Carotenoid diversity in cultivated citrus is highly influenced by genetic factors

Citrus fruits are complex sources of carotenoids with more than 100 kinds of pigments reported in this genus. To understand the origin of the diversity of carotenoid compositions of citrus fruit, 25 genotypes that belong to the 8 cultivated Citrus species were analyzed. Juice extracts of mature fruit were analyzed by high-performance liquid chromatography using a C30 column. The 25 citrus genotypes presented different carotenoid profiles with 25 distinct compounds isolated. Statistical analyses revealed a strong impact of genotype on carotenoid compositions. Two kinds of classifications of genotypes were performed: on qualitative data and on quantitative data, respectively. The results showed that variability in carotenoid compositions was more interspecific than intraspecific. Two carotenoids, cis-violaxanthin and the beta-cryptoxanthin, strongly determined the classification on qualitative data, which was also in agreement with previous citrus variety classifications. These findings provide evidence that, as for other phenotypical traits, the general evolution of cultivated Citrus is the main factor of the organization of carotenoid diversity among citrus varieties. To the authors' knowledge this is the first study that links the diversity of carotenoid composition to the citrus genetic diversity. These results lead to the proposed major biosynthetic steps involved in the differential carotenoid accumulation. Possible regulation mechanisms are also discussed.     

利用SSR标记对中国柚类资源及近缘种遗传多样性研究

利用SSR标记研究了122份我国柚(CitrusgrandisOsbesk)类资源及近缘种遗传多样性。31对SSR引物从供试材料中检测出335个等位基因变异,平均每个位点可检测到9.85个等位基因。位点多态信息量(PIC)变幅为0.1939!0.9073,平均为0.7085。用UPGMA方法将122份材料分成7个组群,110个柚类品种在相似系数0.712,可细分成18个亚组,主要由沙田柚品种群、文旦柚品种群及庞大的杂种柚品种群组成。     

加强Psy基因表达和通过RNAi抑制Lcy基因表达的载体构建

通过基因工程手段加强八氢番茄红素合成酶（Psy）基因的表达和通过RNAi抑制番茄红素β-环化酶（Lcy）基因（该基因是调控番茄红素转化为其他类胡萝卜素的主要的基因）的表达是培育高番茄红素品种的重要手段之一。根据GenBank中拟南芥的Psy基因序列（NM-121729），通过聚合酶链式反应（PCR）从拟南芥的cDNA中扩增出了长1296的Psy基因。根据GenBank中番茄的Lcy基因序列（X86452）和Pds启动子序列（U46919）分别设计特异引物从番茄中扩增出了Lcy基因的高度保守的长302bp的DNA片段和长1790的Pds启动子片段。根据RNAi的原理，将Lcy基因的长302bp的DNA片段以正反两个方向通过一段内含子序列连接在一起形成RNAi片段插入到抗Kan的pVCT2020的表达载体中，并通过具有果实特异性特点的启动子——八氢番茄红素去饱和酶基因（Pds）启动子控制该干扰片段的表达，同时将Psy基因在Pds启动子调控下插入到同一载体中，从而构建成了能同时加强Psy基因的表达和抑制Lcy基因表达的植物表达载体。     

Genetic diversity of wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. et Zucc.) and Japanese cultivated soybeans [<Emphasis Type="Italic">G. max</Emphasis> (L.) Merr.] based on microsatellite (SSR) analysis and the selection of a core collection

Wild soybeans, Glycine soja, are a source of genetic variation to improve soybeans. To improve the efficiency evaluation of conserved germplasm a core or mini-core collection approach that maximizes allelic diversity in a proportion of the whole collection has frequently been advocated. The genetic diversity of a wild soybean collection (1,305 accessions) plus Japanese cultivated soybeans (53 accessions) were analyzed at 20 SSR marker loci. Higher levels of allelic diversity were found in wild soybeans (28 alleles per locus) than Japanese cultivated soybean (five alleles per locus). The genetic distance between wild soybeans from different regions reflected their proximity. Accessions from Russia consisted of a diverse array of alleles resulting in accessions being spread further apart in a PCA plot than accessions from other regions. Accessions of wild soybean from Korea included many rare alleles and thus had a high representation in the core collection. The two core collections developed here, traditional and mini, consisted of 192 accessions with 97% of the allelic diversity (14% of the whole collection) and 53 accessions with 62.4% of the allelic diversity (5% of the whole collection), respectively.     

