Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships

Thirty-two Chinese peach landraces/cultivars, a major subset of the core Chinese peach collection, were fingerprinted using seven pairs of SSR primers to assess their genetic diversity and relatedness. The seven primer pairs detected eight loci and revealed an allele richness of 3.125 (average alleles per locus), an expected heterozygosity (He) of 0.450, and a Shannon index of 0.728 among the landraces/cultivars. This level of genetic diversity is lower compared to other fruit trees and Prunus congenus species (cherry and apricot), but it is comparable to previous reports in peaches. A greater level of genetic diversity was observed in landraces than in cultivars, indicating that peach landraces are valuable for germplasm collection. All cultivars and landraces, except two, were unambiguously identified based on multi-locus genotypes. Eight unique alleles were detected among this group of Chinese peaches. UPGMA clustering analysis separated the 32 cultivars/landraces into two distinct groups, which is generally in accordance with the known pedigree information. The results provide accurate genetic information for defined acquisition policy in the repositories, improving the integrity and efficiency of germplasm management and giving evidences for protection of breeder's intellectual rights.     

基于中国樱桃转录组的SSR分子标记开发与鉴定

对中国樱桃(Prunus pseudocerasus Lindl.)休眠芽转录组数据进行了SSR位点搜索和分析,发现了7 197个SSR位点,总的发生频率为15.62%。SSR重复类型以二核苷酸发生频率最高(58.65%),三核苷酸次之(34.72%)。利用8对多态性引物在24份樱桃种质中进行了引物有效性验证和遗传多样性分析,结果表明有效等位基因数最大值为2.92(Pp SSR2),最小值为1.09(Pp SSR8),平均值为1.73;香农多样性指数变化范围是0.202~1.290,平均值为0.755。观察杂合度和期望杂合度的变化范围分别是0.083~0.917和0.082~0.671;平均值分别为0.391和0.384。香农多样性指数、观察杂合度和期望杂合度3个多样性指数最大值均出现在位点Pp SSR2,最小值出现在位点Pp SSR8。基于SSR标记的24份樱桃种质的聚类结果与经典的形态学分类不完全一致,但聚类结果清晰地划分出5个不同组别,说明浙江省的中国樱桃种质资源遗传多样性丰富。两个龙泉地方品种与其他的浙江樱桃种质资源明显不同,它们与山樱和浙闽樱有着更紧密的遗传关系,该地方品种可以作为樱桃新品种选育的优良材料,有利于拓宽现有樱桃栽培品种的遗传背景。     

Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.)

Here we characterized eight novel polymorphic SSR markers, developed from an enriched genomic library of garlic (Allium sativum L.). These SSRs produced a total of 64 alleles across 90 garlic accessions, with an average of 8 alleles per locus. Values for observed (H_O) and expected (H_E) heterozygosity ranged from 0.16 to 0.77 (mean = 0.44) and from 0.22 to 0.86 (mean = 0.65), respectively. Six loci deviated significantly (P < 0.05) from Hardy–Weinberg equilibrium (HWE). The averages of gene diversity and PIC values were 0.65 and 0.62, respectively. The mean genetic similarity coefficient was 0.4380, indicating that among garlic accessions existed wide genetic variation. Based on 64 alleles obtained by 8 SSRs, a phenogram was constructed to understand the relationships among the 90 accessions. These newly developed SSRs should prove very useful tools for genotypes identification, assessment of genetic diversity and population structure in garlic.     

