Xiaoping Guo

QTL mapping for economic traits based on a dense genetic map of cotton with PCR-based markers using the interspecific cross of Gossypium hirsutum × Gossypium barbadense

A high-density molecular marker linkage map of cotton based entirely on polymerase chain reaction-based markers is useful for a marker-assisted breeding program. Four kinds of markers—simple sequence repeats (SSRs), sequence-related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and retrotransposon-microsatellite amplified polymorphism (REMAP)—were used to assay an F2 population from a cross between “Handan208” (Gossypium hirsutum) and “Pima90” (Gossypium barbadense). Sixty-nine F2 plants were used for map construction using 834 SSRs, 437 SRAPs, 107 RAPDs, and 16 REMAPs. Linkage analysis revealed that 1,029 loci could be mapped to 26 linkage groups that extended for 5,472.3xa0cM, with an average distance between 2 loci of 5.32xa0cM. The corresponding 69 F2:3 families were grown, arranged in two replicates, and scored for eight phenotypes. Quantitative trait loci (QTL) analysis was performed by means of composite interval mapping using WinQtlCart ver 2.0. A total of 52 distinct QTLs were detected: 4 QTLs for lint index, 8 for seed index, 11 for lint yield, 4 for seed cotton yield, 9 for number of seed per boll, 3 for fiber strength, 5 for fiber length, and 8 for micronaire value. The present map and QTL analysis may provide a useful tool for breeders to transfer desirable traits from G. barbadense to the mainly cultivated species, G. hirsutum.

Isolation and Characterization of Genes Associated to Cotton Somatic Embryogenesis by Suppression Subtractive Hybridization and Macroarray

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and preglobular somatic embryos, as the tester, and cDNA from nonembryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 unigenes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, posttranscription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from proembryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pΔgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 108 cells ml−1 (OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l−1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.

Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, but its fiber development mechanism has not been surveyed comprehensively. We constructed a normalized fiber cDNA library (from −2 to 25xa0dpa) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA. We screened Pima 3-79 fiber RNA from five developmental stages using a cDNA array including 9,126 plasmids randomly selected from the library, and we selected and sequenced 929 clones that had different signal intensities between any two stages. The 887 high-quality expressed sequence tags obtained were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20-dpa RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, seven types of expression profiles were elucidated. Furthermore our results showed that phytohormones may play an important role in the fiber development.

Genetic Diversity of Asian Cotton (Gossypium arboreum L.) in China Evaluated by Microsatellite Analysis

Asian cotton (Gossypium arboreum L.) was once widely cultivated in China. It has also been a valuable source of genetic variation in modern cotton improvement. In this study, the genetic diversity of selected G. arboreum accessions collected from different regions of China was evaluated by microsatellite (simple sequence repeats, SSRs) analysis. Of the 358 microsatellite markers analyzed, 74 primer pairs detected 165 polymorphic DNA fragments among 39 G. arboreum accessions examined. Twelve accessions could be fingerprinted with one or more SSR markers. With the exception of two accessions, DaZiJie and DaZiMian, genetic similarity coefficients among all accessions ranged from 0.58 to 0.87 suggesting high level of genetic variation in the G. arboreum collections. The UPGMA dendrogram constructed from genetic similarity coefficients revealed positive correlation between cluster groupings and geographic distances. In addition, comparison of the microsatellite amplification profiles of the diploid G. arboreum and tetraploid Gossypium hirsutum L. found that size distribution of amplified products in G. arboreum was dispersive and that of G. hirsutum was relatively concentrated. The information on the genetic diversity and SSR fingerprinting from this study is useful for developing mapping populations for constructing diploid cotton genetic linkage map and tagging economically important traits.

Mapping and quantitative trait loci analysis of verticillium wilt resistance genes in cotton.

Verticillium wilt is one of the most serious constraints to cotton production in almost all of the cotton-growing countries. In this study, XinLuZao1 (XLZ1), a susceptible cultivar Gossypium hirsutum L. and Hai7124 (H7124), a resistant line G. barbadense, and their F(2:3) families were used to map and study the disease index induced by verticillium wilt. A total of 430 SSR loci were mapped into 41 linkage groups; the map spanned 3,745.9 cM and the average distance between adjacent loci was 8.71 cM. Four and five quantitative trait loci (QTLs) were detected based on the disease index investigated on July 22 and August 24 in 2004, respectively. These nine QTLs explained 10.63-28.83% of the phenotypic variance, six of them were located on the D sub-genome. Two QTLs located in the same marker intervals may partly explain the significant correlation of the two traits. QTLs explaining large phenotypic variation were identified in this study, which may be quite useful in cotton anti-disease breeding.

