Identification of S-alleles in Brassica oleracea

Summary S-alleles of Brassica oleracea were identified using a method which is based on the amplification of S-sequences from genomic DNA, followed by digestion of the PCR products with selected restriction enzymes (PCR-RFLP). A study was made in which the same S-allele was present in the homozygous state in a range of different crop types. This showed that, with minor exceptions, characteristic restriction patterns were obtained, and therefore that it was possible to identify the S-allele. To test whether the method was also suitable for the identification of both the S-alleles present in heterozygotes, a number of S-heterozygotes together with an F₂ population were screened. The results showed that the standard method was not very reliable for the identification of both of the S-alleles. This is because firstly, one of the S-alleles may be amplified preferentially, and secondly, the restriction patterns are not unique to a particular combination of S-alleles. Finally, although it is not possible to identify unequivocally both S-alleles of heterozygotes using a standard technique, the procedure can be modified for particular combinations of alleles to enable the identification to be made.     

Identification of incompatibility genotypes in almond (Prunus dulcis Mill.) using specific primers based on the introns of the S-alleles

Identification of the incompatibility genotypes of almond cultivars is important in breeding programmes for designing crosses and for selecting progeny. This paper describes a novel molecular technique for the identification of S‐alleles in almond based on the use of PCR primers designed from the sequences of the introns without the need for restriction enzyme digestion. Nine specific pairs of primers have been designed for the S1, S2, S5, S7, S8, S9, S10 (putative), S23 and Sf alleles, and these confirmed the S‐allele specificities for 22 of the 23 accessions for which published information is available. This technique provides a precise method for identifying S‐alleles from the genomic DNAs of almond cultivars, and will be useful for confirming the segregation of alleles in breeding progeny.     

Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S _f. However, the amplified fragments of the S _f allele were of similar sizes to those amplified from the S ₃ self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S _f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.     

新疆野扁桃自交不亲和花粉SFB基因的克隆及生物信息学分析

旨在克隆获得新疆野扁桃自交不亲和花粉SFB新基因,以新疆野扁桃花药为试材,应用分子同源克隆及RT-PCR等技术,并进一步采用在线软件分析SFB基因的蛋白质理化性质,实验成功克隆到了PtSFB5与PtSFB6 2个新SFB基因的cDNA全长,2个基因的cDNA全长分别为1124 bp和1072 bp,分别编码了374和348个氨基酸,2个基因都具有F-Box基因结构,属于F-Box基因家族,预测的相对分子量分别为30787.26和44037.74,等电点为5.93和5.43,均属于水溶性不稳定蛋白。     

Determination of self-incompatible genotypes in sweet cherry (Prunus avium L.) accessions and cultivars of the German Fruit Gene Bank and from private collections

Sweet cherries are self-incompatible because of a gametophytic self-incompatibility system. S alleles in the style and pollen determine the crossing relationships. Knowledge of the S allele constitution of cultivars is very important for cherry growers and breeders, and recently, molecular methods have been developed to distinguish the S alleles in sweet cherries. The S allele genotypes of 149 sweet cherry cultivars and clones, including 126 not previously genotyped, were determined by using PCR analysis. Thirteen different S alleles in 40 combinations were distinguished and nine new incompatibility groups were documented. Two new S alleles were identified in five local sweet cherry processing cultivars from southwestern Germany using the second intron primers. The sequence of these alleles was determined and compared to all known sequences available in the NCBI database. The sequences obtained showed high similarities to the alleles S ₁₉ and S ₂₂, previously described only in wild cherries, Prunus avium L.     

仁用杏离体培养技术

本项目主要研究仁用杏离体培养技术,为仁用杏遗传转化提供良好的再生体系。本试验以仁用杏品种‘围选1号’和‘优一’为试验材料,在仁用杏休眠期对其1年生枝进行短截和长放两种修剪处理。分别于5月、6月、7月、8月、9月上旬采集外植体,研究修剪、外植体采集时期、以及外源激素等因素对仁用杏离体培养的影响。结果表明:5、6月份采集外植体,仁用杏品种‘围选1号’和‘优一’的茎段腋芽的诱导率较高,同时短截处理茎段腋芽的诱导率高于长放处理;8、9月份采集外植体,茎段腋芽的诱导率较低,同时短截处理与长放处理相比较,茎段腋芽的诱导率无明显差异。外源激素6-BA、NAA有利于仁用杏品种‘围选1号’和‘优一’茎段腋芽的诱导、生长与分化。培养基中添加1.0 mg/L 6-BA、0.1 mg/LNAA时,茎段腋芽诱导率最高,组培苗生长最健壮。本研究结果为仁用杏离体培养提供科学数据,进而为仁用杏的基因工程育种提供帮助。     

Development of self-incompatible Brassica napus: (I) introgression of S-alleles from Brassica oleracea through interspecific hybridization

Cultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

S-allele identification by PCR analysis in sweet cherry cultivars

Gametophytic self‐incompatibility, governed by the S‐locus, operates in sweet cherry. The knowledge of the S‐genotype of sweet cherry cultivars is therefore essential to establish productive orchards by defining compatible combinations. The isolation of sweet cherry S‐R Nases has allowed the use of different molecular techniques to characterize the S‐genotypes of sweet cherry cultivars. Previously, incompatibility group assignment could only be carried out on mature trees through pollination tests. In this work, PCR analysis with primers designed on the conserved sequences of sweet cherry S‐R Nases has been used to characterize the S‐genotype of 71 sweet cherry cultivars, including 26 cultivars whose S‐allele constitution had not been previously described. This approach has allowed the detection of alleles that had not been amplified by PCR before, to identify six putative new S‐alleles, to define three new self‐incompatibility groups and to compile the standards for a PCR‐based S‐allele typing method in sweet cherry.     

Inheritance of nut and kernel traits in almond (Prunus amygdalus Batsch)

Summary An almond breeding program was initiated in 1966 to develop improved cultivars for arid conditions with irrigation. Nut and kernel traits were evaluated for almond parents and progenies. Highly significant parent/progeny correlation coefficients (0.7–0.9) were found for shell hardness, percentage of kernel, in-shell (nut) and kernel weight, kernel length and width, as well as kernel color and outer shell retention.Double kernels and kernel thickness had low parent/progeny correlation coefficients. Shell hardness and percent kernel were highly correlated, as were shell hardness and outer shell retention, percent kernel and outer shell retention, and kernel width and nut weight.All but two of the evaluated traits (kernel thickness and percent doubles) appear to be highly heritable with mostly additive gene action, although some degree of dominance appeared to be involved in percent kernel, shell hardness and percent doubles.Contribution No. 185-E from Agricultural Research Organization, Israel.     

Differential cold sensitivity of pollen grain germination in two Prunus species

Summary Pollen grains from selected cutivars of almond [Prunus dulcis (Mill.) D. A. Webb] and peach (Prunus persica Batsch L.) were exposed to a range of temperatures between 1°C and 34°C on a thermogradient plate. Pollen germination at temperatures below 9°C was conspicuously greater in almond than peach. Miximal germination percentages were attained at about 16°C (almond) and 23°C (peach). The two species did not differ in their capacity for pollen tube elongation over a broad range of temperatures. Maximal pollen tube elongation occurred at temperatures 5°C to 8°C higher than maximal pollen germination. Species affiliation appeared to be of much greater consequence than chilling requirement or bloom date of the sporophyte in predicting gametophytic response to temperature.     

甜杏仁深加工技术及综合利用初探

依照华北地区甜杏仁的种植和加工现状,探讨甜杏仁深加工技术及综合利用新方向,着重研究甜杏仁蛋白粉和杏肉清汁的加工工艺。介绍在甜杏仁蛋白粉和杏肉清汁制取过程中所需要的主要设备及技术关键点,旨在为甜杏仁的深加工及其综合利用开辟新的途径。     

The production of inbred lines of brussels sprouts with dominant S-alleles

Summary The best sources of Brussels sprout inbred lines with both good agronomic characters and high self-incompatibility are likely to be cultivars of reasonably good agronomic type which have not been too intensively selected. Comparison of three cultivars of different agronomic quality showed that the cultivars of poor and moderate quality had about 55% of plants with a dominant S-allele, but the most highly selected cultivar had only 25% of such plants. A programme of S-allele screening is suggested which incorporates the minimum number of tests required to determine whether or not a particular plant has a dominant S-allele. A survey of S-alleles present in commercial F₁ hybrids showed that the frequency of dominant S-alleles was only 19% in hybrids released prior to the end of 1971, but was 50% in hybrids released since 1971.     

桃EST-SSR引物的开发及通用性分析

本研究基于NCBI-EST数据库开发了4 526对桃EST-SSR引物,探究桃EST-SSR引物在蔷薇科30个物种的通用性,并对上述30个物种进行了聚类分析。结果表明,以单核苷酸、二核苷酸和三核苷酸重复最多,分别占35.80%、36.43%和25.56%;抽选了100对EST-SSR引物在12个桃品种中共扩增出237个条带,其中有79对引物具有多态性;期望杂合度平均为0.38;观察杂合度平均为0.12;引物鉴别力平均为0.41;66.67%的桃EST-SSR引物在30个蔷薇科物种中扩增出多态性条带,说明桃EST-SSR引物在蔷薇科植物中具有较高的通用性。     

Pollen retention following natural self pollination in peach,almond, and peach × almond hybrids

