Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei''s average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Fortification of sorghum (Sorghum vulgare) and pearl millet (Pennisetum glaucum) flour with zinc

Deficiency of zinc is believed to be as widespread as that of iron, with equally serious consequences. Fortification of staple foods with this mineral is a cost-effective method that can be used to combat this deficiency. In the present study, flours of pearl millet and sorghum were evaluated as vehicles for fortification with zinc. Zinc stearate was used as the fortificant, and added at a level that provided 5 mg Zn/100 g flour. The metal chelator EDTA was used as a co-fortificant, the molar ratio of exogenous Zn:EDTA being 1:1. Bioaccessibility of zinc from the fortified flours, both raw and cooked, was determined by an in vitro simulated gastrointestinal digestion procedure. The results of the study revealed that there were differences among these two flours with respect to the feasibility of fortification with zinc. Although fortified pearl millet flour provided a higher amount of bioaccessible zinc, this was attributable to the presence of EDTA, rather than to the fortified zinc. The benefit of fortification with zinc was more evident in sorghum flour, compared to that in pearl millet flour, the increase in bioaccessible zinc content being more than 1.5 times higher as a result of fortification. Fortified sorghum and pearl millet flours were stable during storage for a period of up to 60 days. Thus, millet flours seem to be satisfactory candidates for fortification with zinc, and can be exploited to address zinc deficiency.     

青铜时代中晚期辽西地区农业活动特征

辽西地区城子山遗址和夏家店遗址农作物种子遗存研究显示,城子山地区青铜时代中期以旱作农业种植为主.粟的栽培比例要明显高于黍.青铜时代晚期的夏家店地区属半农半牧区,存在黍与粟为主的旱作农业种植,但黍的栽培比例要远高于青铜时代中期的城子山地区.干旱化可能是导致夏家店地区青铜时代晚期黍栽培比例升高的主要原因.城子山和夏家店地区农业种植结构在青铜时代晚期可能已有所不同,但这2个地区农业种植的差异究竟从何时开始,仍是一个需要继续深入研究的问题.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

糜子叶绿体基因组密码子使用偏性的分析

密码子使用偏性（CUB）是生物体重要的进化特征,对研究物种进化、基因功能以及外源基因表达等具有重要科学意义。本研究利用糜子（Panicum miliaceum L.）叶绿体基因组中筛选出的53条蛋白编码序列,对其密码子使用模式及偏性进行了分析。结果表明,糜子叶绿体基因的有效密码子数（ENC）在37.14~61之间,多数密码子的偏性较弱。相对同义密码子使用度（RSCU）分析发现,RSCU > 1的密码子有32个,其中28个以A、U结尾,表明第3位密码子偏好使用A和U碱基。中性分析发现,GC₃与GC₁₂的相关性不显著,回归曲线斜率为0.2129,表明密码子偏性主要受到自然选择的影响;而ENC-plot分析发现大部分基因落在曲线的上方及周围,表明突变也影响了密码子偏性的形成。进一步的对应性分析发现,第1轴为主要影响因素,解释了17.92%的差异,其与ENC、GC_3S值的相关性均达到显著水平,但与CBI、GC_all不相关。最后,9个密码子被鉴定为糜子叶绿体基因组的最优密码子,糜子叶绿体基因组的密码子使用偏性可能受选择和突变共同作用。     

Effect of crop residues on root growth and phosphorus acquisition of pearl millet in an acid sandy soil in Niger

The effect of long-term (1983–1988) applications of crop residues (millet straw, 2–4 t ha^-1 yr^–1) and/or mineral fertilizer (30 kg N, 13 kg P and 25 kg K ha^-1 yr^-1) on uptake of phosphorus (P) and other nutrients, root growth and mycorrhizal colonization of pearl millet (Pennisetum glaucum L.) was examined for two seasons (1987 and 1988) on an acid sandy soil in Niger. Treatments of the long-term field experiment were: control (–CR–F), mineral fertilizer only (–CR+F), crop residues only (+CR–F), and crop residues plus mineral fertilizer (+CR+F).In both years, total P uptake was similar for +CR–F and –CR+F treatments (1.6–3.5 kg P ha^-1), although available soil P concentration (Bray I P) was considerably lower in +CR–F (3.2 mg P kg^-1 soil) than in –CR+F (7.4) soil. In the treatments with mineral fertilizers (–CR+F; +CR+F), crop residues increased available soil P concentrations (Bray I P) from 7.4 to 8.9 mg kg^-1 soil, while total P uptake increased from 3.6 to 10.6 kg P ha^-1. In 1987 (with 450 mm of rainfall), leaf P concentrations of 30-day-old millet plants were in the deficiency range, but highest in the +CR+F treatment. In 1988 (699 mm), leaf P concentrations were distinctly higher, and again highest in the +CR+F treatment. In the treatments without crop residues (–CR–F; –CR+F), potassium (K) concentrations in the leaves indicated K deficiency, while application of crop residues (+CR–F; +CR+F) substantially raised leaf K concentrations and total K uptake. Leaf concentrations of calcium (Ca) and magnesium (Mg) were hardly affected by the different treatments.In the topsoil (0–30 cm), root length density of millet plants was greater for +CR+F (6.5 cm cm^-3) than for +CR–F (4.5 cm cm^-3) and –CR+F (4.2 cm cm^-3) treatments. Below 30 cm soil depth, root length density of all treatments declined rapidly from about 0.6 cm cm^-3 (30–60 cm soil depth) to 0.2 cm cm^-3 (120–180 cm soil depth). During the period of high uptake rates of P (42–80 DAP), root colonization with vesicular-arbuscular mycorrhizal (VAM) fungi was low in 1987 (15–20%), but distinctly higher in 1988 (55–60%). Higher P uptake of +CR+F plants was related to a greater total root length in 0–30 cm and also to a higher P uptake rate per unit root length (P influx). Beneficial effects of crop residues on P uptake were primarily attributed to higher P mobility in the soil due to decreased concentrations of exchangeable Al, and enhancement of root growth. In contrast, the beneficial effect of crop residues on K uptake was caused by direct K supply with the millet straw.     