Genetic structure of mulberry from different ecotypes revealed by ISSRs in China: An implications for conservation of local mulberry varieties

Mulberry is a perennial and economically important plant that has traditionally been used for feeding the silkworm. Evaluating genetic relationship is important for long-term improvement in mulberry yield, quality and resistance, and for germplasm conservation and identification. Population structure and genetic diversity of 8 mulberry populations from different ecotypes in China were analyzed by ISSR markers. Twelve ISSR primers generated a total of 83 amplification products, of which 50 were polymorphic, revealing 60.24% polymorphism among 66 mulberry local varieties, the mean PIC value was 0.1469. The total heterozygosity (H_T), heterozygosity within population (H_S), diversity between populations (D_ST) were 0.1600, 0.0851 and 0.0749, respectively. The coefficient of population differentiation (G_ST) was 0.4683, indicating that the variations among populations and those within populations contributed 46.8% and 53.2% to the total heterozygosity, respectively. The gene flow (N_m) was 0.5678, suggesting that genetic drift between populations can caused local genetic differentiation and therefore, population divergence. The mean genetic similarity coefficient was 0.8456, genetic similarity coefficient among 8 mulberry populations ranged from 0.8441 to 0.9640, indicating that genetic diversity of different populations existed variation. A dendrogram of all 66 local varieties of mulberry based on the genetic similarity using ISSR markers was generated by UPGMA cluster method. In the dendrogram, most varieties from the same ecotype clustered together.     

福建省梨地方品种的遗传多样性研究

利用叶绿体DNA非编码区序列acc D-psa I和17对SSR引物对原产于福建省的49个梨地方品种的遗传多样性进行分析,旨在为育种利用提供参考。获得49份福建地方品种的acc D-psa I序列,并检测到4个多态性位点,包含5个叶绿体单倍型(Hap1~Hap5),其中核苷酸多态性(Pi)为0.00139,单倍型多样性(Hd)为0.660。TCS网络图显示Hap5是最古老的单倍型,其仅在两个样品中检测到。17个SSR位点共检测到211个等位基因位点,有效等位基因数(A)为5~23个,平均值为12.41;观察杂合度(Ho)和期望杂合度(He)的平均值分别为0.5802和0.7549;17个SSR位点的Shannon’s信息指数(I)值为0.5830~2.7683,平均值为1.8661,表现出较高的遗传多样性。基于Nei和Li的遗传距离的邻接法(Neighbor-Joining,NJ)将49份样品聚为9个组,其中大部分聚类结果与单倍型类型表现的亲缘关系较为一致。叶绿体单倍型和SSR多态性位点信息证实福建地方品种具有较为丰富的遗传多样性。     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

RAPD analysis of genetic diversity among and within Portuguese landraces of common white bean (Phaseolus vulgaris L.)

Seventeen landraces of common beans (Phaseolus vulgaris L.) sampled in the North and Centre of Portugal were analyzed at population level for 689 RAPD loci amplified by using forty random primers. The two different parameters used to estimate the genetic variability in and between samples indicate that the inter-population component of the genetic variability is mainly responsible for the diversity found, since only about a 10% would be of an intra-population nature. In addition, the gametic disequilibrium was estimated and reached an average value of 40% for the different combinations of pairs in the 689 loci studied taking the 17 samples as a whole population. Self-pollination, genetic drift and adaptation would thus be favouring the formation of multilocus associations. In addition, the fingerprinting study suggests that each landrace produced unique amplification products allowing it to be distinguished from the other tested genotypes, but, nevertheless, the landraces show a considerable genetic similarity (56% and 70.5% using two different methods). The Neighbour-Joining dendrogram did not show any relation between the geographical distribution of landraces and genetic distance. The results suggest that these Portuguese landraces conserved an important genetic diversity that can be useful to widen the genetic base of currently cultivated beans.     

