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 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.     

Increasing productivity in sweet almond using selected clones of bitter almond

Summary Eight controlled crosses were made between seven different sweet-kerneled almond cultivars (Prunus dulcis [Mill.] D. A. Webb), and five crosses were made between sweet x highly floriferous bitter almond clones. Sweet x bitter progenies out-yielded the sweet x sweet matings by more than three to one in the first year of production. This was attributable to greater cropping efficiency rather than to tree size or precocity. There was no pleiotropic association or association due to linkage between bitterness and yield. The selected Alnem (bitter) clones appear to be potentially useful progenitors for increasing almond yield capacity by conventional breeding methodology.     

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.     

Potential of marker-assisted selection in hemp genetic improvement

Summary The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.     

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.     

Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups 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.     

Mapping major genes and quantitative trait loci controlling agronomic traits in almond

Six tree traits (self-compatibility, blooming date, blooming density, productivity, leafing date and ripening time) and five pomological traits (kernel taste, in-shell weight, shell hardness, kernel weight and double kernel) were studied in an F₁ almond progeny of 167 seedlings from the cross between the French cultivar 'R1000' and the Spanish cultivar 'Desmayo Largueta'. In addition, a set of 135 codominant microsatellites or simple-sequence repeat (SSR) markers developed from peach, cherry and almond were used for the molecular characterization of the progeny. A genetic linkage map was constructed with 56 of these SSRs. Cosegregation analysis allowed the identification of the map positions of two major genes to be confirmed for kernel taste ( Sk ) in linkage group five (G5) and for self-incompatibility ( S ) in G6. QTLs mapped include two for leafing date ( Lf-Q1 and Lf-Q2 ) in G1 and G4, one for shell hardness ( D-Q ) in G2, one each for double kernel ( Dk-Q ) and productivity ( P-Q ) in G4, one for blooming date ( Lb-Q ) in G4, two for kernel weight ( Kw-Q1 and Kw-Q2 ) in G1 and G4, and two for in-shell weight ( Shw-Q1 and Shw-Q2 ) in G1 and G2. Four SSR loci (BPPCT011, UDP96-013, UDP96-003 and PceGA025) were linked to the important agronomic traits of leafing date, shell hardness, blooming date and kernel taste. Finally, the development of efficient marker-assisted selection strategies applied to almond and other Prunus breeding programmes was also discussed.     

Comparison of homozygous and heterozygous self-compatible seedlings in an almond breeding programme

Homozygous self-compatible almond cultivars have not been reported. It is unclear if they are more inferior than heterozygotes or simply have not yet been detected. To investigate if homozygous individual are generally inferior, the self-compatibility genotype, homozygous or heterozygous, was determined by stylar ribonuclease assay in a population of 241 almond trees obtained by self-fertilisation of self-compatible selections. The resulting zymograms showed that 129 of the seedlings were homozygous and 112 heterozygous. For three years the differences observed between these two classes of self-compatible individuals were analysed with respect to 16 agronomic characteristics. In general, there were no important differences between the two classes. Both showed a low degree of productivity, probably as a result of their inbred origin. Some selected homozygous individuals were used in crosses, which were planned so as to ensure the self-compatibility of 100% of the descendants and to eliminate the laborious task of testing the seedlings for self-compatibility. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance of root traits and phosphorus uptake in common bean (Phaseolus vulgaris L.) under limited soil phosphorus supply

