Germplasm and Genetic Stocks

Cotton is an important cash crop worldwide, accounting for a large percentage of world agricultural exports; however, yield per acre is still poor in many countries, including Pakistan. Diallel mating system was used to identify parents for improving within-boll yield and fiber quality parameters. Combining ability analysis was employed to obtain suitable parents for this purpose. The parental genotypes CP-15/2, NIAB Krishma, CIM-482, MS-39, and S-12 were crossed in complete diallel mating under green house conditions during 2009. The F₀ seed of 20 hybrids and five parents were planted in the field in randomized complete block design with three replications during 2010. There were highly significant differences among all F₁ hybrids and their parents. Specific combining ability (SCA) variance was greater than general combining ability (GCA) variance for bolls per plant (9.987), seeds per boll (0.635), seed density (5.672), lint per seed (4.174), boll size (3.69), seed cotton yield (0.315), and lint percentage (0.470), showing predominance of non-additive genes; while seed volume (3.84) was controlled by additive gene action based on maximum GCA variance. Cultivar MS-39 was found to be the best general combiner for seed volume (0.102), seeds per boll (0.448), and lint per seed (0.038) and its utilization produced valuable hybrids, including MS-39 x NIAB Krishma and MS-39 x S-12. The parental line CIM-482 had high GCA effects for boll size (0.33) and seeds per boll (0.90). It also showed good SCA with S-12 and NIAB Krishma for bolls per plant, with CP- 15/2 for boll size, and with MS-39 for seeds per boll. The hybrids, namely, CP-15/2 x NIAB Krishma, NIAB Krishma x S-12, NIAB Krishma x CIM-482, MS-39 x NIAB Krishma, MS-39 x CP-15/2, and S-12 x MS-39 showed promising results. Correlation analysis revealed that seed cotton yield showed significant positive correlation with bolls per plant, boll size and seeds per boll while it showed negative correlation with lint percentage and lint per seed. Seed volume showed significant negative correlation with seed density. Seeds per boll were positively correlated with boll size and negatively correlated with bolls per plant lint percentage and lint per seed. Similarly, lint per seed exhibited positive correlation with lint percentage and boll size showed significantly negative correlation with bolls per plant. Presence of non-additive genetic effects in traits like bolls per plant, seeds per boll, lint per seed, seed cotton yield, and lint percentage is indicative of later generation selection or heterosis breeding may be adopted. For boll size, seed volume and seed density early generation selection may be followed because of the presence of additive gene action. The parental material used in this study and cross combinations obtained from these parents may be exploited in future breeding endeavors.

EVALUATION OF COTTON GERMPLASM AGAINST BACTERIAL BLIGHT OF COTTON (Xanthomonas axonopodis pv malvacearum) Syed Kamil Husnain and Dr Sabir Hussain Khan Ayub Agriculture Research Institute Plant Pathology Research Institute, Faisalabad Pakistan Corresponding author email: kamiluaf@gmail.com Abstract Most promising cotton varieties from renowned research institutes of Pakistan (Ayub Agriculture Research Institute Faisalabad, National Institute of Biotechnology and Genetic Engineering, Nuclear Institute of Agriculture and Biology) and private sector (Four Brothers Groups) were evaluated against bacterial blight of cotton Xanthomonas axonopodis pv malvacearum under field condition. During the month of July diseased leaves were collected from different cotton growing areas and ground in distilled water. Suspension was made after pressing it in muslin cloth. On the leaves of test entries scratches were made by sand paper and suspension applied by spray method. Out of 28 varieties, 7 varieties found to be susceptible, while three varieties performed high susceptibility against the disease.13 varieties were moderately resistant and five varieties show resistant reaction against the diseases.

The level of genetic diversity is an essential element of sustainable crop improvement. With the main objectives to facilitate direct selective breeding and conservation of germplasm resources, the genetic diversity of 308 elite upland cotton (Gossypium hirsutum L.) genotypes was studied using 195 simple sequence repeat (SSR) markers. In this study, the total number of SSR alleles, average number of polymorphic alleles per locus, polymorphism information content (PIC) per locus, genotype diversity index (H’), pair-wise genetic distance similarity, and UPGMA clustering will be determined and analyzed. The study would provide useful guides for efficient utilization of the existing genetic variations to address the issue of narrow genetic-base challenging improvement of upland cotton for fiber yield and fiber quality worldwide. Key words: Upland cotton, genetic diversity, SSR markers Note:This abstract is part of the ongoing PhD research study which will be completed very soon and becomes available for the ICGI 2014 Conference (25-28 September, Wuhan).

