薏苡种子形态性状多样性评价

薏苡是优质的药食兼用作物。本研究对我国36份薏苡种质的种子形态性状进行了主成分分析和聚类分析。结果表明,36份薏苡种质具有较高的形态性状多样性。不同薏苡种质的4个种子质量性状间存在差异。单因素方差分析显示11个种子数量性状在不同薏苡种质间呈极显著差异。数量性状的变异系数在8.43%~68.01%之间,种壳与外种皮重量的变异系数最大而种仁长度的变异系数最小。主成分分析将36份薏苡种质分为中部及南部栽培种质、北部栽培种质和野生种质3大类,3类种质的形态特征存在明显差异。聚类分析将36份薏苡种质分为5大类群,即中部及南部栽培种质、北部栽培种质和3个类群野生种质。初步筛选出6份可能具有较高抗肿瘤活性的薏苡种质FJNJ、TJ-BK-1、TJ-BK-2、MY-BK-1、MY-BK-2和JYSD。     

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor,cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.     

Production of wheat doubled haploids by pollination with Job's tears (Coix lachryma-jobi. L.)

Wheat (Triticum aestivum L.) haploids were produced by crossing with Job's tears (Coix lachryma-jobi L.) as the pollen parent. Pollination was followed by 2,4-D treatment, detached tiller culture, and embryo culture, as described for maize pollination. The frequency of embryo formation was similar to that obtained by crossing wheat with maize pollen. Job's tears is a perennial plant which forms several stalks and its pollen can be collected throughout the year when the plant is maintained in a controlled environment. Our results indicate that Job's tears can be used as the pollen parent for wheat crosses for haploid production without requiring synchronization of flowering dates.     

Newly developed polymorphic microsatellite markers in Job's tears (Coix lacryma‐jobi L.)

A microsatellite‐enriched library of Job's tears (Coix lacryma‐jobi L. var. Ma‐yuen Stapf) was constructed using a modified biotin–streptavidin capture method. After screening, 17 polymorphic microsatellites were used for diversity analysis among 30 Job's tears accessions. The number of alleles ranged from one to five alleles per locus with an average of 2.8 alleles. Expected heterozygosity (H_E) and polymorphism information content (PIC) ranged from 0 to 0.676 and from 0 to 0.666, respectively. The newly developed microsatellite markers are expected to be very valuable tools for evaluation of genetic diversity among large germplasm collection of Job's tears present in our Korean Gene Bank.     

Effect of inbreeding and selection for vigour and fertility on meiotic behaviour in autotetraploid job's tears,Coix lacryma-jobi L.

Summary Meiotic behaviour of the colchicine-induced raw autotetraploid (4n = 40) Job's tears (Coix lacryma-jobi L.) was compared with that of the tetraploid envolved from it through selection for vigour and fertility over a 4 year period and selfing of the selected plants for 3 generations. A significant decrease in quadrivalent frequency and an increase in bivalent frequency per cell, greater frequency of ring quadrivalents, more cells with regular separation of chromosomes and fewer cells with laggards at anaphase I, fewer irregularities in meiosis II, fewer pollen quartets with micronuclei and fewer aneuploids in the progenies were found in evolved tetraploid. The average chiasma frequency per cell, per quadrivalent and per bivalent were more or less the same in both tetraploids. All these facts indicate that inbreeding and selection for vigour and fertility have brought about a shift towards regular meiosis in the evolved tetraploid. The increase in fertility during the period of selection was not, however, significant, suggesting that its response to selection is slow, that a number of factors are probably involved and that, besides multivalents, genic factors also govern sterility in the tetraploid.     

通过图像分析方法作出的薏苡定量染色体图

薏苡染色体数目为2n=20,由于中期染色体的长度差异不大,相邻染色体难以区分,而薏苡前中期的染色体相对较长,经DAPI染料染色后染色体上显示明显不同的深染区和浅染区,深染区和浅染区分别相当于染色体上染色质纤维较浓缩和较伸展的区域。前中期染色体上染色深浅不一的染色区可以作为识别薏苡特定染色体的重要标志,也可用于辨别同源染色体。用MetaMorph软件定量分析了薏苡每条前中期染色体上(从短臂到长臂)DAPl信号强度的变化,结合染色体的长度和臂比作为辅助参数,构建了薏苡前中期染色体的定量染色体图。该定量染色体图不仅描述了每条染色体不同区域浓缩程度的不同,而且也反应出不同浓缩区域所占染色体长度的比例。因此该定量染色体图是实际的前中期染色体的一种直观模式,可作为识别薏苡基因组中每条染色体的有力依据。     

Microsporogenesis,Megasporogenesis and the Formation of Male and Female Gametophyte in Coix lacryma-jobi L.

