Development of SCAR markers to differentiate between mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.)

Summary

Mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.) are similar in fruit and tree morphology, and exhibit high cross- and graft-compatibility with each other. It is therefore difficult to differentiate mume and apricot cultivars on the basis of morphological and phenotypical characteristics. Molecular markers were developed to differentiate nine mume from ten apricot cultivars. Four dominant, random amplified polymorphic DNA (RAPD) markers that can discriminate between mume and apricot cultivars (designated OPA15₆₂₈, OPO10₅₅₀, OPO20₂₅₉, and OPU03₄₁₅) were identified from 21 decamer primers. Two RAPD markers (OPO10₅₅₀ and OPU03₄₁₅) were developed into dominant sequence-characterised amplified region (SCAR) markers (SCO10 and SCU03). These SCAR markers could differentiate between all mume and apricot cultivars.     

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.     

Development of sequence characterized DNA markers linked to a temperature dependence for flower induction in lychee (Litchi chinensis Sonn.) cultivars

In this paper, we present a method to find DNA markers for traits of interest in lychee cultivars (Litchi chinensis Sonn.) using high-annealing temperature random amplified polymorphic DNA (HAT-RAPD) as an initial screening method. Using 5 arbitrary random primers, a wide range of polymorphic bands ranging from 200 to 5200 bp were produced. Bands of interest were then selected for sequencing and conversion to the more reproducible and robust sequence characterized amplified region (SCAR) markers. Specifically, SCAR markers were found that distinguished lychee varieties requiring a sustained interval at low temperatures for flower induction versus those varieties that do not require such an environment, and another SCAR marker was found that amplified only the economically important Kom cultivar. These sequences shared similarity to known transposons suggesting a mechanism by which the temperature insensitivity may have initially developed.     

Application of simple sequence repeat (SSR) markers in apricot breeding: molecular characterization,protection, and genetic relationships

Seventeen peach simple sequence repeat (SSR) markers have been used in the molecular characterization of 8 apricot (Prunus armeniaca L.) cultivars from Spain, North America, France, and Greece; a new breeding line from the apricot breeding program of INRA (Avignon, France); and 13 breeding lines and 3 new releases from the apricot breeding program of CEBAS-CSIC (Murcia, Spain). DNA fingerprints have been developed establishing the genetic relatedness among cultivars, new releases, and breeding lines. Amplification of SSR loci was obtained for all 17 primer pairs and 14 of them produced polymorphic amplification. The number of presumed alleles revealed by the SSR analysis ranged from one to nine, although most primers revealed three alleles or more. The mean number of alleles per locus was 4.1. Results allowed the molecular identification of all the apricot genotypes assayed. Apricot genotypes clustered into seven principal groups in accordance with their origin and pedigree. The implications of these results for apricot breeding programs in terms of protection of new release and design of new crosses are also discussed.     

Conversion of chromosome-specific RAPDs into SCAR-based anchor markers for onion linkage maps and its application to genetic analyses in other Allium species

Integration of previously developed Allium cepa linkage maps requires the availability of anchor markers for each of the eight chromosomes of shallot (A. cepa L. common group Aggregatum). To this end, eight RAPD markers originating from our previous research were converted into SCAR markers via cloning and sequencing of RAPD amplicons and designing of 24-mer oligonucleotide primers. Of the eight pairs of SCAR primers, seven resulted in the amplification of single bands of the original RAPDs, and the remaining primer set amplified an additional band. The results of Southern hybridization using RAPD amplicons from genomic DNA of Japanese bunching onion (Allium fistulosum L.)—shallot monosomic addition lines indicated that five SCAR markers were single shallot chromosome-specific markers and were not detected in genomic DNA of A. fistulosum. The eight SCAR primer pairs were applied to other Allium species and exhibited three types of amplification profiles, namely RAPD amplicons observed only in shallot, in shallot and Allium vavilovii, and in several Allium species. A mapping study using 65 F₂ plants generated by the selfing of one interspecific cross A. cepa × Allium roylei individual integrated the SCAR marker SAOE17₅₀₀ into chromosome 5 as expected. The results of the present study show that the eight SCAR primer sets specific to shallot can facilitate the mapping in A. cepa and can also serve as anchor points between maps of different Allium species.     

山葡萄性别相关AFLP分子标记的筛选及SCAR标记的转化

 山葡萄是一种重要的抗寒、抗病葡萄种质资源。通过AFLP分子标记方法,应用64对引物组合EcoR I-NNN/Mse I-NNN对山葡萄雌花、雄花和完全花植株基因池进行筛选,引物E-AGC/M-CTG在雄花和完全花植株基因池DNA中扩增出一条分子量为283 bp的特异性条带;经组池个体和其它品系验证,该特异性条带能在雄株和完全植株中稳定出现。将该特异性条带回收、克隆、测序,设计SCAR标记引物,对已知性别的85份山葡萄种质进行盲测,准确率达到97.6％,表明标记转化成功。由于该SCAR标记在绝大多数山葡萄栽培品种和有育种价值的种质资源中得到验证,可利用该SCAR标记对山葡萄杂交后代在苗期进行性别鉴定,提高育种效率。     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.     

