绿豆基因组研究进展

绿豆是亚洲国家重要的经济作物。绿豆基因组的研究工作已开展多年,至今已经发布了6张遗传连锁图谱,然而还未有一张图谱的连锁群数与绿豆(2n=2x=22,n=11)的染色体基数一致。近年来,豆科植物比较基因组学的研究成果,为绿豆遗传连锁图谱的发展提供了新的思路。通过将绿豆遗传连锁图与其他豆类连锁图比较发现,绿豆与小豆、豇豆、普通菜豆、大豆、藊豆以及豆科模式植物—蒺藜苜蓿的基因组间有不同程度的保守性,其中尤以绿豆与普通菜豆基因组间共线性水平高。本文分别从绿豆遗传连锁图谱构建、比较基因组作图以及抗豆象基因定位等方面进行了综述,以期为绿豆遗传研究工作者提供参考。     

柚品种的等位酶变异研究

研究了柚的48个品种的等位酶变异,利用等位酶分析技术对柚的酯酶（EST）,6－磷酸葡萄糖异的酶（PGI）,6－磷酸葡萄糖变位酶（PGM）,莽草酸脱氢酶（SKD）,超氧化物歧化酶（SOD）共5个酶系统的10个等位酶基因座进行了分析,除PGI－1,PGI－2两个基因座外,其它8个均为多态性基因座;10个等位酶基因座共观察到的等位基因25个,平均每个基因座的有效等位基因数目为1．55,基因多样度0．2805,柚的品种间具有较为丰富的等位酶标记遗传多样性,但柚类种质资源群体总的遗传多样性水平偏低。柚的较低的有效等位基因数目与基因多样度可能由于人工选择及资源流失造成。     

Effect of storage on redgram [Cajanus cajan (L.) Millsp] and greengram [Vigna radiata (L.) Wilczek] varieties with particular reference to carbohydrate composition

The effect of storage on carbohydrate composition of three redgram (TTB-7, BRG-1 and ICP-8863) and three greengram (Pusa Bisaki, KDM-1 and China Moong) varieties during storage was studied. The total free sugars, starch and dietary fibre were evaluated both in control and in samples stored for six months. Decrease in percentage of (a) free non-reducing sugars, (b) starch and (c) pentose sugar of dietary fibre was observed during storage. Concomitantly insect infested samples showed increase in reducing sugar, dietary fibre and also hexose sugars of dietary fibre.     

The genomic relationship between cultivated sorghum [Sorghum bicolor (L.) Moench] and Johnsongrass [S. halepense (L.) Pers.]: a re-evaluation

Summary The genomic relationship between cultivated sorghum [Sorghum bicolar (L.) Moench, race bicolor, De Wet, 2n=20] and Johnsongrass [S. halepense (L.) Pers., 2n=40] has been a subject of extensive studies. Nevertheless, there is no general consensus concerning the ploidy level and the number of genomes present in the two species. This research tested the validity of four major genomic models that have been proposed previously for the two species by studying chromosome behaviors in the parental species, 30-chromosome hybrids [sorghum, (2n=20) x Johnsongrass, (2n=40)], 40-chromosome hybrids [sorghum, (2n=40) x Johnsongrass, (2n=40)] and 60-chromosome amphiploids. Chromosome pairings of amphiploids are reported for the first time. Chromosomes of cultivated sorghums paired exclusively as 10 bivalents, whereas Johnsongrass had a maximum configuration of 5 ring quadrivalents with occasional hexavalents and octovalents. In contrast, 40-chromosome cultivated sorghum had up to 9 ring quadrivalents and 1 hexavalent. Pairing in the 30-chromosome hybrids showed a maximum of 10 trivalents, and that in the 40-chromosome hybrids exhibited 8 quadrivalents, 5 of which were rings, together with a few hexavalents. Amphiploid plants showed up to 3 ring hexavalents, 1 chain hexavalent and a chain of 12 chromosomes. The data suggest that cultivated sorghum is a tetraploid species with the genomic formula AAB1B1, and Johnsongrass is a segmental auto-allo-octoploid, AAAA B1B1B2B2. The model is further substantiated by chromosome pairing in amphiploid plants whose proposed genomic formula is AAAAAA B1B1B1B1 B2B2.Contribution no. 87-391-J from the Kansas Agriculatural Experiment Station     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.     