Molecular diversity of Chinese Cucurbita moschata germplasm collections detected by AFLP markers

Cucurbita moschata is an important vegetable crop. Although a total of 1032 landraces of C. moschata are maintained in China, little is known about their genetic diversity. Molecular characterization is needed to facilitate the use of this C. moschata germplasm collection in breeding. Seventy-four Chinese accessions and 15 accessions from other countries were selected for evaluation based upon variation in fruit traits and geographical origin of molecular diversity with AFLP analysis. Nine pairs of EcoRI/MseI primers produced 500 fragments, of which 75.57% were polymorphic, indicating a high degree of diversity. The accessions from China were classified into two clusters, which were clearly differentiated from the accessions originating from Mexico, Guatemala, Honduras, and Ecuador. Chinese group genetically more closely related to other Asian countries group (India and Japan). In general, the accessions from the Americas had a greater number of unique loci than those from China. The differences are probably due to a limited number of introductions and genetic drift. The Americas are the center of origin of C. moschata and therefore more diverse. With AFLP analysis, the accessions did not clearly group according to fruit shape; however, sub-clusters exist in acorn- and dumbbell-shaped accessions. The assessment of genetic distance, along with some unique traits among the different genotypes, could be useful in further genetic studies and the selection of the most adequate accessions for use in breeding programs.     

Novel genomic and EST-derived SSR markers in Japanese chestnuts

A total of 366 novel simple sequence repeat (SSR) markers were developed in Japanese chestnut (Castanea crenata), comprising 220 genomic SSRs derived from enriched genomic libraries and 146 expressed sequence tag (EST)–SSRs obtained from large-scale EST sequencing analysis. Thirty accessions, comprising Japanese, Chinese (C. mollissima), European (Castanea sativa), and American chestnuts (Castanea dentata), were used for evaluation of SSR polymorphism and transferability across species. The EST–SSRs showed less polymorphism than the genomic SSRs and were more transferable. The mean observed heterozygosity (H_O) and the mean expected heterozygosity (H_E) of genomic SSRs in the Japanese chestnuts were 0.63 and 0.68, respectively; those of EST-SSRs were each 0.47. Although about 80% of the genomic SSRs were amplified in all 4 species, more than 95% of the EST–SSRs were transferable across all 4 species. The many novel SSRs developed in this study will be applicable for the construction of genetic linkage maps, QTL analysis of phenotypic traits, high-throughput genotyping of marker-assisted selection, and association genetics.     

Identification and distribution of S haplotypes in Brassica vegetables from China

The amplification efficiency and identification diversity with gene-specific primers derived from S locus glycoprotein gene (SLG) and S locus receptor kinase gene (SRK) were compared, and the geographical distribution for S haplotypes was investigated by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 72 genotypes of 5 Brassica vegetables from China. The amplification efficiency and identification diversity with class I SRK primers were generally higher than that with class I SLG in most crops tested. Class I alleles were found in total 66 genotypes and they were classified into 16, 10, 7 and 10 groups for Chinese cabbage, purple flowering stalk, cauliflower and cabbage respectively. The number of amplification accessions and identification diversity using the primers of class II SLG and SRK were quite similar. Class II alleles were detected in 55 genotypes and further grouped into one type in mustard and three in other crops. The nucleotide sequences showed high similarity between identical S haplotypes determined by reciprocal pollination and PCR-RFLP tests. It demonstrated that the PCR-RFLP analysis was feasible for identification of S alleles, and SRK should be considered as a better marker for the identification of S haplotypes than SLG. The types of S haplotypes are highly diverse in Brassica vegetables from China. Nevertheless, they were geographically limited in some Brassica vegetables, so the exchange of germplasm resources should be enhanced for breeding.     