糜子育成品种苗期抗旱性评价与鉴定指标筛选

在年降雨量不足40 mm的敦煌市,对我国不同地区育成的56份糜子品种,采用反复干旱法进行了苗期抗旱性鉴定,利用反复干旱存活率和抗旱性综合评价值D的聚类结果筛选出一级抗旱品种4份,分别是陇糜5号、吉3、蒙粳糜1号和蒙粳糜7号;配对t测验显示:反复干旱胁迫后糜子苗高、植株含水量、单株叶面积、生物学产量分别降低了16.097 cm、0.393 g/株、10.358 cm2/株、0.075 g/株,差异达到了极显著水平,而根长、相对生长率反向增加了0.555 cm、3.213%;主成分分析中,植株含水量、生物学产量、单株叶面积3个指标在第1主成分中起决定作用(系数均≥0.881),且与两种抗旱评价参数值相关性均达到了极显著水平,因此,确定植株含水量、生物学产量、单株叶面积作为糜子苗期抗旱性鉴定1级指标。     

施用氮、磷肥对作物体内养分含量的影响

２年四季盆栽试验结果表明：（１）施用磷肥能改善作物地上部生物学产量向籽粒分配．在Ｎ、Ｐ胁迫条件下,施Ｎ增加非经济产量与经济产量比值．（２）对于谷子,在Ｎ、Ｐ亏缺条件下,施Ｎ能增加茎叶含Ｎ量,降低Ｎ向籽粒中转移;施磷能改善Ｎ在茎叶与籽粒之间的分配。（３）对于小麦,在Ｎ或Ｐ胁迫条件下,补施Ｐ或Ｎ均能增加冠根吸Ｎ量之比,对于谷子,其对冠根吸Ｎ量之比影响与小麦完全不同．（４）谷子在Ｎ、Ｐ胁迫条件下,施Ｎ能降低Ｐ在籽粒中积累,施Ｐ却能显著地改善磷向籽粒中转移,小麦在Ｎ、Ｐ胁迫条件下,增施Ｐ或Ｎ均能显著改善磷向籽粒中转移。     

Genome-wide development of transposable elements-based markers in foxtail millet and construction of an integrated database

Transposable elements (TEs) are major components of plant genome and are reported to play significant roles in functional genome diversity and phenotypic variations. Several TEs are highly polymorphic for insert location in the genome and this facilitates development of TE-based markers for various genotyping purposes. Considering this, a genome-wide analysis was performed in the model plant foxtail millet. A total of 30,706 TEs were identified and classified as DNA transposons (24,386), full-length Copia type (1,038), partial or solo Copia type (10,118), full-length Gypsy type (1,570), partial or solo Gypsy type (23,293) and Long- and Short-Interspersed Nuclear Elements (3,659 and 53, respectively). Further, 20,278 TE-based markers were developed, namely Retrotransposon-Based Insertion Polymorphisms (4,801, ∼24%), Inter-Retrotransposon Amplified Polymorphisms (3,239, ∼16%), Repeat Junction Markers (4,451, ∼22%), Repeat Junction-Junction Markers (329, ∼2%), Insertion-Site-Based Polymorphisms (7,401, ∼36%) and Retrotransposon-Microsatellite Amplified Polymorphisms (57, 0.2%). A total of 134 Repeat Junction Markers were screened in 96 accessions of Setaria italica and 3 wild Setaria accessions of which 30 showed polymorphism. Moreover, an open access database for these developed resources was constructed (Foxtail millet Transposable Elements-based Marker Database; http://59.163.192.83/ltrdb/index.html). Taken together, this study would serve as a valuable resource for large-scale genotyping applications in foxtail millet and related grass species.