Germplasm and Genetic Stocks

Upland cotton (Gossypium hirsutum) is the most widely cultivated species, with over 90% of the global cotton production. Owing to long-term natural selection and artificial breeding under diverse climatic and cultivated conditions, plentiful germplasm resources have been produced for sustainable genetic improvement. Thus, to detect genetic factors contributing to the important traits in a genome-wide scale is indispensable based on these germplasm resources. In the present study, on the basis of approximately 3.66 million SNPs, we conducted genome-wide association study with 419 accessions in three important traits including days to flowering (FD), fiber length (FL) and strength (FS). The results showed that there were 1199, 1661 and 735 SNPs (P < 10-6) significantly associated with FD, FL and FS. We identified 528 FD, 456 FL and 301 FS genes in the 50-kb region surrounding the associated SNPs. For FD, we observed that 94.6% of the associated SNPs (1,199) were located on Dt03 with a strong peak. Among the identified FL-associated SNPs, 646 (38.9%) and 755 (45.5%) were located in At10 and Dt11 with strong association signals, respectively. For FS, 391 (53.1%) SNPs were located in At07. Therefore, we focused on these chromosomes to detect the new genes. We adopted the method that combines peak SNPs and SNP type in GWAS with transcriptome data, functional annotation of the orthologues in Arabidopsis to rapidly identify candidate genes associated with the traits. Through phenotypic significance analysis of haplotypes, qRT-PCR using two types of varieties with different haplotypes, transgenic method and VIGS technology, we found that GhCIP1 and GhUCE are the new genes contributing to FD and fiber initiation in cotton. GhFL1 in At10 and GhFL2 in Dt11 are the new genes controlling fiber elongation. Gh_A07G1769 is a causal gene underpinning the fiber strength of cotton. These results provide targets for molecular selection and genetic manipulation in cotton improvement.

The U.S. National Plant Germplasm System holds a wealth of valuable cotton germplasm available for breeders. The cotton breeding program at Texas A&M University has actively used numerous lines in its program as parent material over the last 12 years. Both Gossypium hirsutum and G. barbadense lines have been integrated into elite TAMU breeding material. By tracking the fate and value of the lines in terms of performance and creation of genetic variability, results indicate this source of germplasm has substantially benefited the program in terms of improving yield potential, fiber quality and host plant resistance.

Long-term cotton genetic improvement is dependent upon the careful introduction and exploitation of new genetic variation. One of the largely untapped reservoirs of genetic diversity available to cotton breeders resides in the primitive Gossypium hirsutum L. landrace collection. Although most accessions are classified as G. hirsutum and reside within the primary upland cotton gene pool, widespread use of these genetic resources has been minimal due to their photoperiodic nature and perennial growth habit. In our research program, we are deploying two different, but related approaches to introduce and use novel genetic variation present within this genetic resource. First, we are developing breeding populations derived from crosses involving a number of day-neutral converted lines developed by the USDA-ARS Mississippi State, MS research program. Second, we are characterizing a set of naturally occurring day-neutral primitive landrace accessions in an effort to identify novel accessions for use as breeding parents. Preliminary results suggest these genetic resources contain beneficial alleles for fiber quality and climate resiliency.

Fifty cotton cultivars were evaluated for NaCl tolerance in the Department of Plant Breeding and Genetics in the University of Agriculture, Faisalabad. Plants were irrigated with a nutrient solution with an electrical conductivity (EC) of 10dSm-1 and 15dSm-1 with the addition of NaCl from 10-day seedlings to 40-day seedlings. The data for chlorophyll content, root length, shoot length, fresh root weight, fresh shoot weight, shoot dry weight, root dry weight, Na+, K+ and K+/Na+ were taken from the seedlings at the time of harvesting. Analysis of variance indicated that there were significant differences among the genotypes at control and both salt stress levels. Salinity negatively impacted the growth of all cotton cultivars, however, the magnitude of reduction varied among all cultivars. High heritability and the high genetic advance was noticed for the root length, fresh root weight, dry root weight, dry shoot weight and potassium to sodium ratio which indicated the presence of additive gene actions in the expression of these characters. NIAB-824, Mubarak, CIM-612, FH-114, and Kehkshan were conceived as salt tolerant genotypes. The existence of genetic variability in the cotton germplasm exhibited that it can be exploited for the genetic improvement in future breeding programs.

