Identification of Fe and Zn Responsive Genes in Pearl Millet (Pennisetum glaucum L.) through Whole Genome Transcriptome Approach

Micronutrient malnutrition due to iron and zinc deficiencies is a serious public health problem in developing countries. In India alone, about 80% of the pregnant women and 74% of children suffer from iron and zinc deficiency. At present, knowledge of the genes controlling specific steps in the Fe and Zn pathways is still rudimentary hence studying genes of Fe and Zn density has become important. A whole genome RNA-Seq approach was carried out to understand the genes and pathways related to Fe and Zn accumulation in pearl millet. Seedlings of a high Fe and Zn grain density pearl millet inbred ICMB 1505 were exposed to four Fe and Zn stress treatments namely, –Fe –Zn, –Fe +Zn, +Fe –Zn and +Fe +Zn in hydroponics for 12 days (Figure 1). Seedlings were expressed interveinal chlorosis in the leaves due to the non-availability of Fe and Zn compared to control during the stress treatments. At that time, the leaf and root tissues in four treatments were harvested separately. Total RNA was extracted from the treated samples followed by cDNA synthesis. Illumina HiSeq 2500 platform was used to sequence the constructed cDNA libraries. Analysis of differentially expressed genes and pathways associated Fe and Zn is underway. Simultaneously, a set of 40 genotypes with variable Fe and Zn grain density were selected and tissue samples from different plant parts such as seed, flag leaf, root, stem sheath, panicles at anthesis and panicle at milky-stage were collected. RNA was extracted from the samples to validate the genes associated with Fe and Zn homeostasis. The information will be used for accelerated breeding programmes to improve the Fe and Zn density in pearl millet

Construction of genetic linkage map and QTL analysis of sinksize traits in pearl millet (Pennisetum glaucum)

A linkage map, primarily based on SSCP-SNP markers, was constructed using 188 F2:3 (F2-derived F3) mapping population progenies derived from a cross between two pearl millet inbred lines having diverse pedigrees. The parents had large differences for two sink size traits (grain size and panicle diameter), and also differed for panicle length. The skeleton linkage map covered 1019 cM and it comprised of 44 loci (detected with 24 SSCP-SNP, 10 genomic SSR, 6 EST-SSR and 4 STS primer pairs) distributed across the seven linkage groups. Average adjacent-marker intervals ranged from 14 cM on LG1 to 38 cM on LG6, with an overall mean of 23 cM. Using the F2 linkage map and phenotypic data collected from the F2 and F2:3 generations of the mapping population, a total of 18 putative QTLs were detected for the three sink-size components. Eight QTLs explained 42.7% of observed phenotypic variation for panicle length, with individual QTLs explaining 6.1 to 18.2% using the F2:3 data set. For panicle diameter, 5 QTLs explained 45.8% of observed phenotypic variation with individual QTLs accounting for 6.3 to 30.2%. Similarly for grain size, 5 QTLs explained 29.6% of phenotypic variation with individual QTLs accounting for 6.1 to 8.9%. Genomic regions associated with panicle length, panicle diameter and grain size co-mapped on LG6 between Xpsms88 and Xpsms2270, indicating the existence of a gene or gene cluster with major effects involved in the control of significant proportions of the phenotypic variation for all three sink-size traits. The QTLs for panicle length on LG2 and LG6 (LOD>3 in both F2 and F2:3 data sets), for panicle diameter on LG2 and LG3 (LOD>14 in the F2:3 data set) and for grain size on LG3 and LG6 (LOD>3 in both F2 and F2:3 data sets) were identified as promising candidates for validation prior to possible application in marker-assisted breeding

Mapping QTLs Controlling Flowering Time, Plant Height, Panicle length and Grain Mass in Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Pearl millet is an important cereal of arid- and semi-arid regions, and can endure dry conditions but experiences drought stress during post-flowering growth. Exploiting the bold seeded semi-dwarf early flowering genotypes in pearl millet is a key breeding strategy to enhance yield and for adequate food in resource-poor zones. Genetic variation for agronomic traits of pearl millet inbreds can be used to dissect complex traits through QTL mapping. Quantitative trait locus (QTL) mapping for 50% flowering time, plant height, panicle length, and grain mass (self and open pollinated seeds) was performed in recombinant inbred line (RIL) population, ICMB 841-P3 ? 863B-P2. Correlations between traits were also performed and significantly negative association between plant height and TGM was observed. High heritabilities (>0.6) were recorded for all traits. A total of 50 QTLs that affected above traits were detected. Six putative QTLs for 50% flowering time were identified on five chromosomes. One QTL on LG3 were common between flowering time and plant height. Three major QTLs for panicle length, one each on LG1, LG2 and LG6B were detected.The major QTL for TGM_self on LG 6B had a partial R2 of 23.8% and 0.8 additive effects.The total phenotypic variance for 50% FT, TGM_self, and panicle length was 23.2% (LOD- 56.28), 22.3% (LOD- 5.96) and 59.4% (LOD- 52), respectively.A total of 21 digenic interactions were demonstrated for 50%FT (R2=18%-40%) and PL (R2publishersversionPeer reviewe