The development of the anther wall follows the monocotyledonous type. During meiotic stages of prophase I, some cytoplasmic channels are observed on the walls between meiotic cells. which divide synchronously. Cytokinesis in the microspore mother cell is of the successive type and gives rise to iscbilateral tetrad. The cell wall between the generative cell and the vegetative cell in early stage shows PAS positive reaction. The mature pollen grain is of 3-celled type. The development of the female gametophyte follows the polygonum-type; the antipodal cells proliferate to form a multicellular tissue mass. Many starch grains are present in the central cell. The nucleus of the mature egg cell is located at the micropylar end; a great deal of starch grains are in the cytoplasm surrounding the nucleus, while vacuoles of various size distribute throughout its cytoplasm but are more and larger at the chalaza,1 end. The nucleus of the synergid cell is located at the micropytar end where a filiform apparatus is formed and many small vacuoles are present at the chalazal part.     

玉米mir1基因在玉米和薏苡中的比较物理定位

玉米基因mir1编码一种抗秋季黏虫的半胱氨酸蛋白酶。利用RFLP作图mir1基因被定位在玉米第 6号染色体短臂上 ,但它在第 6号染色体短臂上的物理位置还不知道。实验以mir1和 4 5SrDNA为探针 ,通过双色荧光原位杂交技术确定了mir1基因在玉米细胞分裂中期和粗线期第 6号染色体上的物理位置。Southern杂交结果表明 ,在薏苡基因组中存在mir1基因的同源序列 ,进一步利用荧光原位杂交的方法确定mir1基因的同源序列定位于薏苡第 7号染色体长臂的近末端 ,其信号与着丝粒的百分距离为 73 33± 0 15。     

水稻程序性细胞死亡抑制基因Dad-1在薏苡中的定位

基因Dad-1是普遍存在于动物和植物中一个高度保守的程序性细胞死亡抑制基因。以水稻Dad-1基因为探针,采用Southern杂交在薏苡中检出了Dad-1基因的同源序列,并利用荧光原位杂交的方法对其进行了染色体物理定位。在薏苡第9染色体短臂上检测到Dad-1基因的杂交信号,信号与着丝粒的百分距离为67.44±1.45。     

Changes in levels of Proteases and Their Inhibitors during Development of the Seeds of Job's Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) and Their Localization and Interactions

Changes in levels and localization of proteases and a trypsininhibitor (JBTI) in the developing seeds of Job's tears (Coixlacryma-jobi L. var. Ma-yuen Stapf) were followed, and theirinteractions were examined. The JBTI was induced from the middlestage of development (12 DAF, i.e., 12 days after flowering)and increased until the late stage of development (24 DAF),and was localized in the germ. Two groups of proteases (A, thosewith molecular weights of 55–70 kDa; and B, those withmolecular weights greater than 94 kDa) were detected by activestaining of substrate-containing SDS-polyacrylamide gels. Thegroup of larger proteases seems to consist of three members(B₁, B₂₎ B₃, with increasing molecular weights). Band A wasobserved only in the early stage of development (until DAF 9),band B₂ persisted during all stages, while bands B, and B₃ werepresent only during early and late stages, respectively. Thegroup A proteases and one of the group B proteases (probablyB₁) were inhibited by diisopropyl-fluorophosphate (DFP), bytrypsin inhibitors from soybean and rice, and by JBTI. The proteasesthat were present in the seeds of Job's tears at a late stageseemed to be localized in the germ. (Received September 9, 1988; Accepted April 19, 1989)     

Assessment of the Genetic Diversity of Different Job's Tears (Coix lacryma-jobi L.) Accessions and the Active Composition and Anticancer Effect of Its Seed Oil

Job’s tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job’s tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job’s tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job’s tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job’s tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (G_ST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job’s tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job’s tears.     

