Browsing by Person "Bhosale, Sankalp"

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    Association analysis of genes controlling variation of flowering time in West and Central African sorghum
    (2012) Bhosale, Sankalp; Melchinger, Albrecht E.
    Sorghum is extremely important for the food security in the arid to semi-arid regions of West and Central Africa (WCA). A serious constraint to the sorghum production in WCA is the scattered beginning but relatively fixed end of the rainy season among years, forcing farmers to adjust their individual sowing dates according to the start of the rains. Owing to the delayed sowing and fixed end of the rainy season, farmers require varieties that flower at the end of the rainy season, regardless of the sowing date. Photoperiod sensitivity of sorghum accessions is an important adaptation trait that allows flowering or synchronized flowering of the accessions at the end of the rainy season. This is also particularly important in avoiding grain mold, insect and bird damages for early maturing varieties, and incomplete grain filling due to soil water shortage occurring at the end of the season in late maturing varieties. Cultivars with photoperiod sensitivity may have the potential to increase yield and yield stability. Unfortunately, in WCA most of the present day cultivars are photoperiod insensitive. Furthermore, unavailability of simple screening methods in selecting photoperiod sensitive cultivars complicates the situation. Breeding techniques such as marker assisted selection (MAS) by employment of molecular markers would greatly enhance the selection efficiency for this major adaptation trait. Candidate-gene (CG) based association studies can assist in investigating the effect of polymorphisms in flowering time genes on phenotypic variation. Allele-specific molecular markers can be developed after a significant marker-phenotype association is identified. These markers can effectively be used in MAS of photoperiod sensitive sorghum cultivars. In this study we carried out a CG based association analysis to investigate the association between variation for photoperiodic sensitivity of flowering time in sorghum and polymorphisms in six partially amplified genes putatively related to variation in flowering time. Five out of six CGs were known to be involved in photoperiod pathway of flowering time [CRYPTOCHROME 1 (CRY1-b1), CRYPTOCHROME 2 (CRY2), LATE ELONGATED HYPOCOTYL (LHY), GIGANTEA (GI), HEADING DATE 6 (HD6)], and the gene SbD8 was involved in the gibberellic acid (GA) pathway of flowering time. In the first part of the study we determined the presence, the expression and the molecular diversity of genes homologous to the important flowering time gene D8 in maize on a set of 26 sorghum and 20 pearl millet accessions. Homologs of D8 were successfully amplified and tested for their expression in sorghum (SbD8) and pearl millet (PgD8). Pearl millet, because of its autogamous nature, showed higher nucleotide diversity than sorghum, which is an allogamous species. In maize, a 6 bp deletion flanking the SH2-like domain of D8 was found to be significantly associated with flowering by Thornsberry et al. (2001). We found in the PgD8 gene a 3 bp insertion or deletion (Indel) flanking the SH2 domain in the region, which was only conserved between D8 and PgD8. Cluster analysis performed for the D8, SbD8, and PgD8 indicated that maize is more closely related to pearl millet than sorghum. These findings suggest that, similar to maize, the indel in PgD8 flanking the SH2 domain might play an important role in determination of flowering. It is advisable to carry out an association study to reveal the potential role of PgD8 in flowering time control in pearl millet. After successfully amplifying and confirming the expression of SbD8 and PgD8, we carried out the association analysis on the selected CGs. A panel of 219 mostly inbred accessions of sorghum from major sorghum growing areas in WCA was complied. In the second part of the study the association analysis panel of accessions was phenotyped for their flowering response in the field in 2007 in Mali. The entire panel was sown twice (June and July), photoperiod response index (PRI) was estimated as the difference between DFL50% of the two sowing dates of the accessions. The PRI of the accessions showed a wide range from close to zero (photoperiod-insensitive) up to values close to 30 or above (highly-photoperiod sensitive). This result confirmed that the range of response based on the choice of the accessions was appropriate for an association analysis. The plant height reduction observed in accessions sown in July compared to the once sown in June was in accordance with previous studies performed in West African sorghum varieties. The sorghum accessions were genotyped using 27 simple sequence repeat markers. Population structure analysis using software STRUCTURE was carried out to control the false positives in the association analysis. The results showed existence of two subgroups in our sorghum accessions. The first subgroup included mainly race guinea (83%) originating from western West African countries such as Mali and Bukina Faso and the second subgroup included accessions mainly from Nigeria and Niger and also accessions originating from other countries and other major races. The race guinea could clearly be distinguished from the other races. Fisher's exact test for the presence of earliness among subgroups showed that there are significantly (p = 0.06) more early maturing accessions in subgroup one than subgroup two. But there was an absence of a clear structuring pattern. The study suggests that the race, the geographical origin, and maturity of the accessions are the most likely forces behind the observed structuring pattern of the accessions. We found a high level of genetic diversity among the sorghum accessions. Race guinea was found to be the most diverse and race kaura was the least diverse. In general, the estimates of the gene diversity were comparable to previous studies. The results showed that clustering of early-intermediate maturing guinea varieties may have increased the linkage disequilibrium (LD) in subgroup one compared to subgroup two. The differences in the extent of LD between our study and those in the previous studies can be due to the differences in the molecular markers used as well as differences in the racial composition of the accessions studied. In the final part of the study the association analysis was carried out using a mixed-model method. This method takes both population structure and kinship information into account. The candidate genes polymorphism data were obtained by amplifying and sequencing of the chosen genes. The association analysis for the polymorphism found within the CGs was carried out using values of PRI for each accession. From the six genes studied, genes CRY1-b1 and GI had several polymorphic sites which were significantly (p < 0.005) associated with PRI variation in the sorghum panel. The most important polymorphism in the gene CRY1-b1 showed an effect on PRI value of up to -4.2 days. This single nucleotide polymorphism (SNP) at position 722 in CRY1-b1 was located in the flavin adenine dinucleotide binding domain (N-terminal domain) of SbCRY1; hence, this domain appears to be important in photomorphogenesis in sorghum. In the case of the GI gene homolog, SNP888 had the largest effect on PRI of about +8 days. Similar to the studies in rice, the GI gene delayed flowering under June sowing (long-day conditons) and shortened the time to flower in sorghum under July sowing (short-day conditons). Therefore, the action of the GI gene homolog in sorghum might be revealed by a detailed investigation of GI by comparison of sorghum accessions grown under short-day and long-day conditions. In the case of gene SbD8, no significant association with PRI could be found; hence, the potential involvement of this gene in flowering time control of sorghum was not confirmed. Negative Tajima?s D values, of CGs indicated that the genes may have been subjected to adaptive selection as variation in flowering time may confer adaptive advantages in sorghum. The results showed that CG-based association analysis using a mixed model approach can be successfully applied to unravel the genetic variation related to phenotypic variation in flowering time. The polymorphisms significantly associated with PRI can be used to develop cleaved amplified polymorphic sequence markers. Functional markers could also be created directly from the significant SNPs. These molecular markers can serve as powerful tools in MAS for sorghum to identify cultivars sensitive to photoperiod.