Germplasm and Genetic Stocks

Malate dehydrogenase (MDH) is an enzyme of the citric acid cycle that catalyzes the reversible oxidation of malate to oxaloacetate with the concomitant reduction of NADH and plays crucial roles in various cellular processes. Here, we identified 12 malate dehydrogenase (MDH) genes from diploid cotton species Gossypium raimondii, and designated GrMDH1-12. These MDH members were located on 10 chromosomes in G. raimondii with uneven distribution. Their exon-intron numbers and genomic positions were highly conserved in different plant species. A phylogenetic analysis showed that MDH putative proteins can be divided into four groups, MDH I, MDH II, MDH III and MDH IV that contain 2, 3, 3 and 4 members, respectively. Expression analysis indicates that 11 GrMDH genes, except GrMDH 10, are differentially expressed in various cotton fiber, seeds and vegetative tissues. More importantly, eight of 12 GrMDH genes showed higher expression level in the fibers, particularly in early fiber elongation stage, demonstrating the high potential roles of MDH in the cotton fiber cells elongation. Finally, a promoter element analysis of the GrMDH genes indicates redundant but distinctive cis-regulatory elements for fiber cell development and stress responsiveness. This study is the first to characterize MDH gene family in cotton and provides a foundation for future studies on specific functions of these genes in fiber development.

Perennial forms of Gossypium hirsutum are classified under seven races. Five Mesoamerican races would have been derived from the wild race ‘yucatanense’ from northern Yucatán. ‘Marie-Galante’, the main race in the Caribbean, would have developed from introgression with G. barbadense. The racial status of coastal populations from the Caribbean has not been clearly defined. We combined Ecological Niche Modeling with an analysis of SSR marker diversity, to elucidate the relationships among cultivated, feral and wild populations of perennial cottons. Out of 954 records of occurrence in Mesoamerica and the Caribbean, 630 were classified into four categories cultivated, feral (disturbed and secondary habitats), wild/feral (protected habitats), and truly wild cotton (TWC) populations. The widely distributed three first categories cannot be differentiated on ecological grounds, indicating they mostly belong to the domesticated pool. In contrast, TWC are restricted to the driest and hottest littoral habitats, in northern Yucatán and in the Caribbean (from Venezuela to Florida), as confirmed by their climatic envelope in the factorial analysis. Extrapolating this TWC climatic model to South America and the Pacific Ocean points towards places where other wild representatives of tetraploid Gossypium species have been encountered. The genetic analysis sample comprised 42 TWC accessions from 12 sites and 68 feral accessions from 18 sites; at nine sites, wild and feral accessions were collected in close vicinity. Principal coordinate analysis, neighbor joining, and STRUCTURE consistently showed a primary divergence between TWC and feral cottons, and a secondary divergence separating ‘Marie-Galante’ from all other feral accessions. This strong genetic structure contrasts strikingly with the absence of geographic differentiation. Our results show that TWC populations of Mesoamerica and the Caribbean constitute a homogenous gene pool. Furthermore, the relatively low genetic divergence between the Mesoamerican and Caribbean domesticated pools supports the hypothesis of domestication of G. hirsutum in northern Yucatán.

Cotton is facing some serious challenges in the global market from the competition of synthetic fibers. Genetic solutions to this challenge require adequate genetic variation to be present in the breeding germplasm.The exotic gene pools in wild and unadapted species holds the promise of unlocking many potentially valuable genes/alleles in the genetic improvement of Upland cotton (Gossypium hirsutum L.). However, the potential utility and value of specific alien genes is usually compromised by co-inheritance of closely linked genes that have deleterious effects on productivity in conventional method of interspecific crosses. To physically separate the beneficial alien genes from undesirable ones by breeding can thus be extremely difficult in interspecific introgression to improve Upland cultivars. A potentially complementary approach of conventional breeding method is to use chromosome or chromosome segment substitution (CS) lines from alien species. The objective of this paper is to provide a summarized report on the concept, development and utilization of CS lines from G . barbadense, G. tomentosum and G. mustelinum in the genetic analysis and germplasm improvement of Upland cotton. In each CS line, a pair of chromosomes (or chromosome arm-specific segments) of G. hirsutum inbred TM-1 was replaced by the respective pair from the alien species. We also developed chromosome specific recombinant inbred line (CS-RIL) from crosses of a CS line with their common recurrent parent, inbred TM-1. An individual plant of the F2 population from this cross was maintained by selfing via single seed decent method until F6 generation in CS-RIL development. The comparative analysis of such unique genetic materials detected the genetic effects of novel alleles by specific alien chromosome/segment associated with the quantitative traits of importance. This research demonstrated a new strategy to complement conventional interspecific introgression using a novel method of chromosome shuffling among the alien and domesticated species. This study helped in the development of a unique set of germplasm by targeted introgression of desirable genes from wild and unadapted types into genetic backgrounds with reduced linkage drag effects, readily useable by the cotton breeders. Comparative analysis of fiber traits on a chromosome-by-chromosome basis provided new information of valuable genes and some cryptic alleles of alien species that can be useful for the genetic improvement of Upland cotton.