Culture and regeneration of mesophyll-derived protoplasts of sorghum [Sorghum bicolor (L.) Moench]

 A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l^–1 kinetin and 2 mg l^–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity. Received: 7 October 1998 / Revision received: 13 January 1999 / Accepted: 20 January 1999     

Accumulation and Partitioning of Dry Matter in Taro [Colocasia esculenta (L.) Schott]

A field study was conducted as part of an ongoing effort tocollect data on patterns of leaf area development and dry matteraccumulation and partitioning among various plant parts duringgrowth and development of two taro cultivars. Plants were harvestedfor biomass about every 6 weeks during the growing season. Ateach harvest, plants were separated into various plant parts,and their dry matter content was determined. The first 80 dafter planting were characterized by low rates of dry matteraccumulation, with only leaves, petioles, and roots showingsubstantial growth. Afterwards, increases in total dry matterwere mainly the result of corm and sucker growth. Corm bulkingoccurred after the attainment of maximal leaf area indices.The absence of an optimal leaf area index for a longer periodof time may have prevented the realization of higher dry matteryields. The partitioning of dry matter to the corms of bothcultivars remained almost constant especially after 150 d afterplanting. This process was in contrast to the partitioning ofdry matter to the suckers, which increased significantly untilthe end of the growing cycle.Copyright 1995, 1999 Academic Press Taro, Colocasia sp., growth, dry matter partitioning     

Purification and Properties of Cystathionine [gamma]-Synthase from Wheat (Triticum aestivum L.)

Cysthathionine [gamma]-synthase (CS), an enzyme involved in methionine biosynthesis, was purified from an acetone powder prepared from wheat (Triticum aestivum L.). After several chromatographic steps and radiolabeling of the partially purified enzyme with sodium cyanoboro[3H]hydride, a single polypeptide with a molecular weight of 34,500 was isolated by sodium dodecyl sulfate-high performance electrophoresis chromatography. Since the molecular weight of the native enzyme was 155,000, CS apparently consists of four identical subunits. The pyridoxal 5[prime]-phosphate-dependent forward reaction has a pH optimum of 7.5 and follows a hybrid ping-pong mechanism with Km values of 3.6 mM and 0.5 mM for L-homoserine phosphate and L-cysteine, respectively. L-Cysteine methyl ester, thioglycolate methyl ester, and sodium sulfide were also utilized as thiol substrates. The latter observation suggests that CS and phosphohomoserine sulfhydrase might be a single enzyme. CS does not seem to be a regulatory enzyme but was irreversibly inhibited by DL-propargylglycine (Ki = 45 [mu]M, Kinact = 0.16 min-1). Furthermore, the homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 45, 40, and 1.1 [mu]M, respectively.     

Viability and Germination of the Pollen of Sorghum [Sorghum bicolor (L.) Moench]

Previous studies have demonstrated that pollen of sorghum [Sorghumbicolor (L.) Moench] loses capacity to both germinate in vitroand to set seed in vivo soon after being shed. The current studyevaluates the capacity for dehydrated pollen to effect in vitrogermination, reduce tetrazolium chloride, and set seed on cytoplasmicmale sterile plants. Morphological changes during pollen germinationwere examined by scanning electron microscopy (SEM). Close to70% of the pollen germinated in 5 min, or less, when collectedat 80% relative humidity (RH) and stored in sealed glass vials.Pollen tubes elongated autotropically with atmospheric humidityapparently being a controlling factor in the process. Pollendehydrated at 50% RH and 25°C for 15-30 min neither germinatedin vitro, reduced tetrazolium chloride, nor set seed on malesterile plants. Rehydrating the pollen did not restore the capacityfor germination. SEM micrographs demonstrated that elongatingpollen tubes encircled the pollen grain and were contiguousto the surface. A fibrillar-like material existed on the exineof separated pollen grains at the point where the grains hadbeen previously attached.Copyright 1994, 1999 Academic Press Sorghum pollen, germination, seed-set, viability, scanning electron microscopy, Sorghum bicolor (L.) Moench     

Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (PCR) assays were designed that enabled the zygosity of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) to be determined. The two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers, were both accurate and reproducible in the determination of the number of transgenes present in a cell line. In the first assay, in which TaqMan assays were performed on increasing amounts of a plasmid containing the transgene of interest, a linear relationship between the level of fluorescence and the template amount was produced. Using the resultant linear relationships as standard curves, we were able to determine the zygosity of both soybeans segregating for the cry1Ac transgene and that of a T₁ peanut segregating for the hph transgene. In the second assay, a relative determination of copy number (referred to as comparative Ct) was performed on transgenic soybeans by comparing the amplification efficiency of the transgene of interest to that of an endogenous gene in a multiplexed PCR reaction. Both methods proved to be sufficiently sensitive to differentiate between homozygotes and hemizygotes. These assays have numerous potential applications in plant genetic engineering and tissue culture, including the hastening of the identification of transgenic tissue, selecting transformation events with a low number of transgenes and the monitoring of the transmission of transgenes in subsequent crosses.     

Rhizobium inoculant for soybean [Glycine max (L.) Merrill] in Mekong Delta

Summary Rhizobial inoculation trials were conducted in an acid heavy clay soil in Mekong Delta, Viet Nam, using peat based inoculants produced locally and the commercial granular product of Nitragin CCo., Wisconsin, USA. The pH of these soils ranged from 4.5 to 5.1. Two soybean cultivars, MTD₆ and MTD₁₀, were tested as host plants. There were no significant differences between locally made inoculant treated plants and the uninoculated controls in both cultivars. But, the Nitragin inoculation improved all plant characteristics examined in both cultivars. Grain yields of Nitragin inoculated plants of cultivar MTD₆ and cultivar MTD₁₀ were 6.5 and 5.5 times as much as those of the controls; protein content of grain increased 11 and 16 percent, respectively. Well nodulated plants had shorter life cycles, flowering durations, and days to flowering. The Rhizobium symbiosis resulted in an additional 153 kg grain-N/ha. These studies show that a surface coated commercial multistrain inoculant can be used to successfully grow soybeans in the acid, heavy clay soils of the Mekong Delta.     

Boron deficiency in soybean [Glycine max (L.) Merr.] peanut (Arachis hypogaea L.) and black gram [Vigna mungo (L.) Hepper]: Symptoms in seeds and differences among soybean cultivars in susceptibility to boron deficiency

The susceptibility of soybean cultivars (cvs) to boron (B) deficiency was examined in three experiments on a Typic Tropaqualf in Northern Thailand: one experiment also included peanut cv Tainan 9 and black gram cv Regur.Without added B (B0), B deficiency depressed seed yield by 60% in soybean cv NW1 compared with 30% in cv SJ5, 40% in cv 7016, 45% in peanut, and 93% in black gram. B deficiency also induced a localised depression on the internal surface of one or both cotyledons of some soybean seeds resembling the symptom of hollow heart in peanut seeds. It induced 50% hollow heart in peanut, 17% in soybean cv NW1, 5% in SJ5, and 1% in 7016: black gram seeds had no symptoms. Addition of B decreased or eliminated the symptoms.In a comparison of 19 soybean cvs, the incidence of hollow heart symptoms in seeds at B0 varied widely from none in two cvs to 75% in cv Buchanan: cv NW1 showed intermediate susceptibility to B deficiency with 36% hollow heart in its seed while SJ5 was insensitive with 1%. Application of B eliminated hollow heart except from one seed in one cultivar. The results suggest that susceptibility to B deficiency is sufficiently important and variable among soybean genotypes to warrant its inclusion as a selection criterion when breeding cultivars for areas with low soil B.     

Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill]

Somatic embryos and embryogenic tissues were initiated from immature zygotic embryos of soybean [Glycine max (L.) Merrill cv. Fayette]. Zygotic embryos were placed on a medium containing 40 mg/l of 2,4-dichlorophenoxyacetic acid and 6% sucrose. Somatic embryos were first seen 4 weeks after cultures were initiated. Following transfer, secondary somatic embryos proliferated directly from the apical or terminal portions of the older primary somatic embryos. Single somatic embryos or clusters of embryos were seen growing directly from the top of older somatic embryos. Light microscopy revealed that these embryos were of surface or subsurface origin. The apical soybean somatic embryo tissue may represent cotyledonary tissue (which has been shown to be most responsive) at a very young and manipulatable state.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid Salaries and research support were provided by state and federal funds appropriated to OARDC-OSU. Journal Article No. 131-87     