Chromosome substitution backcrossing lines (CS-B) have a pair of chromosomes or chromosome arms from Pima cotton (Gossypium barbadense) which replaced the respective pair of chromosomes or chromosome arms of TM-1 (G. hirsutum). These lines are largely similar to each other, except that each differs by the replacement of a specific homologous pair of chromosomes from Pima 3-79 into Upland genetic background (Texas Marker-1). The aim of this research is to develop chromosome specific recombinant inbred mapping populations using CS-B lines for future performance of fine association mapping of agronomically important QTLs. Development of chromosome specific mapping population based on a set of CS-B lines are of importance to enrich genetic diversity in cotton because it will show wide segregation in a single substituted chromosome from Pima cotton. In this study, the commercial Uzbek cotton cultivar AN-Boyovut-2 was used as a common parent and 17 CS-B lines as donor genotypes. The common parent, An-Boyovut-2 is an elite cultivar, having high yield and salt tolerance but middle fiber quality, was crossed with 17 CS-B donor lines in 2006, followed by self-pollination to develop 17 segregating F2 populations. In order to get the next generations of these populations, 11 segregating F2-3 populations were chosen in which the existence of substituted chromosomes were confirmed by chromosome specific DNA markers. The created chromosome specific recombinant inbred mapping populations will be excellent genetic resource for dissecting and fine mapping superior fiber quality loci.

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A new high density allotetrploid cotton genetic map of simple sequence repeat (SSR) markers was developed by using BC1F1 population CCRI36×(CCRI36×Hai1) including 135 individuals. The genetic map comprised 2292 loci and covered 5115.16 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 2.23 cM. Of the 2292 loci, 1025 (44.72%) were mapped to 13 At-subgenome chromosomes, covering 2420.71 cM (47.32%), and 1268 (55.28%) mapped to 13 Dt-subgenome chromosomes, covering 2694.45 cM (52.68%). 62.92 % duplicated loci sufficiently bridged 13 expected homologous At/Dt chromosomes. 53.14% marker loci were newly discovered in this map compared with the published 5 high-density cotton genetic maps constructed mainly by SSR markers. By comparison with the whole-genome marker map based on the D genome sequence, the overall marker order for 1465 common loci on this new map agree well with the marker order on D genome, indicating high level of homology between the diploid D genome and the tetraploid cotton genetic map. This new independent high density SSR genetic map will be facilities, as a valuable genomic resource, for research on structural and functional genomics of allotetraploid cotton.

Molecular marker technology offers improved selection strategies in plant breeding. In the last decade, the DNA marker technology has been applied for identification QTLs associated with fiber quality and donor lines (with superior quality) for the purpose of developing new cotton lines in cotton breeding. The application of DNA marker technology to cotton for germplasm studies has identified novel quantitative trait loci (QTL) associated with fiber quality and unique donors with superior quality. Twenty six germplasm accessions, as donor lines and more than 10 varieties, as the recipient parents were selected for mobilization of novel QTL alleles from unique donors to the genetic background of commercial Uzbek cotton cultivars through marker assisted backcrossing (MAB). In each backcross generations, allele mobilization to hybrids was controlled via molecular markers, respectively and hybrids with marker bands were selected. Selected hybrid fibers were analyzed for their quality, including length (FL), fiber strength (FS), micronaire (MC), fiber elongation (FE), fiber uniformity (FU) using High Volume Instrument (HVI). The results of HVI analyses were indicated good fiber qualities of selected hybrids. In 2013, between two combinations of F1BC5 generations, two lines named “Ravnaq-1” and “Ravnaq-2” (translated as “Advance”) were selected and sent to the State Variety Testing Committee of Uzbekistan for larger filed trails in different environments. Both varieties possess higher fiber strength and improved length. For instance, “Ravnaq-1” has superior fiber quality with improved fiber strength (37 g/tex) and staple length (38 mm) compared to its recurrent parent “Andijan-35” which has 32 g/tex fiber strength and 35 mm staple length. Similarly, in “Ravnaq-2” molecular markers effectively mobilized superior fiber quality loci that improved fiber strength by 17% and staple length by 9% compared to its recurrent parent “Mekhnat”. In conclusion, our markers and donors have been proved to be useful in MAS to obtain superior cotton cultivars within short times, which bear novel untaped fiber quality loci.

