Sultan Habibullah Khan

The low down on association mapping in hexaploid wheat ( Triticum aestivum L.)

The past few decades have witnessed hundreds of family-based linkage studies mapping for numerous traits but only a limited number of QTLs were actually cloned, tagged, or used for marker-assisted selection. Although providing valuable information, this conventional approach cannot be scaled up to underpin the incredible amount of phenotypic variation in the form of 266, 589 hexaploid wheat accessions maintained in public germplasm collections. Association mapping has recently emerged as an alternative and more powerful mapping approach where a natural population is surveyed to determine marker-trait associations using linkage disequilibrium (LD). After its first application for milling quality in 2006, association mapping studies in hexaploid wheat are being extended to tag yield traits, protein quality, and tolerance to biotic and abiotic stresses. Advances in genotyping technology and statistical approaches greatly accelerated the shift from conventional linkage-based mapping to LD-based association mapping. Association mapping stands out because of simultaneous utilization of a large number of ex situ-conserved natural variation due to historical recombination events accumulated over centuries.

Use of TALEs and TALEN Technology for Genetic Improvement of Plants

Genome editing with engineered nucleases has become a powerful tool of targeted genome modifications providing unprecedented control over animal and plant genetic material for precise, robust and highly specific genome engineering. Precise genome editing has been a long standing goal in the field of biology which has been achieved with the help of engineered nucleases like zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated system. These engineered nucleases consist of a binding and a nuclease domain which are generally used in the form of a pair. The binding domain binds specifically to a DNA sequence whilst the nuclease domain creates double-strand breaks (DSBs) which are further used for non-homologous end joining or homologous recombination repair. Creation of DSBs is the principle of this technology which can be further used for gene addition, deletion and modification in the targeted DNA. Besides nuclease activity, TALE (transcription activator-like effector) proteins have also been used along with other effector domains for different purposes like gene activation, gene repression, epigenetic modifications, etc. The use of TALEs and TALENs for precise genome modifications of plants is now a common practice. So far, tens of crop plants have been modified using engineered nucleases like rice, wheat, tomato, potato, tobacco, maize, barley, cotton, etc. The TALE and TALEN technology is being used for development of biotic and abiotic stress-resistant plants as well as yield and quality improvement. In this article, we will briefly review and discuss TALEs and TALENs, their discovery, binding specificity, designing, functional domains, delivery and use for genome editing specifically in plants.

IDENTIFICATION OF QTLs ON CHROMOSOME 1B FOR GRAIN QUALITY TRAITS IN BREAD WHEAT (TRITICUM AESTIVUM L.).

The present study was designed considering the importance of grain quality traits, genetic diversity and marker-trait association analysis in wheat. A significant amount of genetic diversity was found for various seed traits though the genotypes included in the study were found structured. The extent of polymorphism was high with a range of 2–13 alleles and average of 6.5 alleles per locus. Population structure was detected with 30 unlinked SSRs that divided the population of 92 genotypes in three sub-populations. Extensive LD extent was found on chromosome 1B with 42 SSRs specific for 1B chromosome. Marker-trait associations were determined using mixed linear model, where population structure and kinship calculated on the basis of unlinked markers were covariated with 1B specific markers and traits data. Eight QTLs for five traits including protein, gluten contents, test weight bread and chapati making quality. Protein content, test weight, bread quality and Glu-B1 were found significantly associated with primers WMC419 (32 cM); WMC128 (30 cM), WMC419 (32 cM); WMC818 (17 cM) and WMC416 (44 cM), respectively.

Patterns of trait associations in various wheat populations under different growth environments

Five wheat populations were investigated for two years to explore the pattern of trait associations and their contribution to grain yield. The correlation pattern between two traits and their association with grain yield was similar in CIMMYT and Pakistani germplasm. Indian germplasm had different pattern of trait association from those of CIMMYT and Pakistani germplasm. The number of kernels per plant, number of spikes per plant, spike length and spike dry weight were the major yield contributing traits in CIMMYT, Pakistani and ICARDA genotypes. In Indian and miscellaneous genotypes, the number of kernels per plant and number of spikes per plant were the only traits with a positive effect on grain yield. Furthermore, three traits, the number of kernels per plant, the number of spikes per plant and the spike dry weight appeared to have positive effect on grain yield and other major yield traits. Spike density had a negative effect on grain yield in CIMMYT germplasm in dry season. Chlorophyll contents showed no correlation with grain yield in all populations.

