Genetic dissection of tetraploid cotton resistant to Verticillium wilt using interspecific chromosome segment introgression lines

Verticillium wilt(caused by the pathogen Verticillium dahliae) is of high concern for cotton producers and consumers. The major strategy for controlling this disease is the development of resistant cotton(Gossypium spp.) cultivars. We used interspecific chromosome segment introgression lines(CSILs) to identify quantitative trait loci(QTL) associated with resistance to Verticillium wilt in cotton grown in greenhouse and inoculated with three defoliating V. dahliae isolates. A total of 42 QTL, including 23 with resistance-increasing and 19 with resistancedecreasing, influenced host resistance against the three isolates. These QTL were identified and mapped on 18 chromosomes(chromosomes A1, A3, A4, A5, A7, A8, A9, A12, A13, D1, D2,D3, D4, D5, D7, D8, D11, and D12), with LOD values ranging from 3.00 to 9.29. Among the positive QTL with resistance-increasing effect, 21 conferred resistance to only one V. dahliae isolate, suggesting that resistance to V. dahliae conferred by most QTL is pathogen isolate-specific. The At subgenome of cotton had greater effect on resistance to Verticillium wilt than the Dt subgenome. We conclude that pyramiding different resistant QTL could be used to breed cotton cultivars with broad-spectrum resistance to Verticillium wilt.

Construction of a Bacterial Artificial Chromosome Library of TM-1,a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.

Characterizati0n,development and exploitation of EST-derived microsatellites in Gossypium raimondii Ulbrich

Microsatellite DNA or simple sequence repeats (SSRs) can be derived from expressed se- quence tags (ESTs). These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop EST-SSRs for cotton gene map- ping, we selected and characterized functional markers in Gossypium raimondii, which consisted of 58906 non-redundant EST sequences from NCBI. Among them there were 2620 microsatellite se- quences containing 2818 EST-SSRs, which amoun- ted to 4.45% of the non-redundant starting sequence population. This incidence was equivalent to one EST-SSR in every 14.8 kb of G. raimondii genetic material. Among the different motifs ranging from 1 to 6 bp, trinucleotide repeats were most abundant (38.31%), followed by dinucleotide repeats (24.09%) and mononucleotide repeats (23.35%). Among all identified motif types, A/T had the highest frequency (18.67%), followed by AT/TA (14.83%). Among the compound motifs, tandem trinucleotides occurred with the highest frequency (48.65%). In all, we identi- fied 1554 EST-SSRs primer pair sequences. 300 of them were randomly selected to screen the poly- morphisms between the mapping parents G· hirsutum acc. TM-1 and G· barbadense cv. Hai7124, to con- struct linkage groups in cultivated allotetraploid cot- ton. Among them, 129 (43%) primer pairs were found to have polymorphisms. Using these EST-SSRs we can compare EST-SSR distributions among different cotton species and various chromosomal locations.

Development of Gossypium barbadense chromosome segment substitution lines in the genetic standard line TM-1 of Gossypium hirsutum

Chromosome segment substitution lines (CSSL) consist of a battery of nearisogenic lines that have been developed and cover the entire genome of some crops. With the exception of one homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for genome research and particularly QTL mapping. In the present study, we first developed one set of CSSL lines using G. hirsutum acc. TM-1 (the genetic standard), as the recipient parent and G. barbadense cv. Hai7124 as the donor parent using molecular assisted-selection in BC5S1-3 generations. The CSSL consisted of 330 different lines, in which 1-4 different lines had the same or overlapping substituted segments. The genetic length of the substituted segments covered 5271.9 cM with an average segment distance of 10.9 cM, 1.5 times the total genetic length of Upland cotton (3514.6 cM). The substituted segments of each line varied in length, ranging from 3.5 cM for the shortest segment to 23.2 cM in the longest segment. Our CSSL have not yet to cover the entire tetraploid cotton genome, due to the absence of some donor parent interval segments.

Fine mapping of the dominant glandless Gene Gl_2~e in Sea-island cotton (Gossypium barbadense L.)

Gle2 is a mutant gene that controls glandless trait in cotton plants and seeds. It is an important gene resource to gossypol-free cottonseed breeding. The objective of this research was to develop SSR markers tightly linked with Gle2 by using the F2 segregating population containing 1599 plants derived from the cross of G. hirsutum genetic standard line TM-1 and G. barbadense glandless mutant line Hai-1. Genetic analysis suggested that the Gle2 was an incomplete dominant gene. Based on the backbone of genetic linkage map from G. hirsutum × G. barbadense BC1 published by our laboratory,Gle2 was lo-cated between CIR362 and NAU2251b,NAU3860b,STV033,with a genetic distance 9.27 and 0.96 cM,respectively. This result is useful for cloning Gle2 gene by map-based cloning method.

