Chloroplast Simple Sequence Repeats Research Articles

Genetic diversity in Chinese sorghum landraces revealed by chloroplast simple sequence repeats

A collection of sorghum, including more than 12,000 Chinese landraces, has been constructed and maintained in China. However, the genetic diversity of Chinese sorghum landraces has not been fully investigated, and the origin of Chinese sorghum is still in dispute. In this study, the complete chloroplast genome sequence of sorghum line Tx623B was searched for simple sequence repeats (SSRs). 31 SSR loci with at least 10 mononucleotide repeats or five dinucleotide repeats were identified, and primer pairs for 27 loci were designed. Chloroplast DNA variation in cultivated sorghum was investigated by using these primer pairs on 185 Chinese sorghum landraces and 70 cultivated sorghum accessions from other countries. Among the 27 loci, 14 were polymorphic. The number of alleles per polymorphic locus ranged from 2 to 5 with an average of 2.79. Allelic data at 14 polymorphic loci were combined to give 12 haplotypes. The average allelic diversity index across the 14 polymorphic loci and corresponding haplotype diversity were markedly lower for Chinese sorghum landraces than were those for accessions from other countries. However, Chinese sorghum landraces shared a predominant allele at each polymorphic locus and a predominant haplotype with foreign accessions. Our results indicate that Chinese landraces experienced a severe maternal bottleneck during the introduction process with a predominant haplotype being present in 171 of 185 accessions analyzed. Except for one rare exception, haplotypes found in Chinese landraces were either identical or closely related to those found in foreign accessions and could not be separated clearly from them by cluster analysis. Our results are consistent with the hypothesis of African origin of Chinese sorghum.

Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species

Chloroplast microsatellites, or simple sequence repeats (cpSSRs), are typically mononucleotide tandem repeats. When located in the noncoding regions of the chloroplast genome (cpDNA), they commonly show intraspecific variation in repeat number. Despite the growing number of studies applying cpSSRs, studies of economically important plants and their relatives remain over-represented. Thus, the potential of cpSSRs to offer unique insights into ecological and evolutionary processes in wild plant species has yet to be fully realized. This review provides an overview of the technical resources available to aid cpSSR discovery including a list of cpSSR primer sets available and cpDNA sequencing resources. Our updated analysis of 99 whole chloroplast genomes downloaded from GenBank confirms that potentially variable cpSSRs are abundant in the noncoding cpDNA of plants. Overall variation in the frequency of cpSSRs was extreme, ranging from one to 700 per genome (median=93), while in 81 vascular plants, between 35 and 160 cpSSRs were detected per genome (median=86). We offer five recommendations to aid wider development and application of cpSSRs: (i) When genus-specific cpSSR primers are available, cross-species amplification can often be fruitful. (ii) While potentially useful, universal cpSSR primers at best provide access to only a small number of variable markers. (iii) De novo sequencing of noncoding cpDNA is the most effective and efficient way to develop cpSSR markers in wild species. (iv) DNA sequencing of cpSSR alleles is essential, given the complex nature of the genetic variation associated with hypervariable cpDNA regions. (v) The reliability of cpSSR length based genetic assays need to be validated in all studies.

A new set of universal de novo sequencing primers for extensive coverage of noncoding chloroplast DNA: new opportunities for phylogenetic studies and cpSSR discovery

We present a new set of universal de novo sequencing primers targeting noncoding chloroplast DNA. The set of 107 polymerase chain reaction (PCR) primers span approximately 86% of the noncoding nucleotides in the large single copy region of Nicotiana tabacum, Oryza sativa and the orchid Phalaenopsis aphrodite. PCR tests confirmed the primers are effective in a wide range of monocots and dicots. More than 19.5kb of cpDNA sequence was obtained across representative orchid genera with up to 82 chloroplast simple sequence repeats (cpSSRs) detected per genus. This primer set will facilitate both phylogenetic studies and rapid discovery of cpSSRs for plants, such as orchids, where there are limited genomic resources.

Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats

We analyzed 25 chloroplast simple sequence repeat (cpSSR) loci in 82 accessions, 59 of cultivated radish and 23 of three wild Raphanus species and identified 7 polymorphic loci and 20 haplotypes. The distribution of haplotypes in different species and different geographical areas was assessed. Minimum-spanning network (MSN) was used to identify phylogenetic relationships in cultivated and wild radish. The MSN provides evidence for at least three independent domestication events, including black Spanish radish and two distinct groups of cpSSR haplotypes. One of these two haplotype groups is restricted geographically to Asia. This led Asian cultivated radish haplotypes to higher cpSSR diversity than Mediterranean cultivated radish or wild radish. These data are consistent with the diversity and distribution of agronomic traits in cultivated radish. At the same time, this implies that Asian cultivated radish is not originated from the diffused descendants of European cultivated radish, probably originated from a wild species that is distinct from the wild ancestor of European cultivated radish. Unfortunately we do not know the wild ancestor of Asian cultivated radish.