Identification of AFLP fragments linked to seedlessness in Ponkan mandarin (Citrus reticulata Blanco) and conversion to SCAR markers

Seedlessness is a desirable trait in citrus and has been an important breeding objective. In this study, we employed the amplified fragment length polymorphism (AFLP) technique to find molecular markers linked to the seedless trait in the Ponkan mandarin (Citrus reticulata Blanco). After screening with 72 primer pairs, 5 AFLP markers were identified that putatively correlated with the target trait. Their association was also tested by analyzing the AFLP profile from pooled individual accessions. The five fragments were cloned and sequenced, and BLAST searches showed that four of the markers had high homology to functional genes, providing some promising information that may aid in understanding the molecular mechanism of seedlessness in citrus. Based on the sequence information, eight specific primers were designed and eventually fragments AFLP-2 and AFLP-5 were successfully converted into sequence characterized amplified region (SCAR) markers. Thus, the markers detected could be useful for accelerating citrus breeding programmes by enabling early screening for seedlessness mutations using marker-assisted selection.     

Genetic variation in tomato populations from four breeding programs revealed by single nucleotide polymorphism and simple sequence repeat markers

Genetic variability in modern crops is limited due to domestication and breeding. To investigate genetic variation in different populations, 216 tomato (Solanum lycopersicum L.) cultivars, hybrids, and elite breeding lines from four breeding programs were genotyped using single nucleotide polymorphism and simple sequence repeat markers. Of 47 markers analyzed, 72.3% were polymorphic in the whole collection of 216 genotypes and 51.06–59.57% showed polymorphisms in individual populations. However, genetic variation was narrow in all four populations. Nei's genetic distance varied from 0.0422 to 0.1135 between populations and from 0.0085 to 0.3187 between lines in individual populations. Cluster and principal coordinate analysis indicated that the four populations could be grouped into three clades. Lines from Shenyang Agricultural University and China Agricultural University population formed the first clade, lines from Beijing Vegetable Research Center were in the second clade, and lines from Nunhems were in the third clade. This was further supported by population structure analysis using STRUCTURE2.2, and suggested that a lack of germplasm exchange might exist among breeding programs. It might be the reason that the progress of developing new varieties with significant improvement of horticultural traits in China is slow in recent years.     

Amplified fragment length polymorphism (AFLP) markers for fingerprinting of Argyranthemum frutescens cultivars

Argyranthemum frutescens is a commercially important ornamental species with extensive breeding programmes in several countries. Since it is vegetatively propagated there is a great need for tools for identification of cultivars. Vegetatively propagated species require clean-up from diseases, often performed through meristem-tip cultures. Forty-three cultivars of A. frutescens propagated by meristem-tip culture and traditional cuttings were analyzed for genetic relatedness and possible somaclonal variation using AFLP. Five primer combinations resulted in a relatively high degree of polymorphism. Ten molecular markers generated by one primer combination could distinguish between all 43 cultivars. Differences in fingerprints between meristem-tip culture and cuttings from the same cultivars were found, but the proportion of fragments being specific for either tissue culture or cuttings was relatively low. Some cultivars that did not display somaclonal variation as judged by the AFLP-fingerprints may still be genetically unstable since some morphological variation was observed in the true to type test.     

番茄斑萎病毒TSWV 的鉴定及抗病种质的筛选

 在北京顺义地区番茄栽培温室中随机抽取了15份疑似感染番茄斑萎病毒（TSWV）的植株叶片和果实,利用TSWV试剂盒进行DAS-ELISA检测,结果均为阳性,表明其感染了TSWV。利用来自秘鲁番茄的抗TSWV基因Sw-5的共显性SCAR标记,对442份番茄材料进行分析,筛选出24份含有Sw-5的材料,其中4份为野生番茄,1份为杂交种,16份为高代育种自交系,3份为野生潘那利番茄渐渗系。4份野生番茄综合农艺性状较差,如果实小、产量低、品质和风味差等,不能直接应用于育种;其余20份含Sw-5的材料综合农艺性状较好,可作为番茄抗TSWV的育种材料。     

与大白菜橘红心基因紧密连锁的SCAR标记

利用游离小孢子培养获得的DH系群体和BSA法对与大白菜橘红心基因紧密连锁的分子标记进行了研究, 筛选到与橘红心球色基因or连锁的RAPD标记S1338₆₅₆和AFLP标记P67M54₁₇₂ , 其遗传距离分别为8 cM和13 cM, 并将其成功转化成SCAR标记SCOR₂₀₄和SCOR₁₂₇。对标记的通用性在110个大白菜自交不亲和系育种材料中进行了验证, 与田间鉴定结果的吻合率分别为90%和89.1%。SCAR标记的获得为大白菜橘红心性状的分子标记辅助选择与基因的图位克隆奠定了基础。     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcL gene sequences