Ultrastructure of Transfer Cells in Endosperm of Coix lacryma-jobi

Special attention was paid to the ultrastructure of transfer cells (TCs) in different locations of basal endosperm in Coix lacryma-jobi at 10 and 25 days after pollination. At 10 days after pollination. TCs of the outermost layer had long wall ingrowths (WIs) whereas those of the second layer possessed fewer and shorter Wis. In both layers TCs had a lobed nucleus, abundant mitochondria, rough endoplasmic reticulum (RER), ribosomes, and a certain number of dictyosomes and vesicles which contained dense substance connected with plasma membrane of WIs. Mitochondria were located near or between WIs. The distribution of organelles in TCs of the second layer was similar to that of the outermost layer. Mitochondria had well defined cristae and dictyosomes and RER seemed more numerous than in TCs of the outermost layer. At 25 days after pollination, TCs of the outermost and the second layer were almost filled with Wis but the organelles were recognizable. TCs of the fourth layer had branched and network-like WIs, many mitochondria, starch grain within plastids and lipids locating near WIs and in the interstices of WIs. Dictyosomes were frequently found but less RER fragments were seen. TCs of the fifth layer with short WIs contained large starch grains and small protein bodies. Plasmodesmata were not observed in the walls of TCs of the outermost and second layer at both 10 and 25 days after pollination but were found in the walls of TCs of the fourth and upper layers and also in the network-like WIs at 25 days after pollination. The roles of the organelles and functions of TCs of different layers were discussed.     

Differential genome evolution and speciation of Coix lacryma-jobi L. and Coix aquatica Roxb. hybrid guangxi revealed by repetitive sequence analysis and fine karyotyping

Abstract

Background

Coix, Sorghum and Zea are closely related plant genera in the subtribe Maydeae. Coix comprises 9–11 species with different ploidy levels (2n = 10, 20, 30, and 40). The exclusively cultivated C. lacryma-jobi L. (2n = 20) is widely used in East and Southeast Asia for food and medicinal applications. Three fertile cytotypes (2n = 10, 20, and 40) have been reported for C. aquatica Roxb. One sterile cytotype (2n = 30) closely related to C. aquatica has been recently found in Guangxi of China. This putative hybrid has been named C. aquatica HG (Hybrid Guangxi). The genome composition and the evolutionary history of C. lacryma-jobi and C. aquatica HG are largely unclear.

Results

About 76% of the genome of C. lacryma-jobi and 73% of the genome of C. aquatica HG are repetitive DNA sequences as shown by low coverage genome sequencing followed by similarity-based cluster analysis. In addition, long terminal repeat (LTR) retrotransposable elements are dominant repetitive sequences in these two genomes, and the proportions of many repetitive sequences in whole genome varied greatly between the two species, indicating evolutionary divergence of them. We also found that a novel 102 bp variant of centromeric satellite repeat CentX and two other satellites only appeared in C. aquatica HG. The results from FISH analysis with repeat probe cocktails and the data from chromosomes pairing in meiosis metaphase showed that C. lacryma-jobi is likely a diploidized paleotetraploid species and C. aquatica HG is possibly a recently formed hybrid. Furthermore, C. lacryma-jobi and C. aquatica HG shared more co-existing repeat families and higher sequence similarity with Sorghum than with Zea.

Conclusions

The composition and abundance of repetitive sequences are divergent between the genomes of C. lacryma-jobi and C. aquatica HG. The results from fine karyotyping analysis and chromosome pairing suggested diploidization of C. lacryma-jobi during evolution and C. aquatica HG is a recently formed hybrid. The genome-wide comparison of repetitive sequences indicated that the repeats in Coix were more similar to those in Sorghum than to those in Zea, which is consistent with the phylogenetic relationship reported by previous work.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1025) contains supplementary material, which is available to authorized users.     

Properties of a Trypsin Inhibitor from Job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A heat stable trypsin inhibitor was found in the bran of soft-shelled job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) seeds. This inhibitor seemed to be a simple protein, and the molecular weight was about 12,000. Similar to other heat stable trypsin inhibitors, this inhibitor also contained many cysteine or cystine residues in the molecule. This inhibitor inhibited bovine trypsin at the molar ratio of 1 to 2, showing that it was double-headed. Its activity was stable against the change of pH at the range of 3 to 11 and high temperature of 100°C under certain conditions. However, the degree of heat stability of the inhibitory activity depended highly upon the kind of the solution in which this inhibitor was dissolved.     