Heterosis is an important way to improve yield and quality for many crops. Hybrid rice and hybrid maize contributed to enhanced productivity which is essential to supply enough food for the increasing world population. The success of hybrid rice in China has led to a continuous interest in hybrid wheat, even when most research on hybrid wheat has been discontinued in other countries for various reasons including low heterosis and high seed production costs. The Timopheevii cytoplasmic male sterile system is ideal for producing hybrid wheat seeds when fertility restoration lines with strong fertility restoration ability are available. To develop PCR-based molecular markers for use in marker-assisted selection of fertility restorer lines, two F₂ populations derived from crosses R18/ND36 and R9034/ND36 were used to map fertility restoration genes in the two elite fertility restorer lines (R-lines) R18 and R9034. Over 678 SSR markers were analyzed, and markers closely linked to fertility restoration genes were identified. Using SSR markers, a major fertility restoration gene, Rf3, was located on the 1B chromosome in both populations. This gene was partially dominant in conferring fertility restoration in the two restorer lines. SSR markers Xbarc207, Xgwm131, and Xbarc61 are close to this gene. These markers may be useful in marker-assisted selection of new restorer lines with T. timopheevii cytoplasm. Two minor QTL conferring fertility restoration were also identified on chromosomes 5A (in R18) and 7D (in R9034) in two R-lines.     

Morphological variation within collections of Moroccan almond clones and Mediterranean and North American cultivars

Summary Cultivated almonds (Prunus dulcis (Miller) D.A. Webb) in Morocco are still propagated from seeds by farmers to overcome transplant failure of grafted trees. Almond collections in southern Morocco conducted since 1975 have resulted in the selection of clones planted at 3 experimental stations. Principal component analysis (PCA) was used to compare kernel, nut, leaf, and growth habit characteristics among 67 selected Moroccan clones and 14 introduced cultivars. Clustering of clones from similar countries and collection areas would suggest the existence of different almond populations. The Moroccan clones did not cluster separately from the foreign cultivars. Three Moroccan clones had exceptionally large nuts and kernels while 7 selections had high yield potential due to high spur density. Moroccan selections tended to be characterized by small leaves in comparison to foreign cultivars. No evidence was found to suggest the existence of separate populations within the Moroccan almond germplasm.     

分子标记技术在甜菜育种中的应用

分子标记技术与常规育种技术相互紧密结合能显著提高育种效率。为了更好地阐明分子标记在甜菜育种中的作用,总结了国内外分子标记在甜菜亲缘关系及遗传多样性研究、遗传连锁图谱构建、数量性状基因定位（QTL）、分子标记辅助选择育种、杂种优势及种质鉴定中的研究现状和存在的问题。指出建立相应的高效分子标记辅助选育体系,创造出高产、优质、多抗或具广谱抗性的甜菜种质或品种是甜菜分子育种的研究方向。当前甜菜种质资源鉴定的关键任务是大力开发新型的分子标记进行甜菜种质资源遗传分析,绘制指纹图谱、进一步构建甜菜种质分子身份证。今后应加强对甜菜重要农艺性状基因进行精细定位,充分发掘QTL的信息,构建更为饱和的分子标记连锁图谱。     

On genetically remodeling the almond tree

Summary Dwarf peach trees require only 1/4th the space of standard trees and thus reduce the cost of experiments in which the tree is the experimental unit, by 75%. Or, put another way, they facilitate estimates of tree performance, in experiments of the same size, that are 50% more precise than those obtainable from standard trees. The objective of this study was to determine if the almond tree could be genetically compressed (dwarfed) to facilitate discrimination of genetic and cultural manipulation on its productivity, to facilitate genetic manipulation, and to increase its productive efficiency.The results obtained indicate the almond tree can be dwarfed (compressed), by the dw gene of peach. interspecific crosses of dwarf (dw/dw) peach, P. persica, with almond, P. amygdalus, followed by backcrosses to almond revealed: 1. Plant stature and node density vary widely among the dwarf (dw/dw) inter species hybrids. The heritabilities of these traits are high. 2. Spur density also varies widely. Its mean is high and its heritability is very high. 3. The dwarf inter species hybrids produce flowers copiously. However, at 22 months of age the flowers of most seedlings are sterile, apparently due to abnormal pistils. Consequently, average productivity is very low. However, some of these dwarf P. persica × P. amygdalus hybrids do express moderate to high productivity. Further, the heritability of productivity, among the seedlings expressing some degree of fertility, is very high. Consequently, further backcrosses to almond are expected to rapidly restore fertility and productivity. The yield potential of dw/dw dwarf almonds will remain unknown until fertility is restored.     