Amplified fragment length polymorphism (AFLP) and partial rbcL gene sequencing were used to investigate genetic diversity among various longan (Dimocarpus longan Lour) accessions as well as a presumed closely related species Dimocarpus confinis How et Ho and litchi (Litchi chinensis Sonn). No significantly shared AFLP fragment was found between the three species, indicating that D. confinis and litchi are very far in genetic distance from any longan accession studied. Partial rbcL sequences of 501 bp from the first coding site in these species were obtained, which revealed several substitutes. One such DNA base pair substitute resulted in an amino acid difference between longan and litchi. Furthermore, another 4 bp resulted in a two amino acid difference between longan and D. confinis, which was consistent with AFLP results and indicated that D. confinis should be excluded from the longan genus, Dimocarpus. Within the longan species, no DNA substitute was found. Using nine primer combinations, a total of 66 AFLP markers were obtained from 41 longan accessions. One non-Chinese longan accession ‘Miaoqiao’ was distinctly different from all other longan cultivars collected in China, indicating that more genetic resources of longan might be collected also from longan production regions outside of China. AFLP markers might be developed to identify longan cultivars as well as expedite progeny screening in breeding programs of this perennial fruit tree.     

番茄Ty-1、Ty-2和Ty-3基因的多重PCR技术鉴定

试验探索在同一个反应体系中完成3个SCAR标记的多重PCR扩增,为利用分子标记辅助番茄黄化曲叶病抗性育种提供一种省时、省力、经济有效的方法。对分别与番茄黄化曲叶病毒病抗性基因Ty-1、Ty-2、Ty-3紧密连锁的共显性SCAR标记同时进行扩增筛选,扩增的特异性片段与单引物扩增片段一致。     

黄瓜全雌性基因连锁的AFLP和SCAR分子标记

 本研究以全雌品种‘戴多星’自交系和弱雌品种‘北京截头’自交系为双亲杂交获得F₁ ,然后得到F₂ 性型分离群体, 利用分离群体分组分析法(Bulked Segregant Analysis, BSA) 构建全雌和弱雌两个基因池, 筛选了64对AFLP选择性引物EcoR I-NN +Mse I-NNN组合, 发现EcoR I-TG +Mse I-CAC引物组合在全雌基因池中扩增出一条分子量为234 bp的特异带。经F₂ 代单株验证, 该特异条带能在全雌单株中稳定出现。以MAP MAKER (Version 310) 软件分析, 该标记与全雌性位点的连锁距离在617 cM。命名该连锁标记为TG/CAC₂₃₄。将该特异条带回收、克隆、测序, 设计特异SCAR引物, 再对F₂ 代单株基因组DNA进行扩增, 仅在全雌单株中扩增出1条分子量为166 bp 的特异带, 表明已成功地将与黄瓜全雌性连锁的AFLP标记转化为操作简便、表现稳定的SCAR标记, 该标记命名为SA₁₆₆。     

S-RNase based S-genotyping of Japanese plum (Prunus salicina Lindl.) and its implication on the assortment of cultivar-couples in the orchard

Determination of the genetic compatibility between self-incompatible cultivars is crucial in agriculture. The Rosaceae family carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. Each haplotype is conferred by an S-locus. The S-locus contains two highly polymorphic genes, S-RNase and SFB, which are characteristic of each haplotype and therefore these genes are ideal markers for molecular S-genotyping. In this study 43 Japanese plum cultivars grown in Israel were S-genotyped based on their S-RNase gene sequences. Four alleles, S^b, S^c, S^e and S^h are widespread and together are responsible for 87% of the S-haplotypes therefore many of the cultivar combinations are semi-compatible. In Israel semi-compatibility was shown to correlate with low yield. However, two cultivars, ‘Wickson’ S^fS^k and ‘Shiro’ S^fS^g carry rare S-haplotypes and, therefore, are fully compatible with most of the analyzed cultivars.     

大葱细胞质雄性不育基因的SCAR 标记开发

 以大葱（Allium fistulosum L.）雄性不育系和保持系为试材,开发了一个能够鉴别细胞质雄性不育类型的SCAR 标记。此标记在大葱雄性不育系（S 型细胞质）中扩增出１条607 bp 片段,而在保持系（N 型细胞质）中没有扩增出此片段,将此片段命名为S607。对5 组不同遗传背景的不育系和相应保持系,以及4 份杂交组合进行验证,SCAR 标记S607 鉴定结果与实际细胞质类型完全相符。     

与黄瓜M基因连锁的三个共显性标记

 利用黄瓜近等基因系纯雌株WI1983G（基因型为FFMM）与两性花株WI1983H（基因型为FFMM）为亲本，以WI1983H为回交亲本构建了BC₁代分离群体，采用SSR及SCAR分子标记技术，获得了3个与M基因紧密连锁的标记SSR23487、SCAR123和SSR19914，它们与M基因的遗传距离分别为0.28 cM、0.94 cM和3.20 cM，两侧标记SSR23487和SCAR123将M基因定位在1.22 cM的遗传区间内。这3个分子标记的获得不仅可以用于分子标记辅助选择育种，而且为最终M基因的克隆打下了基础。     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.