Studies on Endosperm Development and Deposition of Storage Reserves in Coix lacryma-jobi

The transition from free nuclear to cellular endosperm of Coix lacryma-jobi was eompleted 2 days after pollination. By 3 days after pollination the central cell was filled with endosperm cells. At first all cells of endosperm underwent division, later cell division was limited mainly in the peripheral region. 10 days after pollination the epidermal layer ceased its periclinal division and became the aleurone layer. Cell division persisted in the subepidermal 'cambium-like layers until the caryopsis nearly matured. Ceils of the inner region of endosperm became enlarged. Several layers of transfer cells were formed at the basal part of the endosperm. Starch grains appeared in endosperm cells on the 9th day after pollination. 10 days after pollination, lipid bodies occurred in the aleurone layer and the underlying layers. 13 and 15 days after pollination, the small vacuoles of aleurone cells contained protein and 20 days after pollenation they became aleurone grains. By 15 days after pollination pro tein bodies were formed in starch endosperm. Storage reserve deposition continued until the grain ripened. A correlation between endosperm and emoryo development was also observed.     

Studies on Embryo Development and Deposition of Storage Reserves in Coix lacryma-jobi

Embryo development in Coix lacryma-jobi is classified into the following stages: proembryo before club-shaped, club-shaped, coleoptilar, I-leafed, 2-1eared, 3-1eared, 4-1eared, 5-leafed and 6-leafed (mature embryo). The 3-, 4-, 5-leafed embryos have 1, 2 and 3 adventitious roots (seminal roots) respectively, and the matrue also has 3. These seminal roots are arranged in a longitudinal row parallelling with the radicle. The storage reserves first deposit in the scutellar cells. 9 days after anthesis (l-leafed stage), the starch grains are accumulated in cells of scutellum, coleoptile and mesocotyle. When the embryo matures, starch grains are deposited throughout its cells. The increase in size and amount of starch grains correlates with the initiation and growth order of the embryonic organs. But the amount in the scutellar cells decreases from later to mature stage. 10 days after anthesis (2-leafed stage), protein bodies containing crystals, of protein and phytin are present in the scutellar cells. They subsequently become larger and abundant druses. At the same time some protein bodies without crystals are also formed. Later, the protein bodies containing crystals disappear, while those without crystals increase until the embryo matures. 13 days after anthesis (3- leafed stage) protein bodlies are formed in the upper coleoptile cells. Protein bodies are rich in the cells of mature embryo, but the earlier the organ of embryo occurs, the more and the larger protein bodies it contains. 10 days after anthesis, lipid bodies appear in the scutellar cells and increase in size and quantity rapidly as the embryo develops. The correlation of the length of caryopsis and scutellum with embryo development is also observed.     

Characterization of anti-complementary acidic heteroglycans from the seed of Coix lacryma-jobi var. ma-yuen

The anti-complementary polysaccharides, CA-1 and CA-2, were purified from the seed of Coix lacryma-jobi L. var. ma-yuen. CA-1 consists of rhamnose, arabinose, xylose, galactose, galacturonic acid and glucuronic acid in molar ratios of 1.8:43.8:10.8:33.2:3.2:7.2, and CA-2 consists of rhamnose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid and glucuronic acid in molar ratios of 2.4:37.0:11.8:1.7:35.6:2.9:2.6:6.0. CA-1 and CA-2 contained 8 ∼ 11% protein. Their M_rs were estimated to be 160 000 in CA-1 and 70 000 in CA-2 by gel filtration. CA-2 showed more potent anti-complementary activity than CA-1 in low dose.Methylation analysis of CA-1, its carboxyl-reduced products (reduced CA-1a and CA-1b) and CA-2 were carried out by the use of GC/MS and the results suggested that CA-1 has a very complicated and highly branched structure, and CA-2 is also composed of the same glycosidic linkages as CA-1 in different molar proportions. The results of exo α-L-arabinofuranosidase treatment and partial acid hydrolysis suggested that CA-1 and CA-2 contained arabino 3,6-galactan moiety and most of the arabinose was present as an α-L-furanosyl residues in the non-reducing terminals and (1 → 5)-linked side chains which mostly attached to the O-3 of (1 → 6)-linked galactopyranosyl residues. The results also suggested that CA-1 and CA-2 contained rhamnogalacturonan moiety which has a main chain consisting of (1 → 4)-linked galacturonic acid and (1 → 2)-linked rhamnose, and arabino 3,6- and 4-galactan might be attached to the rhamnosyl residue at the O-4. All glucuronic acid residues were present at the non-reducing terminals.