The identification of the cotton monosomics of Uzbek cytogenetic collection using SSR markers Marina Sanamyan1,3, Dilrabo Ernazarova2, Abdusalom Makamov3, Dilshod Usmonov3, Sukumar Saha4, David Stelly5, Ibrokhim Abdurakhmonov3. 1National University of Uzbekistan, Tashkent, Uzbekistan. 2Institute of Genetics and Plant Experimental Biology, Academy of Sciences, Tashkent, Uzbekistan. 3Center of Genomics and Bioinformatics, Academy of Sciences of Uzbekistan, Ministry of Agriculture and Water resources, and “UzCottonIndustry” Association, Tashkent, Uzbekistan. 4USDA-ARS, Crop Science Research Laboratory, Mississippi State, MS, USA. 5Texas A&M University, College Station, TX 77843 USA In Uzbekistan, Cytogenetic collection National University (CCNUz), a total of 94 primary monosomics of G. hirsutum L. from the common genetic background of the highly inbred line L-458 were developed through irradiation of cotton seeds by thermal neutrons or pollen gamma-irradiation in M1, M2 and M3 generations. Some of these monosomics were characterized for morphological traits and cytogenetic characteristics. Further, several monosomics were identified using a well-defined tester set of translocation lines of Cytogenetic collection of the USA. Considering a lot of DNA markers have already been assigned to the individual chromosomes of G. hirsutum L., we aimed to utilize chromosome specific simple sequence repeat (SSR) markers to identify and reconfirm the chromosome specificity of all cytogenetic resources in CCNUz collection. Toward this goal, we crossed our monosomics lines with doubled haploid Pima 3-79 line (G. barbadense) and isolated F1 hybrid plants that are monosomic for substituted Pima chromosomes using cytogenetic analysis. Assignment of SSR markers was straightforward and in a manner described in previous reports that utilized a PCR amplification of chromosome specific markers in the genomic DNAs of hybrid plants. We used chromosome specific SSR markers to identity BC0F1 aneuploid plants from CCNUz collection. Results suggested that we identified monosomic lines associated with chromosome 2 (Mo11, Mo16 and Mo19), chromosome 4 (Mo70, Mo71, Mo76, Mo81, Mo89 and Mo90), and chromosome 6 (Mo13 and Mo67). Molecular identification of remaining cytogenetic stocks is in progress and will be discussed.

Interspecific cotton hybrids are included both the theoretical and practical interest for systematics and evolution of the genius Gossypium L. There are problems of the distant hybridization experiences such as crossability of parental species and sterility of F1 hybrids. The causes of these unfavorable phenomena can be revealed through the cytological method. In thise work, microsporogenesis of F1 hybrids between representatives of different species - G.hirsutum L. (AD1) and G.barbadense L. (AD2) has been examined. Cytological results pointed out that F1 hybrids G.hirsutum L. x G.barbadense L. involved minor alterations in M1 of meiosis. In F1 hybrids, G.hirsutum L. x G.hirsutum L. microsporogenesis takes its normal with extremely rare anomalous microspores. The pollen fertility of the hybrid plants were 90,9 and 95,5%, whereas in other hybrids (G.hirsutum ssp.punctatum var.gambia x G.hirsutum ssp.punctatum, G.hirsutum ssp.punctatum var.gambia x G.hirsutum ssp.purpurascens) the pollen fertility was shown 82,2 and 83,2%. In first generation of hybrids G.hirsutum L. x G.barbadense L. microsporogenesis occurred normally as intraspecific crosses. Anomalous microspores were identified extremely rare in interspecific hybridization. The meiotic index and pollen fertility were consisted to 90,7 and 94,9%, respectively, whereas in reciprocal hybrids (G.barbadense ssp.darwinii х G.hirsutum ssp.purpurascens var.el-salvador and G.hirsutum ssp.purpurascens var.el-salvador x G.barbadense ssp.darwinii) the meiotic index was shown as 87,6 and 86,7%, respectively. Cytological studies of microsporogenesis indicated phylogenetic relationship between species G.hirsutum L. and G.barbadense L. The quadrivalent chromosome associations at the M1 meiosis were detected among hybrids G.barbadense ssp.darwinii х G.hirsutum ssp.purpurascens var.el-salvador and G.barbadense ssp.vitifolium f.brasiliense х G.hirsutum ssp.mexicanum var.nervosum (Jucatan)). Thus, cytological studies confirm the regular character of meiosis in the tetraploid hybrids.

