-4 and 3426-5) were developed by MAS of background and introgressed segments. In diverse field trials, these QTLs consistently and significantly offered additive effects on the target phenotype. Furthermore, we also pyramided two segments from different CSILs (IL080-D6-1 and IL019-A2-6) into cultivar 0768 to accelerate breeding process purposefully with MAS. The improved lines pyramided by these two introgressed segments showed significant additive epistatic effects in four separate field trials. No significant alteration in yield components was observed in these modified lines. In summary, we first report that these CSILs have great potential to improve fiber qualities in Upland cotton MAS breeding programs.     

A BIL Population Derived from G. hirsutum and G. barbadense Provides a Resource for Cotton Genetics and Breeding

To provide a resource for cotton genetics and breeding, an interspecific hybridization between Gossypium hirsutum cv. Emian22 and G. barbadense acc. 3–79 was made. A population of 54 BILs (backcross inbred lines, BC₁F₈) was developed with the aim of transferring G. barbadense genes into G. hirsutum in order to genetically analyze these genes’ function in a G. hirsutum background and create new germplasms for breeding. Preliminary investigation of the morphological traits showed that the BILs had diverse variations in plant architecture, seed size, and fuzz color; the related traits of yield and fiber quality evaluated in 4 environments also showed abundant phenotypic variation. In order to explore the molecular diversity of the BIL population, 446 SSR markers selected at an average genetic distance of 10 cM from our interspecific linkage map were used to genotype the BIL population. A total of 393 polymorphic loci accounting for 84.4% MAF (major allele frequency) > 0.05 and 922 allele loci were detected, and the Shannon diversity index (I) was 0.417 per locus. The average introgression segment length was 16.24 cM, and an average of 29.53 segments were introgressed in each BIL line with an average background recovery of 79.8%. QTL mapping revealed 58 QTL associated with fiber quality and yield traits, and 47 favored alleles derived from the donor parent were discovered. This study demonstrated that the interspecific BIL population was enriched with much phenotypic and molecular variation which could be a resource for cotton genetics and breeding.     

The distribution of Gossypium hirsutum chromatin in G. barbadense germ plasm: molecular analysis of introgressive plant breeding

Cotton is unusual among major crop plants in that two cross-fertile species are widely cultivated for a common economic product, fiber. Both historical evidence and classical genetic studies suggest that many improved forms of Gossypium barbadense (Sea Island, Egyptian, and Pima cottons) may include chromatin derived from G. hirsutum. Using 106 restriction fragment length polymorphism (RFLP) loci well distributed across the cotton genome, we revealed the amount and genomic distribution of G. hirsutum chromatin in 54 G. barbadense collections from around the world. The average G. barbadense collection was comprised of 8.9% alleles apparently derived from G. hirsutum. Pima cultivars (7.3 %) had fewer G. hirsutum alleles than Sea Island (9.0%) or Egyptian (9.6%) cultivars. G. hirsutum alleles were not randomly distributed, as 57.5% of the total introgression observed was accounted for by five specific chromosomal regions that span less than 10% of the genome. The average length of an introgressed chromosome segment was 12.9 cM. Overlap of introgressed chromatin in different breeding programs hints that retention of these G. hirsutum chromosomal segments may impart a selective advantage to G. barbadense genotypes. Although cluster analysis generally grouped germ plasm from common classes and/or breeding programs together, no 2 genotypes were identical — thus differences in the length and repertoire of introgressed chromosome segments also permit DNA fingerprinting of G. barbadense cultivars.     

Identification and expression analysis of phosphatidy ethanolamine-binding protein (PEBP) gene family in cotton

The phosphatidy ethanolamine-binding proteins (PEBP) play an important role in controlling flower development and phase change. Here, a total of 61 PEBP genes were identified, in which 20, 21, 10, and 10 were from tetraploid Gossypium hirsutum (AD1) and G. barbadense (AD2), and diploid G. raimondii (D5) and G. arboreum (A2), respectively. In G. hirsutum, 20 identified PEBP genes were unevenly distributed on 12 chromosomes. The identified PEBP genes were classified into four groups (TFL1, MFT, FT and FT-like). Among those, FT-like group are unique to cotton. The majority of PEBP genes had similar intron/exon distribution, whereas the divergence of PEBP genes suggests the possibility of functional diversification. The expression of PEBP genes varied among different tissues. This study brings new insights into the integrated genome-wide identification of PEBP genes in cotton and provides a foundation for breeding cotton cultivars with early maturation.     

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum

Summary We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.     

Some observations on photoperiodism and the development of annual forms of domesticated cottons

Observations made over the last fifteen to twenty years have discovered no primitive forms of G.barbadense L. orG. hirsutum L. (other than G.hirsutum racelatifolium Hutchinson) which are capable of flowering during the long summer days of temperate latitudes. Annual Upland cottons, grown in the southern United States since the mid-eighteenth century, were probably derived from perennial day-neutral forms of Mexican racelatifolium, though not necessarily by direct introduction from Mexico into the United States. The origin of Sea Island cottons (day-neutral forms of G.barbadense), which were formerly grown on the southeastern seaboard of the United States since the mid-eighteenth century, remains obscure. No primitive forms of this species, capable of flowering during the long summer days of temperate latitudes, have been found. An experiment is reported in which it has been possible to synthesize a day-neutral form of G.barbadense through introgression between primitive short-day sensitive forms of G.barbadense and G.hirsutum, both native to the Caribbean region. It is suggested that the ancestors of Sea Island cottons may have originated naturally by a similar mechanism involving the same species.     

Quantitative trait locus analysis of Verticillium wilt resistance in an introgressed recombinant inbred population of Upland cotton

Verticillium wilt (VW) of Upland cotton (Gossypium hirsutum L.) is caused by the soil-borne fungal pathogen Verticillium dahlia Kleb. The availability of VW-resistant cultivars is vital for control of this economically important disease, but there is a paucity of Upland cotton breeding lines and cultivars with a high level of resistance to VW. In general, G. barbadense L. (source of Pima cotton) is more VW-resistant than Upland cotton. However, the transfer of VW resistance from G. barbadense to Upland cotton is challenging because of hybrid breakdown in the F2 and successive generations of interspecific populations. We conducted two replicated greenhouse studies (tests 1 and 2) to assess the heritability of VW resistance to a defoliating V. dahliae isolate and identify genetic markers associated with VW resistance in an Upland cotton recombinant inbred mapping population that has stable introgression from Pima cotton. Disease ratings at the seedling stage on several different days after the first inoculation (DAI) in test 1, as well as the percentages of infected and defoliated leaves at 2 DAI in test 2, were found to be low to moderately heritable, indicating the importance of a replicated progeny test in selection for VW resistance. With a newly constructed linkage map consisting of 882 simple sequence repeat, single nucleotide polymorphism, and resistance gene analog–amplified fragment length polymorphism marker loci, we identified a total of 21 quantitative trait loci (QTLs) on 11 chromosomes and two linkage groups associated with VW resistance at several different DAIs in greenhouse tests 1 and 2. The markers associated with the VW resistance QTLs will facilitate fine mapping and cloning of VW resistance genes and genomics-assisted breeding for VW-resistant cultivars.