Fine mapping of the red plant gene R_1 in upland cotton (Gossypium hirsutum)

Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chro-mosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R1 gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R1 by using the F2 segregating population containing 1259 plants derived from the cross of Sub 16 and T586 and the backbone genetic linkage map from G. hir-sutum×G. barbadense BC1 newly updated by our laboratory. Genetic analysis suggested that the seg-regation ratio of red plants in the F2 population fit Mendelian 1:2:1 inheritance, confirming that the red plant trait was controlled by an incomplete dominance gene. Preliminary mapping of R1 was conducted using 237 randomLy selected F2 individuals and JoinMap v3.0 software. Then, a fine map of R1 was constructed using the F2 segregating population containing 1259 plants, and R1 was located between NAU4956 and NAU6752, with only 0.49 cM to the nearest maker loci (NAU6752). These results pro-vided a foundation for map-based cloning of R1 and further development of cotton cultivars with red fibers by transgenic technology.

Mining,characterization,and exploitation of EST-derived microsatellites in Gossypium barbadense

Simple sequence repeats(SSRs) have been widely applied as molecular markers in genetic studies.However,the number of expressed sequence tags(ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species.In this study,EST-SSR distribution from G.barbadense was characterized and new G.barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G.barbadense.By mining 9697 non-redundant ESTs,a total of 638 SSR loci derived from 595 ESTs were observed.In G.barbadense,the frequency of ESTs containing SSRs was 6.13%,with an average of 1 SSR in every 10.4 kb of EST sequence.Furthermore,trinucleotide was found to be the most abundant repeat type among 2-6-nucleotide repeat types.It accounted for 26.6% of the total,followed by the hexanucleotide(26.0%) and pentanucleotide repeats(25.9%).Among all the repeat motifs,(AAG)n accounted for the highest proportion.EST-SSR primer pairs were developed using the Primer3 program,and the redundant primers were removed using the virtual PCR approach.As a result,380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G.hirsutum 'TM-1' and G.barbadense 'Hai7124' for constructing linkage groups in cultivated allotetraploid cotton.Out of these,98(25.8%) primer pairs detected polymorphisms.Finally,95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map;of these,42 were mapped into the A subgenome and 53 into the D subgenome.The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.

Establishment of a Multi-color Genomic in situ Hybridization Technique to Simultaneously Discriminate the Three Interspecific Hybrid Genomes in Gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.

Functional diversifications of Gh ERF1 duplicate genes after the formation of allotetraploid cotton

Whole genome duplication, a prevalent force of evolution in plants, results in massive genome restructuring in different organisms. Roles of the resultant duplicated genes are poorly understood both functionally and evolutionarily. In the present study, differentially expressed ethylene responsive factors(GhERF1 s), anchored on Chr-A07 and Chr-D07 were isolated from a high-yielding cotton hybrid(XZM2)and its parents. The GhERF1 was located in the B3 subgroup of the ethylene responsive factors subfamily involved in conferring tolerance to abiotic stress Nucleotide sequence analysis of 524 diverse accessions together with quantitative real-time polymerase chain reaction analysis, elucidated that de-functionalization of GhERF1-7 A occurred due to one base insertion following formation of the allotetraploid cotton. Our quantitative trait loci and association mapping analyses highlighted a role for GhERF1-7 A in conferring high boll number per plant in modern cotton cultivars. Overexpression of GhERF1-7 A in transgenic Arabidopsis resulted in a substantial increase in the number of siliques and total seed yield. Neo-functionalization of GhERF1-7 A was also observed in modern cultivars rather than in races and/or landraces, further supporting its role in the development of high-yielding cotton cultivars. Both de-and neofunctionalization occurred in one of the duplicate genes,thus providing new genomic insight into the evolution of allotetraploid cotton species.

Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene

A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous lines in T3 were obtained. Southern blot and Northern bolt demonstrated that the fibroin gene was inserted into the genome of these 6 lines, stably inherited and expressed. Compared to the control, the surface structure of mature fiber in the 6 lines was significantly distorted and an increased number of convolution was observed by scanning electron microscopy (SEM). Fiber quality traits analysis indicated that fiber elongation of the 6 homozygous lines was all increased and fiber strength of 3 lines was enhanced. These results indicated that fibroin expression influenced cotton fiber structure and quality, suggesting that fibroin has great potential for improving cotton fiber quality by genetic engineering.