The identification of the cotton monosomics of Uzbek cytogenetic collection using SSR markers Marina Sanamyan1,3, Dilrabo Ernazarova2, Abdusalom Makamov3, Dilshod Usmonov3, Sukumar Saha4, David Stelly5, Ibrokhim Abdurakhmonov3. 1National University of Uzbekistan, Tashkent, Uzbekistan. 2Institute of Genetics and Plant Experimental Biology, Academy of Sciences, Tashkent, Uzbekistan. 3Center of Genomics and Bioinformatics, Academy of Sciences of Uzbekistan, Ministry of Agriculture and Water resources, and “UzCottonIndustry” Association, Tashkent, Uzbekistan. 4USDA-ARS, Crop Science Research Laboratory, Mississippi State, MS, USA. 5Texas A&M University, College Station, TX 77843 USA In Uzbekistan Cytogenetic collection National University (CCNUz), a total of 94 primary monosomics of G. hirsutum L. from the common genetic background of the highly inbred line L-458 were developed through irradiation of cotton seeds by thermal neutrons or pollen gamma-irradiation in M1, M2 and M3 generations. Some of these monosomics were characterized for morphological traits and cytogenetic characteristics. Further, several monosomics were identified using a well-defined tester set of translocation lines of Cytogenetic collection of the USA. Considering a lot of DNA markers have already been assigned to the individual chromosomes of G. hirsutum L., we aimed to utilize chromosome specific simple sequence repeat (SSR) markers to identify and reconfirm the chromosome specificity of all cytogenetic resources in CCNUz collection. Toward this goal, we crossed our monosomics lines with doubled haploid Pima 3-79 line (G. barbadense) and isolated F1 hybrid plants that are monosomic for substituted Pima chromosomes using cytogenetic analysis. Assignment of SSR markers was straightforward and in a manner described in previous reports that utilized a PCR amplification of chromosome specific markers in the genomic DNAs of hybrid plants. We used chromosome specific SSR markers to identity BC0F1 aneuploid plants from CCNUz collection. Results suggested that we identified monosomic lines associated with chromosome 2 (Mo11, Mo16 and Mo19), chromosome 4 (Mo70, Mo71, Mo76, Mo81, Mo89 and Mo90), and chromosome 6 (Mo13 and Mo67). Molecular identification of remaining cytogenetic stocks is in progress and will be discussed.

In our study, 8 upland cotton varieties (lines), which were selected from Yangtze River valley and Yellow River valley in recent years, were used as parents. Hybrid crosses were made according to Griffing’s diallel cross design method (4). 8 parental lines, 28 F1 and 28 F2 hybrids in two different ecological environments were analyzed for main agronomic and fiber traits by the genetic model of additive- dominance- additive× additive epistasis with environment interaction (ADAAE model, http://ibi.zju.edu.cn/software/qga/ ). The main findings were as follows: Genetic analysis of main agronomic and fiber traits showed that, growth period, flower and boll stage, plant height, fruit branch number, seed index and fiber elongation were mainly controlled by dominance by environment effect, with large environmental impact. Yield and its component traits existed significant dominance effect, and the proportion of dominance variance in the phenotypic variance varied from 20.9% to 59.2%. What’s more, lint index, fiber uniformity index and micronaire were also mainly controlled by dominance effect. Fiber strength and upper half mean length were mainly controlled by additive effect. Correlation analysis among all kinds of traits showed that, phenotypic and genetic correlation coefficients between lint yield and plant height, fruit branch number, boll weight, lint percent, lint index, seed cotton yield, fiber uniformity or micronaire were positively significant or highly significant. What’s more, lint yield, which had low heritability, could be selected by fruit branch number and lint percent indirectly. For fiber quality traits, fiber upper half mean length and strength were easily to achieve simultaneous improvement, but fiber elongation and upper half mean length or strength had highly significant negative phenotypic and genetic correlation coefficients. The evaluation of all parents and hybrids: P1 (612085-1), P4 (sGK958), P5 (YOU lu272), P6 (3392154-55), P7 (380027-030) and P8 (2028) showed outstanding general combining ability. Analysis of dominance and dominance by environment effect of lint yield in 28 hybrid crosses showed that, there were 18 crosses with high and positive dominance effect. What’s more, dominance by environment effect had the same direction with the dominant main effect for most hybrid crosses, so hybrid crosses in our research had stable lint yield. Heterosis analysis results showed that, seed cotton yield and lint yield of F1 and F2 exhibited heterosis. 9 heterosis crosses were selected from 28 hybrids, and F2 population heterosis over parent better of these 9 crosses ranged from 16.3% to 26.3%. Competitive advantage analysis of 9 heterosis crosses in Yellow River valley showed that, competitive advantage of lint yield of F2 varied from -10.6% to 11.2%, but there were no significant difference between each F2 combination and check variety Zhongzhimian2 for lint yield. Through two years of experimental verification, yield heterosis in cross P1╳P8, P3(ZHONG 41)╳P5 and P3╳P7 could be used to F2 generation, so these crosses should be further investigated.