Relationships among Avena species as revealed by consensus chloroplast simple sequence repeat (ccSSR) markers

Consensus chloroplast simple sequence repeat (ccSSR) makers were used to assess the genetic variation and genetic relationships of 80 accessions from 25 taxa of the genus Avena. Fifteen out of 16 ccSSR markers (93.75%) were polymorphic. A total of 51 alleles were detected at the 16 ccSSR loci. The number of alleles per locus ranged from 1 to 6, with an average of 3.2 alleles. Among these ccSSR loci, the highest polymorphism information content (PIC) value was 0.754, while the lowest PIC value was 0. The mean genetic similarity index among the 80 Avena accessions was 0.545, ranging from 0.188 to 1.000. To assess the usefulness of ccSSRs in separating and distinguishing between haplome (genome) groups, we used ordination by canonical discriminant analysis and classificatory discriminant analysis. Although discriminant analysis separated the haplome groups unequivocally, it was up to 69% predictive of correctly classifying an individual plant whose haplome(s) is unknown in the case where it belonged to the A haplome group, 75% where it belonged in the AC group, and almost 80% where it belonged in the ACD group. The analysis of genetic similarity showed that diploid species with the A haplome were more diverse than other species, and that the species with the As haplome were more divergent than other diploid species with the A haplome. Among the species with the C haplome, A. clauda was more diverse than A. eriantha and A. ventricosa. In the cluster analysis, we found that the Avena accessions with the same genomes and/or belonging to the same species had the tendency to cluster together. As for the maternal donors of polyploid species based on this maternally inherited marker, A. strigosa served as the maternal donor of some Avena polyploidy species such as A. sativa, A. sterilis and A. occidentalis from Morocco. A. fatua is genetically distinct from other hexaploid Avena species, and A. damascena might be the A genome donor of A. fatua. Avena lusitanica served as the maternal parents during the polyploid formation of the AACC tetraploids and some AACCDD hexaploids. These results suggested that different diploid species were the putative A haplome donors of the tetraploid and hexaploid species. The C genome species A. eriantha and A. ventricosa are largely differentiated from the Avena species containing the A, or B, or D haplomes, whereas A. clauda from different accessions were found to be scattered within different groups.

Use of ccSSR markers for the determination of the purity of alloplasmic wheat in different Hordeum cytoplasms

AbstractWe tested three different consensus chloroplast simple sequence repeat (ccSSR) primers to identify amplified polymorphic products in Hordeum chilense, Hordeum vulgare and other Triticeae species with a double aim. First, to use chloroplast‐specific primers as an indirect method for the assessment of wheat cytoplasmic male sterile purity in seedlings during multiplication and in the development of alloplasmic lines. Second, to analyse cytoplasmic relationships among H. chilense accessions and between H. chilense and other members of the Triticeae tribe. The products from primer ccSSR‐4 were easily discriminated using agarose gel electrophoresis. Based on the lengths of amplification products, three groups were identified: the first included almost exclusively H. chilense accessions, the second contained H. vulgare accessions and the third comprised the wheat and the rest of the analysed accessions. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the expansion/contraction of the microsatellite repeat length. Data analyses of sequenced fragments revealed six groups of accessions among the material studied. No significant differences were found among H. chilense accessions.

Genetic and morphological evidence for localised interspecific gene flow in Phormium (Hemerocallidaceae)

Gene flow between two species of Phormium, P. tenax and P. cookianum, was investigated by analysis of morphology, AFLP profiles, and chloroplast simple sequence repeats (cpSSRs). We examined 44 wild samples of Phormium, focusing on Okiwi Bay (Marlborough, New Zealand), where the two species grow in close proximity. Two genetic groups corresponding with P. tenax and P. cookianum are evident in analyses of AFLP profiles from Okiwi Bay. The profiles of Okiwi Bay plants were also compared with profiles from three hypothetically pure P. cookianum populations from Marlborough, and a representative sample of P. tenax combined from sites throughout New Zealand. Bayesian analysis of genetic admixture suggests that all of the plants resembling P. tenax and most of the plants resembling P. cookianum sampled from Okiwi Bay are admixed (i.e., of hybrid origin). Neighbour Net and principal component analyses reveal that P. cookianum and P. tenax from Okiwi Bay are genetically intermediate between “pure” populations of the species. Further, all of the Okiwi Bay samples share the same chloroplast haplotype. Analysis of morphological characters comparing a small number of Okiwi Bay P. tenax plants to selected P tenax plants from throughout New Zealand under common garden conditions confirm that the Okiwi Bay population includes plants with unusually small stature and fruit characters approaching P cookianum. These results support a hypothesis of local introgression between P. tenax and P. cookianum.