S_allele identification and genetic diversity analysis of apricot cultivars

In this study, the sexual incompatibility and S-allele diversity of 24 Turkish apricot cultivars, Paviot and Sak?t-1 as parents and 127 F₁ progenies were identified by polymerase chain reaction (PCR) and sequencing techniques. Additionally, genetic diversity and relatedness among the 24 cultivars were determined using 18 simple sequence repeat (SSR) markers from the genus Prunus. PCR for S_alleles identified nine different S-RNase alleles in the 24 apricot cultivars, namely S_c, S₁, S₂, S₈, S₉, S₂₀, S₂₄, S₅₂, and S₅₃. All primers amplified only one S_allele in the cultivars Adilcevaz-1, Adilcevaz-3, Ethembey, Pasamismisi, Canakkale, and Soganci. Most of the Turkish cultivars were self-incompatible. The S_c allele was present in only three cultivars (Canakkale, Ethembey, Imrahor) that are, therefore, self-compatible. The S_alleles of cultivars Paviot and Sak?t-1 displayed homology with the S_c, S₂ and S₂₀, and S₅₂ alleles. In the 127 F₁ genotypes, the two S_alleles of Paviot were inherited by roughly half of the offspring, while about 76% of the offspring inherited the S₅₂ allele from Sak?t-1, and less than 24% inherited S₂₀. The amplification using all SSR 18 primers was successful and produced 128 polymorphic alleles with an average of 7.11 alleles per locus. Among the apricot cultivars studied, expected heterozygosity (He) ranged from 0.33 to 0.72, observed heterozygosity (Ho) ranged from 0.42 to 1.00, PIC values were between 0.28 and 0.89, and similarity rates were between 0.30 and 0.68. The cultivars Levent and Ozal were genetically closest (0.68) while cultivars Sak?t-3 and Soganc? were the most distinct (0.30).     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

海南椰子栽培品种的SSR标记分析

应用简单重复序列（SSR）标记方法,对海南的11个椰子栽培品种进行了遗传多样性分析。选取30对引物用于PCR扩增,有23对引物扩增出有效多态性片段136条,平均多态性百分率为95.77%,每对引物扩增出的带数2~12条不等,平均为6.17条,每个SSR位点的多态信息量（PIC）变化于0.173~0.896之间,平均为0.561。11份材料之间遗传相似系数变化范围0.061~0.861,说明海南椰子栽培品种之间存在丰富的遗传多样性。UPGMA聚类分析结果表明,11个椰子栽培品种分为四个类群和两个亚群。SSR标记反映出的品种间亲缘关系与形态学研究的分类结果并不完全吻合。     

Genetic diversity of Rehmannia glutinosa cultivars based on sequence-related amplified polymorphism markers

SRAP analytic system was used to assess genetic diversity of Rehmnnia glutinosa. Twenty-three Rehmnnia glutinosa cultivars were screened with 288 primer combinations, of which 13 produced stable and reproducible amplification patterns in three repetitive experiments. Among a total of 338 amplified fragments, 306 (90.5%) were polymorphic, with an average of 23.5 fragments for each primer combination. The percentage of polymorphic bands for each primer combination varied from 58.3 to 100%. The cultivars had a similarity ranging from 0.335 to 0.713 with a mean of 0.518. Shannon's diversity index and expected heterozygosity were 0.3217 and 0.2008, respectively. Based on the cluster, which were conducted on the similarity matrix of SRAP marker data, the cultivars were divided into four groups at the 20 rescaled distance cluster combine. The results demonstrated that SRAP is a stable marker technique for the assessment of genetic diversity of Rehmannia glutinosa cultivars, and that the level of genetic diversity among them from different production areas was relatively high.     

Osmotic adjustment,proline accumulation and cell membrane stability in leaves of Cocos nucifera submitted to drought stress

The role of drought-induced proline accumulation in coconut leaves is still unclear. With the objective of evaluating the impact of water shortage on leaf osmotic potential, proline accumulation and cell membrane stability in young plants of two Brazilian Green Dwarf coconut ecotypes from contrasting areas (Brazilian Green Dwarf from Una, Bahia, UGD, and from Jiqui, Rio Grande do Norte, JGD), a pot experiment was conducted under greenhouse conditions. Three drought cycles consisting of suspension of irrigation until the net photosynthetic rate (A) approached zero and rewatering until recovery of A to 85% of the irrigated control plants. Pre-dawn leaf water potential (Ψ_PD) reached −1.2 MPa at the point of maximum stress (PMS). Dry matter production and leaf area were severely reduced by drought treatment in the two ecotypes. Corrected values of osmotic potential were significantly reduced in stressed plants of the two ecotypes. Green dwarf coconut palm showed low osmotic adjustment (from 0.05 to 0.24 MPa) and significant accumulation of proline (from 1.5 to 2.1 times in relation to control) in leaflets in response to water deficit. Considering the growth reduction observed in both ecotypes, proline was not associated to osmoregulation. On the other hand, the absence of membrane damage, as indicated by electrolyte leakage method, suggests that the protective role of proline in this specie can be more important. The two ecotypes of Green dwarf coconut palm behaved similarly in the present experiment for most traits evaluated. Slight differences among the ecotypes were observed with respect to the response to treatments, such as higher proline accumulation in JGD.     

Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.)

A growing interest in the use of the Strawberry tree (Arbutus unedo L., Ericaceae) has been recently reported for industrial, pharmaceutical and chemical fields. However, the bulk material comes from natural populations because of the lack of selection of interesting cultivars. In Tunisia, A. unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, no conservation or improvement programs are attempted to preserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of nine Tunisian populations of A. unedo L. from different bioclimates, using 65 polymorphic RAPD loci. The analysis of the genetic variation within and among populations is primordial to elaborate conservation and improvement programs. A low genetic diversity within a population estimated by both Nei's (He) and Shannon's diversity (H′) indices (0.155 < He < 0.248; 0.229 < H′ < 0.364) was observed due to genetic drift and selfing. At the species level, the amount of the within population variation estimated by Shannon's index (H_POP/H_SP = 0.686) and the molecular variance (80.67%) was higher than that among populations. A moderate genetic differentiation among populations (Φ_ST = 0.193 and G_ST = 0.314) which could be attributed to the long seed distance dispersal was detected. The UPGMA dendrogram based on Φ_ST values showed three clusters each including populations without relationship to bioclimatic or geographical origin indicating that differentiation occurs at a local space scale. The in situ protection measures should be made appropriately according to a population within bioclimates. The ex situ conservation and the selection of genotypes should involve extensive collection of seeds or cuttings from the within populations rather than among them.     

Isozyme polymorphism in Curcuma alismatifolia Gagnep. (Zingiberaceae) populations from Thailand

Over-collection of Curcuma alismatifolia Gagnep. and habitat destruction had greatly reduced the number of individuals in their natural habitat resulting in genetic erosion. We conducted the isozyme analyses to reveal the genetic diversity among natural populations compared with the cultivated populations. Out of seven enzyme systems analyzed in this study, five enzymes (ADH, GDH-1, LAP-1, GPI-2, PGM) which gave reproducible and consistent bands were used; DIA and EST were excluded due to their complex and inconsistent bands. The GPI-2 loci showed the most variable with six allozymes and 10 zymogram patterns followed by GDH-1 with 5 and 5, ADH with 3 and 5, PGM with 4 and 4, and LAP-1 with three allozymes and four zymogram patterns. Cultivated populations from Japan (cJ) and Thailand (cT) had the lowest percentage of polymorphic loci (P=40–60%), alleles per locus (A_l=1.8), alleles per polymorphic locus (A_p=2.33–3.00), and gene diversity (H_s=0.216–0.304) compared with two lowland populations (L1, L2) with P=100%, A_l=3.0–3.2, A_p=3.0–3.2, H_s=0.465–0.496; and six highland populations (H1–H6) with P=80–100%, A_l=2.4–3.8, A_p=2.4–3.8, H_s=0.342–0.659. Within population, H1 (the highest elevation sampled in this study) had the greatest genetic diversity (H_s=0.659). Mean genetic diversity over all loci across all populations was 0.444. Mean genetic identity between cultivated populations (I_C), lowland populations (I_L), among highland populations (I_H), and across all populations (I_SP) were 0.950, 0.947, 0.944, and 0.922, respectively. Using UPGMA cluster analysis, H1 and cJ were separated first from the rest into distinct groups. Two lowland populations were placed together with H6, while cT was grouped in the same cluster of H2–H5.