Plant type is a key factor that influences planting density, fruiting and harvest. Cytokinins (CKs) are plant hormone regulating cell division, and play an important role in organogenesis. To investigate the dynamic change of endogenous CKs in cotton plant and its effect on morphogenesis of cotton, a synthetic cytokinin-inducible promoter TCS fused with GUS reporter gene was introduced into upland cotton through agrobacterium-mediated transformation. qPCR analysis showed that only CKs could induce expression of GUS gene, and the expression level was increased with the enhancement of CKs. Histochemical staining showed that GUS activity was strongly detected in shoots meristem and vascular tissue of stems and leaves. In flower buds, GUS signal mainly appeared in floral primordium of early stage when floral organs, including bracts, calyxes, stamens and carpels, began differentiation. The distribution of CKs was further confirmed by in situ immuno-hybridization using antibodys against ZR and iPR, respectively. Then, we used TCS::GUS cotton to analyze interaction of IAA and CKs in fruiting stems. Shoots of fruiting branches were decapitated and detected the distribution of CKs in the decapitated stems. The decapitation led to a considerable increase of GUS signal. LC-MS determination showed that the decapitation of fruiting stems resulted in a significant decrease of IAA, while a noticeable increase of CKs in the stem. Meanwhile, the expression of the key cytokinin biosynthesis genes (GhIPTs) was significantly up-regulated. In addition, application of IAA to the apexes of decapitated stems could counter the effect of decapitation. These results suggested that decapitation leads to a decrease of IAA, which in turn up-regulates the expressions of GhIPTs. Taken together, TCS::GUS is convenient for monitoring the change of endogenous CKs and studying the role of CKs in cotton. Key words: cotton; cytokinins; TCS promoter This work was supported by NSFC (31130039 and 31201248) and NSF of Chongqing (cstc2012jjjq0011)

Gossypium hirsutum (Upland cotton) accounts for the majority of cotton fiber production world-wide. The global marketplace places value on longer and stronger fibers, mandating that U.S. breeders develop cultivars to meet this demand. One challenge that breeders face concerning the improvement of fiber quality traits is low genetic diversity among elite, agronomically acceptable genotypes of G. hirsutum. The use of marker-assisted selection (MAS) could help breeders access unexploited genetic diversity as well as decrease the cost of phenotyping for fiber quality traits. Linkage (bi-parental) and association mapping studies have led to the discovery of hundreds of quantitative trait loci (QLT) for fiber length and strength, and many of these QTL detected show promise for use in MAS. However, there are few reports of public programs utilizing MAS for the improvement of fiber quality traits. In general, there has been inconsistency among QTL discovered across studies, which has been one of the major obstacles preventing the use of MAS for fiber quality. This inconsistency could be due to QTL being mapped in different genetic backgrounds and environments as well as experimental error. Therefore, the objective of this study was to validate previously reported microsatellite markers (SSRs) for fiber length and strength in different genetic backgrounds. Two intra-specific populations (G. hirsutum x G. hirsutum) and one inter-specific population (G. hirsutum x G. tomentosum/G. mustelinum) were selected for the study based on high levels of polymorphism. Within the three selected families, 285 individual F2:3 plants were genotyped for approximately 250 SSRs, hand harvested, and sent for high volume instrument (HVI) analysis of fiber quality traits. These data will be used to evaluate associations between SSRs and fiber length and strength and to identify robust candidate markers for MAS.

The present study was carried out to investigate the stability performance in yield, adaptability and genotype × environment interactions of four promising advanced lines viz.; Cyto-124, BH-177, RH-650 and FH-326 along with one commercial cultivar FH-942. Seed cotton yield performance of five genotypes was evaluated over six locations (environments) in Punjab, Pakistan during 2013-14, using randomized complete block design with three replications. Data obtained were subjected to analysis of variance and stability analysis of Eberhart and Russell regression and evaluated on the basis of mean performance, linear regression (b), deviation from regression (S2d) and coefficient of variation (CV%). Genotype × environment interactions revealed that advance line FH-326 produced highest seed cotton yield, significant b value along with above stability performance (b<1). Results showed that FH-326 not only performed best under variant environments but also remained stable genotype in all tested locations. Furthermore FH-942 (standard cultivar) had general adaptability in all the tested locations for seed cotton yield.