Identification of AFLP and RAPD markers linked to anthracnose resistance in grapes and their conversion to SCAR markers

Resistance to anthracnose or black spot ( Elsinoe ampelina ), a serious fungal pathogen in viticulture and table grape production, was investigated on 25 grape cultivars. Bioassays performed with culture filtrates produced by the pathogen revealed 14 resistant genotypes. In most plants resistance originated from Vitis labrucsa but also genotypes with V. rupestris and V. riparia  ×  V. rupestris background showed resistance. Genetic analysis was conducted in F₁, S₁ and BC₁ plants developed from various cultivars. In total, 326 F₁ plants were evaluated, 172 genotypes proofed to be resistant, whereas 154 were susceptible to anthracnose. A Mendelian segregation ratio of 1 : 1 (χ² = 0.30–0.65) indicating that anthracnose resistance is controlled by a single dominant gene. To facilitate the use of marker-assisted selection in grape-breeding PCR-based markers were developed by random amplified polymorphic DNA and amplified fragment length polymorphism in bulk segregant analysis. Finally, OPB 15₁₂₄₇ was developed as a sequence characterized amplified region marker being diagnostic for the locus of resistance to anthracnose in all resistant genotypes tested. Within the 25 grape cultivars OPB 15₁₂₄₇ is diagnostic in the genetic background of both V. labrucsa and V. rupestris and V. riparia  ×  V. rupestris .     

Identification of self-incompatibility (S) alleles in Latvian and Swedish sweet cherry genetic resources collections by PCR based typing

The Latvian and the Swedish sweet cherry (Prunus avium L.) genetic resources collections comprise valuable material for breeding. The collections represent local Latvian and Scandinavian genetic resources: semi-wild samples, landraces, and cultivars developed in local breeding programmes, as well as diverse germplasm from the northern temperate zone. The objective of this investigation was to determine which S ₁ –S ₆ alleles are most important in the sweet cherry genetic resources collections and to compare the identified allelic and genotypic frequencies in material of different origin. Accessions in the two collections were screened for the presence of the self-incompatibility (S) S ₁ to S ₆ alleles, using PCR based typing. Significant differences (P < 0.05) between screened collections were found in frequencies of S ₄ and S ₅ alleles. Analysis of allele combinations identified the high occurrence of selections with the S-genotype S ₃ S ₆ in both collections. Compared to the S-allele frequencies published for over 250 sweet cherry cultivars from Western and Southern Europe, the Latvian and Swedish germplasm appeared to have a high frequency of the S ₆ allele in both collections, and a relatively high frequency of the S ₅ allele in Latvian germplasm. This study represents the first comprehensive S-allele screening for the sweet cherry genetic resources collections in Latvia and Sweden. Both sweet cherry collections contain high proportion of accessions adapted to north central European growing conditions, not typical for the majority of the documented sweet cherry genetic resources, which explains differences in certain S-allele occurrence.     

转耐盐基因水稻的PCR检测方法及其分子辅助育种

研究采用改良CTAB法和磁珠自动提取法提取水稻种子基因组DNA,通过对水稻内源SPS基因、ThST3和Ubiquit-in启动子间构建特异序列进行PCR扩增,扩增产物结合排枪凝胶电泳实现快速检测。其中PCR扩增内源SPS基因的结果表明,采用改良CTAB法和磁珠自动提取法可用于市售水稻种子和转基因种子的DNA提取。实验合成的构建特异引物以及建立的PCR扩增反应体系能特异性地检测转耐盐基因水稻Theli。该方法检测灵敏度高,绝对检测低限达17.3×10-2ng,相对检测低限为0.41%,能有效地对转基因水稻ThST 3进行鉴定;稳定性好,可完全满足转基因水稻的定性检测、监督和标识管理需要。同时可用于对转基因水稻的辅助选择(MAS)育种。     