Mapping Quantitative Trait Loci Controlling High Iron and Zinc Content in Self and Open Pollinated Grains of Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Pearl millet is a multipurpose grain/fodder crop of the semi-arid tropics, feeding many of the world’s poorest and most undernourished people. Genetic variation among adapted pearl millet inbreds and hybrids suggests it will be possible to improve grain micronutrient concentrations by selective breeding. Using 305 loci, a linkage map was constructed to map QTLs for grain [Fe] and [Zn] using replicated samples of 106 pearl millet RILs (F6) derived from ICMB 841-P3 × 863B-P2. The grains of the RIL population were evaluated for Fe and Zn concentration using atomic absorption spectrophotometer. Grain mineral concentrations ranged from 28.4-124.0 ppm for Fe and 28.7-119.8 ppm for Zn. Similarly,grain Fe and Zn in open pollinated seeds ranged between 22.4-77.4 and 21.9-73.7 ppm, respectively. Mapping with 305 (96 SSRs; 208 DArT) markers detected seven linkage groups covering 1749 cM (Haldane) with an average intermarker distance of 5.73 cM. On the basis of two environment phenotypic data, two co-localized QTLs for Fe and Zn density on LG 3 were identified by composite interval mapping (CIM). Fe QTL explained 19% phenotypic variation, whereas the Zn QTL explained 36% phenotypic variation. Likewise for open pollinated seeds, the QTL analysis led to identification of two QTLs for grain Fe density on LG 3 and 5, and two QTLs for grain Zn density on LG 3 and 7. The total phenotypic variance for Fe and Zn QTLs in open pollinated seeds was 16% and 42%, respectively. Analysis of QTL × QTL and QTL × QTL× environment interactions indicated no major epistasis

QTLs controlling yield and stover quality traits in pearl millet

Stover of pearl millet [Pennisetum glaucum (L.) R. Br.] is a major source of dry-season maintenance rations for livestock in traditional smallholder crop-livestock production systems on the semi-arid margins of the Thar desert in South Asia and in the Sahelian region of West and Central Africa (Renard 1997)

QTLs Controlling Yield and Stover Quality Traits in Pearl Millet

Stover of pearl millet [Pennisetum glaucum (L.) R. Br.] is a major source of dry-season maintenance rations for livestock in traditional smallholder crop-livestock production systems on the semi-arid margins of the Thar desert in South Asia and in the Sahelian region of West and Central Africa (Renard 1997). Crop residues, especially those produced in marginal environments, have poor nutritional value. Although nutritional supplements can enhance the feed quality of crop residues, they may not provide a long-term solution because of difficulties in adapting them to local conditions, limited availability, and expense. Genetic enhancement of crop-residue nutritional quality is an alternative approach that can naturally, cost-efficiently, and permanently improve productivity of these crop-livestock systems. Expression of stover quality traits in pearl millet is complex (Hash et al. 2003), and selection based on conventional breeding methods is potentially difficult considering that conventional wet-lab analysis of nutritional value is tedious and time-consuming. Identification of genomic regions associated with stover quality traits would permit application of marker-assisted backcrossing (MABC) to improve the feed value of crop residues of elite hybrids and their parental lines. Preliminary assessment of genetic variability of stover quality traits and stover yield in pearl millet has demonstrated the presence of considerable variation for cell wall digestibility and stover yield (Blümmel et al. 2003). The pearl millet mapping population based on the cross ICMB 841 × 863B used in this study is one of several developed for drought tolerance. It exhibits only limited variation for flowering time and plant height while varying substantially for many other plant traits and maintaining a reasonable degree of agronomic eliteness. Parents of this population exhibit inherent variation for many stover quality traits, so this population provides ample scope for quantitative trait locus (QTL) mapping and markerassisted selection (MAS) (Hash et al. 2003). The present study had the following objectives: mapping QTLs for stover nutritional quality, assessing relationships amongstover yield components and quality traits, and selecting target QTLs for MABC. This is the first detailed report of QTLs mapped for pearl millet stover ruminant nutritional quality traits

Phenotypic and molecular diversity-based prediction of heterosis in pearl millet ( Pennisetum glaucum L. (R.) Br.)

Genetic distances between hybrid parents based on phenotypic traits and molecular markers were investigated to assess their relationship with heterosis for grain and stover yield and other traits in pearl millet (Pennisetum glaucum [L.] R. Br.). Fifty-one hybrids developed using 101 hybrid parents (B and R lines) and showing a wide range of genetic distance between their parents based on eight phenotypic traits and 28–38 SSRs were evaluated in two sets for two seasons. The correlation between Euclidean distance (phenotypic distance, ED) and simple matching distance (molecular distance, SM) for parents of both sets was low but positive and significant (r = 0.2, P < 0.001).The correlation of ED in parents with better-parent heterosis for grain yield was similar in both sets (r = 0.38, P < 0.05). SM was not correlated with heterosis for grain yield in either set of hybrids. The results showed that phenotypic distance could be a better predictor of heterosis than molecular distance. The correlation between phenotypic distance and heterosis was not strong enough to permit the use of phenotypic diversity among parents as a major selection criterion for selection of parental lines displaying high levels of heterosis for grain and stover yield in pearl millet