A high-density integrative linkage map for Gossypium hirsutum

Sequence-related amplified polymorphism (SRAP) combined with SSRs, RAPDs, and RGAPs was used to construct a high density genetic map for a F2 population derived from the cross DH962 (G. hirsutum accession)xa0×xa0Jimian5 (G. hirsutum cultivar). A total of 4,096 SRAP primer combinations, 6310 SSRs, 600 RAPDs, and 10 RGAPs produced 331, 156, 17 and 2 polymorphic loci, respectively. Among the 506 loci obtained, 471 loci (309 SRAPs, 144 SSRs, 16 RAPDs and 2 RGAPs) were assigned to 51 linkage groups. Of these, 29 linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 3070.2xa0cM with a mean density of 6.5xa0cM per locus. The segregation distortion in this population was 9.49%, and these distorted loci tend to cluster at the end of linkage groups or in minor clusters on linkage groups. The majority of SRAPs in this map provided an effective tool for map construction in G. hirsutum despite of its low polymorphism. This high-density linkage map will be useful for further genetic studies in Upland cotton, including mapping of loci controlling quantitative traits, and comparative and integrative analysis with other interspecific and intraspecific linkage maps in cotton.

Mapping QTLs of traits contributing to yield and analysis of genetic effects in tetraploid cotton

Fiber yield and yield components – including lint index (LI), seed index (SI), lint yield (LY), seed cotton yield (SCY) and number of seeds per boll (NSPB) – were investigated on the farm of Huazhong Agricultural University in a population of 69 F2 individuals and corresponding F2:3 families derived from a cross between high-fiber-yield Gossypium hirsutum CV Handan 208 and a low-fiber-yield Gossypium barbadense CV Pima 90. On the basis of the genetic map constructed previously from the same population by Lin et al. (Plant Breed., 2005), quantitative trait locus (QTL) analysis was performed with the software QTL Cartographer V2.0 using composite interval mapping method (LOD ≥ 3.0). A total of 21 QTLs were identified, which were located in 15 linkage groups. The number of QTLs per trait ranged from one to seven. Of these QTLs detected, one affecting LI explained 24.3% of phenotypic variation (PV), five influencing SI explained 16.15–39.21% of PV, seven controlling LY explained 13.01–28.35% of PV, and two controlling SCY explained 22.76 and 39.97% of PV, respectively. Simultaneously, the detected six QTLs for NSPB were located on five linkage groups, which individually explained 28.01–38.32% of the total phenotypic variation. The results would give breeders further insight into the genetic basis of fiber yield.

Somatic embryogenesis and plant regeneration from different wild diploid cotton ( Gossypium ) species

Calli were successfully induced from hypocotyls of eight wild diploid cotton species (Gossypium) on MSB (MS salts and B5 vitamins) medium supplemented with 0.09xa0μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.32xa0μM KT (kinetin). Plant growth regulator (PGR) combinations, adding GA3 (Gibberellic acid), high inorganic salt stress, and PGR-free media were used to induce embryogenic calli from nonembryogenic calli. Embryogenic cultures were induced from G. aridum S. (D4 genome), G. davidsonii K. (D3-d genome), G. klotzschianum A. (D3-k genome), G. raimondii U. (D5 genome), and G. stocksii M. (E1 genome). We then observed somatic embryogenesis in the five species while calli of G. africanum V. (A1-2 genome), G. anomalum W. (B1 genome), and G. bickii P. (G genome) remained nonembryogenic. Somatic embryogenesis was adjusted by changing sugar sources, regulating combinations of PGRs, and using cell suspension culture. Embryos at various developmental stages produced mature and germinating embryos when cultured on filter paper placed on the media containing different sugar sources. The utility of different sugar sources promoted globular embryos developing into cotyledonary stage and increased the frequency of cotyledonary embryos developing into normal plants. Normal plantlets were regenerated from G. davidsonii, G. klotzschianum, G. raimondii, and G. stocksii. Only abnormal plantlets were obtained in G. aridum. This work will contribute to broadening the number of regenerable cotton species and provide foundations for somatic hybridization in cotton to create new germplasm.