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy Citrus cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.

Characterization of a New Natural Periclinal Navel–Satsuma Chimera of Citrus: ‘Zaohong’ Navel Orange

‘Zaohong’ navel orange [Citrus sinensis (L.) Osbeck + C. unshiu Marc.], a new strain of citrus from a graft chimera, was discovered in China. It was diploid and arose at the junction where a ‘Robertson’ navel orange scion was top-worked onto a Satsuma mandarin (C. unshiu). Some characteristics determined by the L1 cell layer, such as juice sacs of fruit and stoma length, were similar to those of Satsuma mandarin, while others, including leaf index, fruit shape, navel, and color and aroma of the rind, were determined by the L2 cell layer, were similar to ‘Robertson’ navel orange. High-performance liquid chromatography analysis of the carotenoid extracts of the flesh of ‘Zaohong’ navel orange indicated that it had the carotenoids profile of Satsuma mandarin with β-cryptoxanthin as the predominant component in the juice sacs in mature fruit. Simple sequence repeats (SSR) and chloroplast simple sequence repeats (cpSSR) analysis showed that both nuclear and chloroplast genomes of ‘Zaohong’ navel orange were composed of both donor plants. On the basis of these facts, ‘Zaohong’ navel orange was found to be a periclinal chimera consisting of L1 derived from Satsuma mandarin and L2/L3 from ‘Robertson’ navel orange. It combined the valuable traits of both donor plants, matured ≈1 month earlier than the present navel orange cultivars, and therefore had good potential in citrus fresh market.

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

AbstractSomatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.

Inter-generic mitochondrial genome recombination in somatic hybrids between Citrus and Poncirus

SummaryThe genetic composition of somatic hybrids between ‘Page’ tangelo (Citrus reticulata × C. paradisi) and trifoliate orange (Poncirus trifoliata) were analysed in fifteen stochastically selected, fusion-derived plants. Flow cytometry revealed that all plants were tetraploids, and nuclear simple sequence repeats (SSR) showed that all plants possessed SSR loci from both fusion parents. Organellar genome compositions were examined using cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphism (RFLP) analyses. Mitochondrial genome (mtDNA) analyses indicated that all 15 hybrids had inherited regions of mtDNA common to both parents, and some specific bands from both fusion parents were also present in the hybrids, indicating the occurrence of mtDNA recombination in these somatic hybrids. No chloroplast genome (cpDNA) polymorphisms were detected by RFLP or CAPS analyses; however, random segregation of cpDNA was revealed by chloroplast simple sequence repeat (cpSSR) analyses. To evaluate genetic variation in mtDNA among the hybrids and both fusion parents, a dendrogram based on the RFLP data for 15 polymorphic mtDNA markers was generated by the Unweighted Pair Group Method with Arithmetic mean. The results showed that the 15 hybrids, and both parents, were located in three distinct clusters, and no variation was detected among these hybrids. The hybrids showed closer genetic similarity coefficients to the suspension cell parent than to the mesophyll cell parent, which demonstrated biased transmission of mtDNA. This is the first report of mtDNA recombination between Citrus and Poncirus by somatic fusion.

Simple sequence repeats in organellar genomes of rice: frequency and distribution in genic and intergenic regions

Simple sequence repeats (SSRs) are abundant across genomes. However, the significance of SSRs in organellar genomes of rice has not been completely understood. The availability of organellar genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. We have analyzed SSRs in mitochondrial and chloroplast genomes of rice. We identified 2528 SSRs in the mitochondrial genome and average 870 SSRs in the chloroplast genomes. About 8.7% of the mitochondrial and 27.5% of the chloroplast SSRs were observed in the genic region. Dinucleotides were the most abundant repeats in genic and intergenic regions of the mitochondrial genome while mononucleotides were predominant in the chloroplast genomes. The rps and nad gene clusters of mitochondria had the maximum repeats, while the rpo and ndh gene clusters of chloroplast had the maximum repeats. We identified SSRs in both organellar genomes and validated in different cultivars and species.

