Development and mapping of Simple Sequence Repeat markers for pearl millet from data mining of Expressed Sequence Tags

<p>Abstract</p> <p>Background</p> <p>Pearl millet [<it>Pennisetum glaucum </it>(L.) R. Br.] is a staple food and fodder crop of marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. It is also a summer forage crop in the southern USA, Australia and Latin America, and is the preferred mulch in Brazilian no-till soybean production systems. Use of molecular marker technology for pearl millet genetic improvement has been limited. Progress is hampered by insufficient numbers of PCR-compatible co-dominant markers that can be used readily in applied breeding programmes. Therefore, we sought to develop additional SSR markers for the pearl millet research community.</p> <p>Results</p> <p>A set of new pearl millet SSR markers were developed using available sequence information from 3520 expressed sequence tags (ESTs). After clustering, unigene sequences (2175 singlets and 317 contigs) were searched for the presence of SSRs. We detected 164 sequences containing SSRs (at least 14 bases in length), with a density of one per 1.75 kb of EST sequence. Di-nucleotide repeats were the most abundant followed by tri-nucleotide repeats. Ninety primer pairs were designed and tested for their ability to detect polymorphism across a panel of 11 pairs of pearl millet mapping population parental lines. Clear amplification products were obtained for 58 primer pairs. Of these, 15 were monomorphic across the panel. A subset of 21 polymorphic EST-SSRs and 6 recently developed genomic SSR markers were mapped using existing mapping populations. Linkage map positions of these EST-SSR were compared by homology search with mapped rice genomic sequences on the basis of pearl millet-rice synteny. Most new EST-SSR markers mapped to distal regions of linkage groups, often to previous gaps in these linkage maps. These new EST-SSRs are now are used by ICRISAT in pearl millet diversity assessment and marker-aided breeding programs.</p> <p>Conclusion</p> <p>This study has demonstrated the potential of EST-derived SSR primer pairs in pearl millet. As reported for other crops, EST-derived SSRs provide a cost-saving marker development option in pearl millet. Resources developed in this study have added a sizeable number of useful SSRs to the existing repertoire of circa 100 genomic SSRs that were previously available to pearl millet researchers.</p

Whole-plant mesotrione dose-response of Palmer amaranth at different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night) as measured by (a) plant height 3 weeks after treatment (WAT), (b) visual injury 3 WAT, (c) leaf chlorophyll index 2 WAT, and (d) photochemical efficiency of PSII (<i>F</i>_<i>v</i><i>/F</i>_<i>m</i>) 2 WAT.

<p>Palmer amaranth plants (10 to12 cm tall, 8 to 10 leaf stage) were treated with 0, 3.28, 6.563, 13.125, 26.25, 52.5, 105, and 210 g ai ha^-1 mesotrione with 1% v/v crop oil concentrate (COC) and 0.85% w/v ammonium sulphate (AMS). Curves for height and visual injury, and chlorophyll index and <i>F</i>_<i>v</i><i>/F</i>_<i>m</i> data were fitted using three parameter log-logistic and Weibull model, respectively, as described by Knezevic et al. (2007).</p

Time course of <i>HPPD</i> expression levels (a) relative to <i>CPS</i> and (b) <i>β</i>-tubulin in mesotrione-treated (105 g ai ha^-1, 0.85% w/v AMS, and 1% v/v COC) Palmer amaranth leaves under three different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night).

<p>Total RNA was extracted from a bulked sample of Palmer amaranth leaves. Data represent means of two to three biological samples. Error bars represent SE.</p

Photographs of mesotrione-treated Palmer amaranth plants grown under (a) LT (25/15°C, day/night), (b) OT (32.5/22.5°C, day/night), and (c) HT (40/30°C, day/night) conditions (15/9 h day/night).

<p>Plant to plant variability was observed within the growth temperature and mesotrione rate. These are the representative plants for each dose and temperature. The photographs were taken 4 weeks after treatment and all photographs were taken under the same magnification.</p

Representative reverse-phase HPLC chromatograms of mesotrione metabolism in Palmer amaranth plants grown under (a) LT (25/15°C day/night), (b) OT (32.5/22.5°C day/night, and (c) HT (40/30°C day/night) conditions (15/9 h day/night) at 48 h after treatment.

<p>Peak retention time around 18.3 min represents the parent mesotrione compound. Palmer amaranth plants (10 to 12 cm tall) were treated with 8- x 2.5-μL droplets (1.6548 mM mesotrione, 0.85% w/v AMS, and 1% COC) containing 7.2 kBq of [¹⁴C] mesotrione on the upper surface of fourth and fifth youngest leaves. Numbers above the peaks represent retention time (min).</p

[¹⁴C] mesotrione absorption (a), translocation (b), total recovery (c), and translocation to treated-leaf (d), above treated-leaf (e) and below treated-leaf (f) at three different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night).

<p>The upper surface of fourth youngest leaf of Palmer amaranth plants (10 to 12 cm tall, 8 to 10 leaf stage) were treated with 4- x 2.5-μL droplets (1.6548 mM mesotrione, 0.85% w/v AMS, and 1% COC) containing 3.3 kBq of [¹⁴C] mesotrione. Significant differences (within harvest time) between the OT and LT (blue asterisks) or HT (red asterisks) plants are indicated with asterisks (*, P ≤ 0.05; **, P < 0.01). Error bars represent SE.</p