Development of two multiplex PCR assays targeting improvement of bread-making and noodle qualities in common wheat

Wheat quality properties are genetically determined by the compositions of high and low molecular weight glutenin subunits, grain hardness, polyphenol oxidase (PPO) activity and starch viscosity. Two multiplex PCR assays were developed and validated using 70 cultivars and advanced lines from Chinese autumn‐sown wheat regions. Multiplex PCR I includes molecular markers for genes/loci ω‐secalin, Glu‐B1‐2a (By8), Glu‐D1‐1d (Dx5), Glu‐A3d, Glu‐B3 (for non‐1B·1R type) and Pinb‐D1b targeting improved gluten parameters and pan bread quality. Multiplex PCR II comprises markers for genes/loci Ppo‐A1, Ppo‐D1 and Wx‐B1b targeting improved noodle quality. The results were consistent with those achieved by SDS‐PAGE and RP‐HPLC, indicating that the two multiplex assays were highly effective, with good repeatability and low costs enabling their use in wheat breeding programmes. In total, nine alleles (subunits) at locus Glu‐B1, four at Glu‐D1 and five at Glu‐A3 locus were identified, and the alleles (subunits) Glu‐B1b (7 + 8), Glu‐B1c (7 + 9), Glu‐D1a (2 + 12), Glu‐D1d (5 + 10), Glu‐A3a, Glu‐A3c and Glu‐A3d were most frequently present in the cultivars and lines tested. The 1B·1R translocation was present in 28 (40.0%) lines, whereas the Wx‐B1 null allele for better noodle quality was present in only seven (10.0%) cultivars and advanced lines, and 37 (52.9%) lines had Pinb‐D1b associated with hard grains. The allele Ppo‐A1b on chromosome 2AL associated with lower PPO activity was present in 38 (54.3%) genotypes, whereas the less effective allele Ppo‐D1a on chromosome 2DL, also associated with low PPO activity was present in 45 (64.3%) of genotypes. These two multiplex PCR assays should be effective in marker assisted selection targeting improved pan bread‐making and noodle qualities.     

A practical method for almond cultivar identification and parental analysis using simple sequence repeat markers

Early and accurate identification of almond [Prunus dulcis (Mill.) D.A. Webb] cultivars is critical to commercial growers and nurseries. Previously published simple sequence repeat loci were examined for their ability to distinguish commonly grown almond cultivars. Twelve highly polymorphic loci were selected for their ability to uniquely identify a set of 18 almond cultivars commonly grown in California, many of which are closely related. These markers also allow an accurate assessment of parent/progeny relationships among cultivars. This system can reliably identify at an early stage of development all major California almond cultivars in current production.     

黄淮麦区Fhb1基因的育种应用

小麦赤霉病是由禾谷镰孢菌引起的一种世界性重要病害,严重威胁小麦生产安全。黄淮麦区作为我国小麦主产区,赤霉病危害日趋严重,因缺乏半冬性抗源,抗赤霉病育种进展缓慢。Fhb1基因是迄今发现的效应最大、抗性最稳定,也是被广泛应用于全球小麦赤霉病抗性育种的主效基因,但Fhb1基因在黄淮麦区尚未被广泛应用。本研究以感病品种矮抗58为轮回亲本, H35为Fhb1基因供体亲本,通过有限回交和分子标记辅助选择,同时利用双单倍体育种和传统系谱选育两种方法,培育出了一批综合性状较好、具有Fhb1基因的优良新品系,其中徐麦DH9和徐麦17252经多年鉴定均达到中抗水平。在以徐麦36和徐麦2023为杂交父本的后代品系中,含Fhb1基因的家系赤霉病平均抗性明显优于感病对照。Fhb1基因的导入显著提高了赤霉病抗性,但部分家系对赤霉病仍旧表现出高感水平,说明赤霉病抗性还受到Fhb1基因以外其他遗传因素的显著影响。本研究为Fhb1基因在黄淮麦区抗赤霉病小麦育种中的应用提供了成功的经验。