The development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for further genome research and crop improvement through MAS. We had developed BC5F3:5 population with the donor parent Hai1 and the recurrent parent CCRI36. In this study，300 CSSLs and their two parents CCRI36 and Hai1 were planted in a randomized complete block design with 2 replications in two Ecological locations(Anyang and Xinjiang) in 2015 and 2016, respectively. Phenotypic evaluation included Verticillium wilt disease resistance(disease index), fiber yield(plant height,boll weight, lint percentage and seed index) and fiber quaility(fiber length, fiber strength, micronaire,fiber uniformity and fiber elongation). Verticillium wilt disease resistance were collected at the time of July and August.A total of 597 pairs SSR markers screened from 2292 pairs of markers in the whole genome high density map from a BC2F1 population of G. hirsutum×G. barbadense were used to identify the polymorphisms among the BC5F3:5 lines.A total of 56 QTLs for Verticillium wilt disease resistance were detected, 30 of them are stable.,38 QTLs (68%) had negative additives effects, which indicate that the G. barbadense alleles increased Verticillium wilt resistance and decrease DI by about 2.64 to 13.23. The highest number of QTLs (15) for Verticillium wilt disease resistance was detected on Chromosome 05. By meta-analysis, 30 QTL hotspot regions for VW resistance were identified and 13 of them were new, unreported hotspot regions.191 QTLs have been detected for fiber yield and fiber quality, 98 for the fiber quality traits and 93 for the yield related traits, 54 of them are stable(12 for fiber length, 8 for fiber strength, 5 for micronaire,2 for fiber elongation, and 6 for plant height,10 for boll weight,6 for lint percentage and 5 for seed index). Three chromosomes Chr05, Chr10 and Chr20 contained more QTLs.30 clusters with disease index and fiber related traits were identified on 16 chromosomes.Most of the fiber traits were clustered with the disease index stable QTLs. We found 6 clusters namely, C01-cluster-1, C05-cluster-4, C07-cluster-1, C19-cluster-2, C22-cluster-1 and C22-cluster-2, which had positive correlation between VW resistance and fiber quality traits. Two clusters, C10-cluster-1 and C25-cluster-1 had also positive correlation between VW resistance and yield related traits( boll weight and lint percentage). One cluster, C20-cluster-1 is important for VW resistance, fiber quality and fiber yield.So, these clusters and related QTLs are very important for breeding improvement of fiber quality and yield, VW disease resistance. Key words: CSSL, Verticillium wilt disease index, fiber quality, yield, QTL, SSR markers. Correspondence: shiyuzhen@caas.cn,yuanyouluyuan@caas.cn

G. hirsutum-G. australe chromosome exchanges were very limited, impeding the stable transference of useful genes from G. australe (G2G2 genome) into the most cultivated cotton, G. hirsutum (AADD). In the present report, the pollen from a pentaploid (2n=AADDG2) of G. hirsutum-G. australe was irradiated with seven different doses ranging from 10 to 40 Grays and used to pollinate emasculated flowers of G. hirsutum over three consecutive years. Our results indicated that irradiation greatly increased the genetic recombination rates of the G. hirsutum and G. australe chromosomes and a total of 107 chromosome introgression individuals in 192 GISH-negative (with no GISH signal on chromosome) survived individuals, 11 chromosome translocation individuals (containing 12 chromosome translocation events) and 67 chromosome addition individuals were obtained in 70 GISH-positive (with GISH signal(s) on chromosome(s)) survived individuals, which are invaluable for mining desirable genes from G. australe. Multicolor genomic in situ hybridization results showed that there were three types of translocation, whole arm translocation, large alien segment translocation and small alien segment translocation, and that all translocations occurred between the G2-genome and the A-subgenome chromosomes in G. hirsutum. We also found that higher doses induced much higher rates of chromosome variation but also greatly lowered the seed viability and seedling survivability. Therefore, we concluded that irradiation can be used to induce chromosome introgressions and chromosome translocations and promote chromosome exchanges between cultivated and wild species. In addition, by balancing the rates of chromosome introgression and translocation to those of seed set, seed germination, and seedling rates in the M1 generation, we suppose that the dosage of 20 Grays is the most suitable. The established methodology may guide the utilization of the tertiary gene pool of Gossypium species such as G. australe in cotton breeding in the future.