Pectobacterium punjabense sp. nov., isolated from blackleg symptoms of potato plants in Pakistan

Pectobacterium isolates SS95T, SS54 and SS56 were collected from a potato field in the Chiniot district in the plains of the Punjab province, Pakistan. Sequencing of the gapA barcode revealed that these strains belong to a novel phylogenetic group separated from P.ectobacterium wasabiae and Pectobacterium parmentieri species. Furthermore, multilocus sequence analyses of 13 housekeeping genes (fusA, rpoD, acnA, purA, gyrB, recA, mdh, mtlD, groEL, secY, glyA, gapA and rplB) clearly distinguished the type strain, SS95T, from its closest relatives, i.e. P. parmentieri RNS 08-42-1AT and P. wasabiae CFBP3304T, as well as from all the other known Pectobacteriumspecies. In silico DNA-DNA hybridization (<44.1 %) and average nucleotide identity (<90.75 %) values of strain SS95T compared with other Pectobacterium type strains supported the delineation of a new species. Genomic and phenotypic comparisons permitted the identification of additional traits that distinguished the Pakistani isolates from all other known Pectobacterium type strains. The name Pectobacterium punjabense sp. nov. is proposed for this taxon with the type strain SS95T (=CFBP 8604T=LMG 30622T).

Patterns of Allelic Diversity in Spring Wheat Populations by SSR-Markers

Precise assessment of diversity in available breeding germplasm helps to preempt epidemics and abrupt environmental changes. Spring wheat germplasm consisting of 84 accessions including cultivars, breeding lines and landraces from various origins was scanned with 44 SSRs. For allele frequencies, allelic patterns, heterozygosity and polymorphism the selected population was divided in three subpopulations: (i) pre-green revolution (pre-1965), (ii) post-green revolution (post-1965), (iii) post-veery (post-2000). Alleles produced in pre-1965, post-1965 and post-2000 subpopulations were 115, 144 and 131, respectively. Mean PIC values for pre-1965, post-1965 and post-2000 subpopulations were 0.48, 0.52 and 051, respectively. Allelic patterns showed no locally common alleles in any of the subpopulation. The pre-1965 subpopulation had also no private allele, however, average number of private alleles decreased from post-1965 to post-2000 subpopulation. In case of effective alleles and Shannon’s information index trend was increasing from pre-1965 to post-1965 and then decreasing from post-1965 to post-2000. The decreasing trend alarms the reduced genetic diversity in wheat varieties developed after 2000. PCA and cluster analysis didn’t clearly differentiated subpopulations, though pre-1965 genotypes showed higher genetic distance from post-1965 and post-2000 subpopulations. The decreasing measures of genetic diversity in post-2000 wheat genotypes should be a concern for wheat breeders, therefore, all sources of broadening genetic diversity should be exploited.

Improvement of Cotton Crop Performance by Estimating Optimum Sowing and Picking Time

Performance of seed is determined by interactive component effect of genetics, physiological quality and the environment. Climate change and introduction of Bt genotypes uncertain the conventional sowing time of cotton responsible for low emergence that proceeds to delayed emergence and stand failure. To predict the emergence potential of cotton, Bt (FH-142) and non-Bt (FH-942) genotypes were sown in field with 15 days interval from 15 March to 15 June, 2013. Post planting observations recorded during crop production were morphological and phenological characteristics along with seed cotton picking during September, October and November. Early sowing delayed time to start emergence and sowing of FH-142 at 15 April and FH-942 at 1 June exposed the seed to optimum temperature finally with the quick and maximum emergence percentage. Cotton plants emerged during 15 March to 15 April compensated their delayed emergence by achieving maximum height, more number of bolls, monopodial and sympodial branches as well as early blooming and with higher economic returns for October picking. Thus, sowing from 15 March to 15 April assures high cotton production through good morphological development and subsequently picking during October provide good quality cottonseed.

Disruption of Phytoene Desaturase Gene using Transient Expression of Cas9: gRNA Complex

Engineered nucleases have emerged as a powerful tool for site specific gene manipulation in plants. Based on Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR associated (CRISPR/Cas) system, engineered Cas9:gRNA complex can be used to cleave specific DNA sequences in the genome. In the present study, Nicotiana benthamiana Phytoene Desaturase (NbPDS) gene was targeted by CRISPR/Cas9 system. The plant codon optimized (pcoCas9) along with guided RNA (gRNA) was cloned in plant expression vector pGreen0029, to target NbPDS gene. The NbPDS gene was disrupted transiently by agroinfiltration of pcoCas9-gRNA complex. Visible albino spots were observed on agro-infiltrated leaves of N. benthamiana plants after 7 days of infiltration. The observed albino spots were analyzed through PCR amplification of gRNAtarget, fluorescent microscopy and chlorophyll contents measurements. Our results support the notion that CRISPR/Cas9 system is a swift, robust and useful tool for targeted gene disruption, deletion and editing.