Genetically-modified, insect-resistant Bt cotton has been adopted extensively across Pakistan’s cotton-growing regions during the past decade, though formal approval for its release was only granted in 2010. As a consequence, Pakistan has seen the widespread adoption of unapproved Bt cotton. The main quality criteria for Bt cotton is the stable and sufficient expression of the transgene that confers resistance to lepidopteran pests such as bollworms. This paper examines Bt cotton performance with reference to transgene expression in plants cultivated under farmers conditions. Leaf and bolls samples from 940 plants were collected at 70 and 120 days after sowing (DAS) from 560 randomly selected households located in 19 districts concentrated in Punjab’s cotton belt. Qualitative analysis of these samples using immunoblot strip testing showed that 35% samples were not synthesizing Cry1Ac protein up to a detectable limit, and none of them was synthesizing the Cry2Ab protein. The quantitative profiling of transgene expression in leaves collected at 70 DAS showed that 59%, 30% and 8% samples are synthesizing toxin below 1, 2 and 3 ug/g of fresh tissue weight, respectively, while expression in the leaves of same plants at 120 DAS was below 1, 2 and 3 ug/g in 74%, 21% and 4% of the samples respectively. The expression level was lower in boll samples as compared to leaf samples with 82% and 79% samples having toxin less than 1ug/g at 70 and 120 DAS respectively. Correlating the expression data with temperature and humidity data collected at the field sites showed a positive and negative relation with humidity and temperature respectively. The conclusion from these results is that there are temporal, meteorological, and other factors affecting expression of the Bt transgene in farmers’ fields and these factors may result in ineffective resistance to lepidopteran pests. Ineffective resistance, in turn, may contribute to both crop losses for farmers and natural selection in favour of lepidopteran resistance to the toxic Bt proteins. This further suggests that the continued proliferation of unapproved and ineffective Bt cotton in Pakistan may have significant ecological, environmental and economic consequences.

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A new high density allotetrploid cotton genetic map of simple sequence repeat (SSR) markers was developed by using BC1F1 population CCRI36×(CCRI36×Hai1) including 135 individuals. The genetic map comprised 2292 loci and covered 5115.16 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 2.23 cM. Of the 2292 loci, 1025 (44.72%) were mapped to 13 At-subgenome chromosomes, covering 2420.71 cM (47.32%), and 1268 (55.28%) mapped to 13 Dt-subgenome chromosomes, covering 2694.45 cM (52.68%). 62.92 % duplicated loci sufficiently bridged 13 expected homologous At/Dt chromosomes. 53.14% marker loci were newly discovered in this map compared with the published 5 high-density cotton genetic maps constructed mainly by SSR markers. By comparison with the whole-genome marker map based on the D genome sequence, the overall marker order for 1465 common loci on this new map agree well with the marker order on D genome, indicating high level of homology between the diploid D genome and the tetraploid cotton genetic map. This new independent high density genetic map will be facilities, as a valuable genomic resource, for research on structural and functional genomics of allotetraploid cotton.

The long-term sustainability of worldwide cotton production is at risk from several fronts, but one of the most formidable is synthetic fibers. A proven and effective approach to address this risk is support for research focused into sustainable germplasm improvement for improved lint yield and fiber quality. This presentation will cover examples from the past decade showing how steady support for research has resulted in greater understanding of cotton’s plant biology using knowledge gained from such investments. Tangible results from this understanding include seminal publications for the larger community, better cooperation between researchers, and improved germplasm for growers.

Global cottonseed output is estimated around 35 million tons in the recent past. Major producers of cotton in the world also dominate the oil sector with China, U.S., India, Pakistan and Brazil are leading. Out of this, nearly 27 million tons are used for oil extraction. Global trade in cotton oil is estimated around 1 million tons. Australia, U.S. and Mexico are the leading exporters of cottonseed oil while Europe and Japan are mail importers. Cottonseed oil is estimated to contribute nearly a fifth of the global vegetable oil production. Cotton seed is a byproduct derived through process called ginning. Cotton seed oil is extracted from cottonseed, which contain around 18-20 per cent oil. This pale yellow oil is generally used for cooking. Cottonseed oil is further refined to remove gossypol, a naturally occurring toxin that protects the cotton plant from insect damage. Oil thus obtained, is used in combination with other oils to produce many vegetable oil products. Composition of cotton seed oil is about 26% Palmitic acid (C16:0) 15 per cent oleic acid (C18:1) and 58 per cent linoleic acid (C18:2). Even through palmitic acid provides stability to the oil during deep frying applications but it leads to low den try lipoproteins subsequently to the greater risk of cardiovascular disease. High level of linoleic acid (50%) is also undesirable because of its oxidative instability. It is therefore it is desirable to develop cottonseed oil higher mano unsaturated fatty acid with thermal stability and better shelf life which is more suitable for deep frying without any rancidity and off flavors. In this study, an attempt was made to develop construct for silencing of 12 desaturase gene, and analyse its expression in cotton plants. A 600 bp DNA fragment was amplified using 12 desaturase gene specific primer from cotton ovules after 30 days pollination. The amplicon was cloned in pTZ57R/T and transformed into E. coli DH5a. Transformants were confirmed through PCR and restriction analysis. The analysis of the sequence revealed maximum of 100% homology at nucleotide and 100% homology at amino acid level with reported sequence in the database (AF270425). Partial sequence of delta 12 desaturase gene was cloned in both sense and antisense direction in generic ihp vector. The expression cassette carrying insect from generic ihp vector was then subcloned into plant transformation vector pCAMBIA 1305.1 to facilitate plant transformation. The recombinant clones were then mobilized into Agrobacterium tumefaciens LBA4404 by tri parental mating then transformed into cotton plants. Callus was analyzed through histochemical analysis of gus gene expression against control plants.