Understanding the genetic diversity and genetic divergence in population structure of germplasms is important when selecting parents for crop breeding. The genomic changes that occurred during the domestication and improvement of upland cotton remain poorly understood, and the available genetic resources from improved cotton cultivars are very limited. By applying restriction site-associated DNA marker sequencing (RAD-seq) technology to 582 tetraploid cotton accessions, we detected two distinct genomic signatures had the highest divergence on chromosomes A06 and A08 which dramaticly affect the population structure and genetic diversity in modern improved upland cotton populations. Based on integrating the pedigree of the accessions with previously reported QTLs, genome-wide association study (GWAS) and introgression analyses, we suggest that the two divergent regions resulted from the introgression of exotic lineages from G. hirsutum races and wild relatives. Genetic divergence of these regions were the typical genomic characteristic might be responsible for maturity in central Asia ecotype cotton population (Group-2) A06 and fiber quality for offspring resulting from interspecific hybridization developing mainly in southern China (Group-1) (A08), and several candidate genes were screened by integrating transcriptome data. Additionally, both genomic regions are located in putative pericentromere regions, implying that their application will be challenging. In the study, based on high-density SNP markers, we firstly reported two genomic signatures on chromosome A06 and A08 which could both distinguish ecotypes and be responsible for important agronomic traits in upland cotton. These results provides new insights for understanding the genomic basis of exotic introgressions in modern cotton cultivars.

DNA methylation, an important component of epigenetics induced usually by adversity, plays a vital role in the response to various stresses including drought and salt. A methylation-sensitive amplification polymorphism method based on capillary electrophoresis was used to explore the epigenetic mechanisms of salt tolerance and heterosis in Upland cotton (Gossypium hirsutum L.), and the results indicated that hypermethylation and demethylation could be an important mechanism to resist the stresses. And the demethylation could be the mechanism to explain heterosis in cotton hybrid. The results of whole genome methylation sequencing showed high DNA methylation density usually occurs in promoter regions and transposons areas. Methylated cytosines in different sequence contexts (CG, CHG and CHH) have different functions and methylation levels. And the results also showed methylated cytosines in asymmetric CHH sequence context are dynamic, being mostly related to stresses. Combined with transcriptome data, we found long non-coding RNAs (lncRNAs) may involve in the regulation of DNA methylation in response to drought stress. All these results could provide theoretical reference value for the mechanism research of tolerance in cotton. Keywords: cotton, salt, draught, methylation

The cotton genus (Gossypium) is remarkable for its extraordinary natural diversity as well as its importance to humankind. More than 50 species are recognized, including several recently described, which collectively are distributed in arid to semi-arid regions of the tropics and subtropics. Diversity in Gossypium has been promoted by two seemingly unlikely processes: trans-oceanic, long-distance dispersal, and wide hybridization among lineages that presently are widely separated geographically. Included are four species that were independently domesticated for their seed fiber, two diploids from Africa-Asia and two allopolyploids from the Americas. As Gossypium spread worldwide, it experienced remarkable morphological, cytogenetic and genomic diversification, spawning eight monophyletic groups of diploid (n = 13) species (“genome groups” A through G, and K) and 8 allopolyploids (n = 26; AD-genome), the latter resulting from an improbable trans-oceanic dispersal of an A-genome taxon to the New World 1-2 million years ago and subsequent hybridization with an indigenous D-genome diploid. The extraordinary diversity in the genus represents a largely untapped genomic reservoir for agronomic exploration. From a genomic perspective, cotton has a marvelously complex evolutionary history. We now understand that modern allopolyploid species are descended from diploid ancestors which themselves were once polyploid, with this cycle of polyploidization followed by diploidization being repeated many times. This history of “genomic superimpositions” will be described, as will some of the many implications that derive from this understanding.