Genetic Diversity in Maintainer and Restorer Lines of Pearl Millet

Molecular markers facilitate rapid and environment-neutral characterization of the pattern of genetic diversity. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) at Patancheru, India, has developed a large number and diverse range of maintainer lines (B-lines) and restorer lines (R-lines) of pearl millet [Pennisetum glaucum (L.) R. Br.] in last three decades. In the present study, 213 lines (98 B-lines and 115 R-lines) were genotyped using high throughput assay and 38 polymorphic simple sequence repeat (SSR) markers, which detected 308 alleles averaging 8.1 alleles per locus. Restorer lines were genetically more diverse than B-lines, as higher average gene diversity was detected among R-lines (0.62) than among B-lines (0.50). A neighbor-joining tree based on simple matching dissimilarity distance matrix clearly differentiated B-lines from R-lines into two clusters, with further subdivision of B-lines into four subclusters and R-lines into five subclusters. Seven B-lines clustered with R-lines, and only two R-lines clustered with B-lines. Few unique alleles (1 to 3) were detected in 14 B- and 30 R-lines, which could be used to distinguish them from each other as well as from the remaining lines used in this study. The SSR alleles of each of the parental lines along with distinctness, uniformity, and stability (DUS) characterization may enable ICRISAT to protect these lines from any kind of infringement by the multiple users who have received these lines for hybrid development

Morphological, Chemical and In Vitro Traits for Prediction of Stover Quality in Pearl Millet for Use in Multidimensional Crop Improvement

The paper reports on the variations in organic matter digestibility (OMD), organic matter intake (OMI), digestible organic matter intake (DOMI) and nitrogen (N) balances of 40 pearl millet stover fed to sheep ad libitum and investigated relationships between these in vivo variables and morphological, chemical and in vitro stover quality traits. Highly significant differences (P<0.0001) were observed for all in vivo variables. Plant height and stem diameter were consistently significantly (at least P=0.0002) inversely related to OMD, OMI, DOMI and N balance. Except for OMD (P=0.16) stover nitrogen content was significantly (at least P=0.0005) positively related to OMI, DOMI and N balance. The fiber constituents neutral (NDF) and acid (ADF) detergent fiber and acid detergent lignin (ADL) were consistently inversely related to OMD, OMI, DOMI and N balance. (at least P=0.0005). Stover in vitro digestibility, metabolizable energy content and extent and rate of in vitro gas production were highly positively (at least P<0.0009) related to the in vivo variables while lag and half time of in vitro gas production were negatively associated (at least P=0.0004) with the in vivo variables. Combined morphological, chemical and in vitro variables in stepwise multiple regressions accounted for 70 to 84%% of the variations observed in the in vivo variables. Application of stringent cross validation procedures reduced the variation in OMD, OMI, DOMI and N balance accounted for by the combined morphological, chemical and in vitro variables mostly moderately to 71, 49, 79 and 76%, respectively. Relatively simple traits can predict stover quality in breeding programs for dual-purpose pearl millet cultivar

Patterns of Molecular Diversity in Current and Previously Developed Hybrid Parents of Pearl Millet [Pennisetum glaucum (L.) R. Br.]

ICRISAT’s pearl millet (Pennisetum glaucum (L.) R. Br.) breeding program at Patancheru, India, has developed genetically diverse hybrid parents since 1980s. The present study investigated genetic diversity pattern between two groups of parents in this program, bred till 2004 and developed during 2004-2010. Combined analysis of 379 hybrid parents (current 166 parents and 213 previously developed hybrid parents) carried out using a set of highly polymorphic 28 SSRs detected 12.7 alleles per locus. An average of 8.5 and 8.7 SSR alleles per locus were found in previously developed and current parents, respectively, indicating marginal improvement in the levels of genetic diversity of hybrid parents in this program. Distance matrix differentiated these current and previously developed hybrid parents into 2 separate clusters, indicating infusion of new genetic variability over time as reflected by development of more genotype-specific alleles. Also, the seed and restorer parents were found clearly separated from each other in both the sets with few crossovers, indicating existence of two diverse and broad-based pools in hybrid parents of pearl millet. Restorer parents (R-lines) were found more diverse than seed parents (B-lines), as higher average gene diversity was detected among R-lines (0.70) than B-lines (0.56), though variation between B- and R-lines was found reduced in newly developed lines to 9.22% from 16.98% in previously developed lines. Results suggested that newly developed lines were as much divergent when compared with previously developed lines, indicating that current ICRISAT pearl millet breeding program was moving towards development of diverse new hybrid parental lines. The study suggested use of trait-specific donors in B- and R-lines separately to maintain sufficient genetic distance between seed and restorer breeding lines. It was pointed out to cross parents having higher genetic distance within the seed (B-lines) and restorer (R-lines) breeding programs to derive diverse and productive hybrid parental lines in future