Genetic relationships among poplar species in section Tacamahaca ( Populus L.) from western Sichuan, China

Although western Sichuan is regarded as a natural distribution and variation center for the Tacahamaca section of the Populus species in China, little is currently known about the majority of poplar species occurring in this region. In the present study, molecular data were utilized to determine the genetic relationships among Populus species in section Tacamahaca in western Sichuan. The chloroplast simple sequence repeat (cpSSR)- and nuclear SSR-based UPGMA analyses were performed for 7 Populus species, with 50 specimens representing 10 accessions. The results provided molecular evidence suggesting that P. schneideri is genetically highly similar to P. kangdingensis, as also indicated by previous studies based on their morphological characters. Similarly, the combination of cpSSR and nuclear SSR analyses indicated that P. trinervis and P. simonii have a close genetic affinity, which is supported by morphological traits and similarities in the range of natural habitats. Although, based on nuclear SSR analysis, P. yunnanensis is distinct from the other species, it shared the same cpSSR profiles with P. simonii and P. trinervis. This result reflects similar maternal phylogeny among the three species. In addition, the cluster analyses clearly showed a close relationship among accessions of the same species and suggested monophyly in P. przewalskii and P. cathayana. Our results demonstrated that the combination of cpSSR and nuclear SSR molecular markers enables a clear estimation of genetic relationships among Populus species and they provided information for preliminary conclusions that will be helpful in guiding future research.

Cross-species microsatellite amplification inVasconcelleaand related genera and their use in germplasm classification

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.

Chloroplast DNA Variation Confirms a Single Origin of Domesticated Sunflower (Helianthus annuus L.)

Although sunflower was long thought to be the product of a single domestication in what is now the east-central United States, recent archaeological and genetic evidence have suggested the possibility of an independent origin of domestication, perhaps in Mexico. We therefore used hypervariable chloroplast simple-sequence repeat markers to search for evidence of a possible Mexican origin of domestication. This work resulted in the identification of 45 chloroplast haplotypes from 26 populations across the range of wild sunflower as well as 3 haplotypes from 15 domesticated lines, representing both primitive and improved cultivars. The 3 domesticated haplotypes were characterized by 1 primary haplotype (found at a frequency of 6.7% in the wild) as well as 2 rare haplotypes, which are most likely the products of mutation or introgression. One of these rare haplotypes was not observed in the wild, bringing the total number of haplotypes identified to 46. A principal coordinate analysis revealed the presence of 3 major haplotype clusters, one of which contained the primary domesticated haplotype, the 2 rare domesticated variants, as well as haplotypes found across much of the range of wild sunflower. The Mexican haplotypes, on the other hand, fell well outside of this cluster. Although our data do not provide insight into the specific location of sunflower domestication, the relative rarity of the primary domesticated haplotype in the wild, combined with the dissimilarity between this haplotype and those found in the Mexican populations surveyed, provides further evidence that the extant domesticated sunflowers are the product of a single domestication event somewhere outside of Mexico.

High genetic diversity in a rare and endangered sunflower as compared to a common congener

Determining the genetic structure of isolated or fragmented species is of critical importance when planning a suitable conservation strategy. In this study, we use nuclear and chloroplast SSRs (simple sequence repeats) to investigate the population genetics of an extremely rare sunflower, Helianthus verticillatus Small, which is known from only three locations in North America. We investigated levels of genetic diversity and population structure compared to a more common congener, Helianthus angustifolius L., using both nuclear and chloroplast SSRs. We also investigated its proposed hybrid origin from Helianthus grosseserratus Martens and H. angustifolius. Twenty-two nuclear SSRs originating from the cultivated sunflower (Helianthus annuus L.) expressed sequence tag (EST) database, and known to be transferable to H. verticillatus and its putative parental taxa, were used in this study thereby allowing for statistical control of locus-specific effects in population genetic analyses. Despite its rarity, H. verticillatus possessed significantly higher levels of genetic diversity than H. angustifolius at nuclear loci and equivalent levels of chloroplast diversity. Significant levels of population subdivision were observed in H. verticillatus but of a magnitude comparable to that of H. angustifolius. Inspection of multilocus genotypes also revealed that clonal spread is highly localized. Finally, we conclude that H. verticillatus is not of hybrid origin as it does not exhibit a mixture of parental alleles at nuclear loci, and it does not share a chloroplast DNA haplotype with either of its putative parents.

Molecular analysis revealed autotetraploid, diploid and tetraploid cybrid plants regenerated from an interspecific somatic fusion in Citrus

More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo ( Citrus reticulata Blanco × C. paradisi Macf) and mesophyll protoplasts of rough lemon ( C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed.