Control of Seed Growth in Soya Beans [Glycine max (L.) Merrill]

The seed is the primary sink for photosynthate during reproductivegrowth and an understanding of the mechanisms controlling therate of seed growth is necessary to understand completely theyield production process. The growth rate of individual seedsof seven soya bean [Glycine max (L.) Merrill] cultivars withgenetic differences in seed size varied from 10.8 to 3.9 mgseed^–1 day^–1. The growth rates were highly correlatedwith final seed size. The growth rate of cotyledons culturedin a complete nutrient medium was highly correlated with thegrowth rate of seeds developing on the plant and with finalseed size. The number of cells per seed in the cotyledons variedfrom 10.2 to 5.7 x 10⁶ across the seven cultivars. The numberof cells per seed in the cotyledons was significantly correlatedwith final seed size and the seed growth rate both on the plantand in the culture medium. The data suggest that genetic differencesin seed growth rates are controlled by the cotyledons and thenumber of cells in the cotyledons may be the mechanism of control. Glycine max L., soya bean, seed size, growth rate, cell number, sink activity     

A first insight into peach [Prunus persica (L.) Batsch] SNP variability

Three factors may have reduced the diversity at both individual gene and whole genome levels in cultivated peach: its self-compatible mating system, the narrow genetic basis of most commercial cultivars, and the recent strong selection towards agronomically interesting traits. Previous diversity analyses with markers such as simple sequence repeats (SSRs) have revealed low levels of genetic variability. Here, we sequenced 23 genome-wide distributed DNA fragments in 47 occidental peach varieties, also observing reduced variability levels. On average, there was one single nucleotide polymorphism (SNP) every 598 bp and one indel every 4,189 bp. As expected, variability was higher in non-coding than in coding regions (one SNP every 390 non-coding bp versus one in 1,850 bp in coding DNA). In general, SNPs were observed at relatively high frequency, mean minor allele frequency?=?0.225, meaning that a large proportion of the SNPs discovered by sequencing similar germplasm will be useful for other purposes, such as association mapping. The average heterozygosity of the varieties was 0.28, with a low correlation between SSR and SNP heterozygosity. The whole sequence of two candidate genes, a pectate lyase 1 candidate for fruit firmness (CGPAA2668) and a sucrose synthase 1 candidate for sugar content (CGPPB6189), in the 47 varieties revealed that they both may have suffered a process of balancing selection.     

Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck]

Citrus sinensis chromosomes, although small in size, present a remarkable differentiation of bands with the fluorochromes CMA and DAPI. These bands suggest that some heteromorphisms are fixed in this species. To investigate the extension of these heteromorphisms, ten cultivars of C. sinenesis were analysed with CMA/DAPI staining and, in some of them, the 18S–5.8S–25S rRNA and 5S rRNA genes were located by in situ hybridization. CMA/DAPI staining showed exactly the same CMA⁺/DAPI⁻ banding pattern for all cultivars. In situ hybridization revealed three 18S–5.8S–25S rRNA gene sites, two proximally located on two similar chromosomes and one terminally located on a third non-related chromosome. Two 5S rRNA gene sites were observed in this species, with one located proximal to the telomeric 18S–5.8S–25S rDNA site. Both cytological approaches revealed an invariable, heterozygotic karyotype among sweet orange cultivars. Based on these data, the putative hybrid origin of the species is discussed. Received: 9 April 1999 / Accepted: 22 June 1999     

Diallel analysis for aluminium tolerance in tropical soybeans [Glycine max (L.) Merrill]

The soybean is a major crop in the agricultural systems of the Brazilian Cerrados (Savannahs), whose soils are acidic, devoid of nutrients and need to be amended before they are cultivated. However, below the ploughed layer there is a scarcity of nutrients and toxic aluminium (Al). These limit root growth, subsequently causing nutritional imbalance and drought stress. Our aim in the investigation described here was to identify genetic differences in the aluminium tolerance of soybeans by a 9 × 9 diallel cross among contrasting varieties grown in high-Al areas and in hydroponics. Combining ability analysis indicated predominantly additive gene effects, and the additive-dominance model explained most of the genetic differences in this germ plasm for mineral element absorption and root growth under aluminium stress. The relationship between the two factors suggest that conjugation hydroponics and field evaluations in breeding programmes would further improve soybeans with respect to yield stability under tropical cultivation conditions.