Summary Retention of pollen grains following natural self-pollination was evaluated in 15 cultivars (cvs.) of almond, 4 peach and 2 nectarine cvs., and 37 interspecific peach × almond hybrids compared to 7 almond seedlings. The level of pollen retention was presumed to reflect and integrate the degree of homogamy, the amount of pollen produced by the flower, the extent of anther-stigma contact during anthesis, and the level of pollen germination. Pollen retention averaged 5 times greater in the peach and nectarine cvs. than in the almond cvs. The greater pollen retention, characteristic of the peach, was dominant in expression in the interspecific F₁ hybrids over the lower levels of pollen retention, characteristic of the almond. Thus, gametophytic self-incompatibility is not the only trait supporting outcrossing in the almond. Our data are consistent with the concept of co-evolution of floral traits relating to different breeding strategies. The level of pollen retention could often be anticipated at anthesis on the basis of blossom phenotype. That is, stigma-anther contact was observed frequently in the blossoms of peach, nectarine, and the peach × almond F₁ hybrids, but only infrequently in almond.     

Independence of self-compatibility and potentiality for self-pollination in peach x almond hybrids

Summary The almond of commerce (Prunus dulcis (Mill.) D.A. Webb) is self-incompatible (SI) and requires honey-bees to effect the transfer of pollen among cultivars that flower simultaneously. Four year old trees from the F₂ generation of several peach x almond hybrids were studied to determine whether self-compatibility (SC) and the potentiality for natural, i.e., abiotic, self pollination (NSP) are genetically related or are inherited independently. Both SC and the high potentiality for NSP are characteristic of peach (Prunus persica (L.) Batsch) but not almond. Forty percent of SC genotypes exhibited adequate NSP (SC, +NSP) for good fertility i.e., without insect-mediated pollination. The remaining 60% of SC genotypes (SC,-NSP) exhibited an average 61% reduction in fruit set on limbs bagged to exclude honeybees during anthesis relative to fruit set on open pollinated limbs. Our data are consistent with the concept that fertility is dependent upon the load of compatible pollen deposited on the stigma. Fruit set reduction on bagged limbs, compared with bagged and self-pollinated limbs, was presumably due to a) lack of/insufficient pollination for fertilization and/or b) post-zygotic abortion of genetically inferior recombinants. Selection following manual self-pollination may result in SC genotypes with or without the capacity for NSP. In contrast, significant fruit set on limbs enclosed during pistil receptivity necessitates that the genotype selected express both SC and the potentiality for NSP.     

Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes

The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S‐RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S1‐S₂3 and Sf. In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite‐like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S₁₀ was shown to be a ‘new’ allele and ascribed to S₂₄ and evidence of five more ‘new’S alleles was found, for which the labels S₂₅‐S₂₉ are proposed. This PCR approach should be useful for genotyping in other Prunus crops.     

李属植物果实成熟软化研究进展

李属植物果实营养丰富,为人类提供了大量的营养物质,但是其果实采后迅速软化,导致果实品质下降、不耐贮藏及货架期短。为了解李属植物果实成熟软化的研究概况,本研究归纳总结了李属植物果实成熟过程中包括呼吸作用、乙烯释放、品质相关物质变化在内的生理变化,细胞壁结构、物质成分变化、细胞壁降解相关酶在内的细胞壁变化,果实成熟软化相关的基因及果实成熟软化蛋白质组学研究进展,并提出了存在的问题及未来研究趋势。指出目前李属植物果实成熟软化研究集中于果实采收后或贮藏期间细胞壁物质成分、结构变化及细胞壁降解相关酶,如多聚半乳糖醛酸酶、β-半乳糖苷酶等的活性变化,及这些酶的基因克隆、功能分析,指出结合转录组学、蛋白质组学、代谢组学和基因组学等几种组学将是李属植物果实成熟软化研究的发展方向。     

欧李发酵茶加工工艺研究

欧李作为我国特有的一种珍贵经济植物,具备产业化开发的潜力。本试验在采用传统发酵茶加工工艺的基础上,利用正交试验研究了漂烫时间、发酵罐装罐质量及发酵温度对成品品质的影响。结果表明,漂烫杀青5 min,稍凉后手揉捻,待茶条紧索装罐发酵,装罐250 g以紧实度较紧为好,然后在40℃下恒温发酵1周后干燥,得到的成品质量较好。并利用高效液相色谱测定了最优条件发酵茶成品中苦杏仁苷的含量,为欧李发酵茶的开发生产提供参考。     

RFLP variability in apricot (Prunus armeniaca L.)

The level of polymorphism of the restriction fragment length polymorphisms (RFLPs) detected by 33 almond genomic and cDNA probes was studied in a set of 52 European and North-American apricot cultivars. Eighteen of these probes were polymorphic and yielded a total of 48 scorable bands, allowing the identification of 45 different phenotypes. Most cultivars (43) had an individually distinguishable RFLP phenotype, and three of the five clusters with the same phenotype contained cultivars that were likely to be synonymous. The group of Spanish cultivars (25) had a lower level of polymorphism than the others, suggesting that bottlenecks may have occurred in the recent history of the apricot that have eroded its genetic variability.