Genetic diversity and population structure of Quercus rubra (Fagaceae) in old-growth and secondary forests in southern New England

The genetic diversity and structure of Quercus rubra L. (Red Oak) in five secondary populations regenerated on old pastures and five paired old-growth populations were compared to determine whether secondary populations may have lost genetic diversity present in the presettlement forest. Old-growth samples had significantly greater nuclear allelic richness than secondary stands at the four microsatellite, simple sequence repeat (SSR), markers tested, but there was no significant difference at three chloroplast SSRs. There was however tenfold greater differentiation (high FST) among secondary forests than among old-growth forests for the chloroplast loci, suggesting greater population isolation at the time of their founding. The young age class in secondary stands had significantly greater chloroplast allelic richness than the old age class, suggesting some recovery as the forest cover increased. Forest cover and patch size at peak deforestation was not correlated with allelic richness in secondary stands, but the power of this test was low. The current size of old-growth stands is, however, positively correlated with levels of genetic diversity. Results suggest that deforestation for agriculture may reduce genetic diversity of forest trees, particularly in the first generation after deforestation, but that some recovery of this diversity is likely in these patchy landscapes. Wind dispersed pollen flow may increase in these areas, but animal dispersed seed flow may be reduced.

Molecular phylogeny ofCucumisspecies as revealed by consensus chloroplast SSR marker length and sequence variation

To investigate phylogenetic relationships in the genus Cucumis, 9 consensus chloroplast simple sequence repeat (ccSSR) primer pairs (ccSSR3, 9, 11, 13, 14, 17, 20, 21, and 23) were employed for DNA fragment length variation and 5 amplified fragments, ccSSR4, 12, 13, 19, and 20, were sequenced using total DNA from 13 accessions representing 7 African Cucumis species (x = 12), 3 Cucumis melo L. (x = 12) accessions, 2 Cucumis sativus L. (x = 7) accessions, and 1 Cucumis hystrix Chakr. (x = 12) accession. A Citrullus lanatus (Thunb.) Matsum. & Nakai (x = 11) accession was used as an outgroup. While fragment length analysis revealed the existence of 3 major species clusters (i.e., a group of African Cucumis species, a group composed of C. melo accessions, and a group containing C. sativus and C. hystrix species), sequence variation analysis identified 2 major species clusters (i.e., a group of African Cucumis species and a group composed of C. melo, C. sativus, and C. hystrix species). Comparative analysis using nuclear DNA (previous studies) and cpDNA sequence substitution data resulted in the placement of C. melo and C. sativus in different cluster groupings. Thus, both nuclear and cytoplasmic DNA should be employed and compared when a putative progenitor or specimens of an ancestral Cucumis species lineage is investigated. In addition, C. ficifolius (2x) and C. aculeatus (4x) of the African Cucumis species clustered together in this study. This result does not agree with reported isozyme analyses, but does agree with previously characterized chromosome homologies between these 2 species. Although African Cucumis species and C. hystrix do not share a close relationship, genetic affinities between C. sativus and C. hystrix are considerable. Combined evidence from previously published studies and data presented herein lend support to the hypothesis that C. hystrix is either a progenitor species of C. sativus or that they at least share a common ancestral lineage.

Genetic diversity in mandarin landraces and wild mandarins from China based on nuclear and chloroplast simple sequence repeat markers

SummaryLandraces and wild germplasm are important citrus genetic resources. Knowledge of the genetic diversity of this germplasm provides an opportunity both to mandarin breeding programmes and to germplasm conservation strategies. Genetic diversity of 33 loose-skin mandarin landraces and 19 wild mandarin accessions were analysed with ten nuclear simple sequence repeat (nSSR) and eight chloroplast simple sequence repeat (cpSSR) markers. Our analysis of nuclear and chloroplast genome polymorphism showed that the genetic diversity in wild populations was slightly higher than that found in landraces, with polymorphic information content values of 0.6138 and 0.5091 at nSSR loci, and 0.4921 and 0.3769 at cpSSR loci, respectively. Overall, 60 nSSR alleles and 19 cpSSR haplotypes were identified, and all but seven accessions were unambiguously discriminated by nSSR markers. Five groups of landraces and two groups of wild mandarins were identified based on the nSSR data, and two groups each of landraces and wild mandarins were identified at the cpSSR loci. Different distribution patterns of landraces and wild mandarins were observed between dendrograms constructed using these two marker systems, and hybrid origins for several landraces were suggested by these results. Combined analysis also suggested that, among wild mandarin, ‘Chongyi’ showed the closest relationship to landraces, and that landrace ‘Chazhigan’ was the primary genotype among landraces.