山西芝麻种质资源SSR遗传多样性及群体结构分析

为探明山西芝麻种质资源的遗传特性,本研究利用30对简单重复序列标记(SSR)对71份山西芝麻种质资源进行遗传多样性分析及群体结构分析.结果 表明,30对SSR标记共检测到144个等位基因位点,平均每个SSR标记4.800个等位基因;有效等位基因数在1.058 ～5.149之间,平均2.805个;Shannon指数变幅为...     

Assessment of genetic diversity in Egyptian barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) genotypes using SSR and SNP markers

A barley core collection can be studied extensively and the derived information can be used to identify loci/genes for the genetic improvement of quantitative and qualitative traits. To assess genetic diversity, allelic variation and population structure of Egyptian barley, 134 barley genotypes collected from a different region along with 19 cultivated genotypes obtained from of the Egyptian Agricultural Research Center. All genotypes were analyzed with 261 polymorphic SSR and SNP alleles. The mean number of alleles per locus was 4 and PIC was 0.49, while the level of genetic diversity was 0.55 ranging from 0.03 to 0.82. The genotypes were assigned to three subpopulations that were consistent with their origins. The genetic variation within population was higher (51%) than among population at the molecular levels (F_ST =?0.491 when P?<?0.10). The level of polymorphic variation was highest in subpopulations-II, due to collected from different regions with different ear-types thus, expected to contain more diversity than local genotypes in subpopulations-I and subpopulations-III. The structured study found that the 153 barley genotypes are in harmony with clustering approaches using the SSR and SNP genotypic data in a neighbor-joining tree and principal components analysis, which identified three subpopulations. These results demonstrated genetic diversity among the Egyptian barley genotypes can be applied to suggest approaches, such as association analysis, classical mapping population development, and parental line selection in breeding programs. Therefore, it is necessary to use the exotic genotypes as the genetic resources for developing new barley cultivars in Egypt.     

Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion

Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.     

我国部分冬小麦新品种（系）SSR标记遗传差异的研究

本研究利用53对SSR引物对全田1999－2000年北方冬麦区及黄淮冬麦区观察圃中选出的48个新品种（系）进行遗传差异研究，共检测出58个SSR位点上的367个等位变异，平均每个位点有6.33个等位变异，其中B组每个位点的等位变异最多，这表明B基因组化更快，分化更大。48个品种（系）在全基因组及A、B、D基因组聚类结果表明这些品种的相似系数聚类的范围较小，为0.75－0.98。全基因组聚类结果与品种的系谱来源及育成地区相吻合。研究结果表明我国冬小麦品种的种质基础相对较狭窄。加强不同来源种质的利用和特异亲本类型的培育对我国冬小麦遗传改良非常重要，利用5个多态性高的SSR标记就可以将这48个小麦新品种（系）区分开，每个品种（系）都有各自独特的指纹图谱。     

Genetic diversity,population structure and differentiation of rice species from Niger and their potential for rice genetic resources conservation and enhancement

Rice genetic resources conservation and evaluation is crucial to ensure germplasm sources for further crop breeding. We conducted a wide collection of Oryza species in Niger and characterize its diversity with microsatellites (or simple sequence repeats, SSR). The aims of this research were to get a better understanding of the extent of genetic diversity, its structure and partition within rice eco-geographical zones of Niger. There were 264 accessions found in farmers’ and other fields: 173 O. sativa (Asia’s rice), 65 O. glaberrima (Africa’s rice), 25 O. barthii, and 1 O. longistaminata (weedy perennial rice), which were genotyped with 18 SSR. A total of 178 alleles were detected, with a mean of 9.89 alleles per locus. The polymorphism information content was 0.65 and heterozygosity was estimated as 0.14. Two main well-differentiate genotypic groups, which correspond to Asian and African rice species, were identified. The SSR set divided the Asia’s rice group (solely indica) into irrigated and floating rice, with rainfed lowland rice in between. The African rice species group was composed of O. glaberrima, O. longistaminata and O. barthii accessions, but without any clear genetic differentiation among them likely due admixtures within the samples of O. barthii. Five accessions that could be natural interspecific hybrids were too admixed for assigning them to any of the two well-differentiated groups. The partitioning of the overall diversity showed that maximum variation was within genotypic groups and subgroups or cropping ecologies, rather than between eco-geographical zones. The eco-geographical distribution of the diversity suggests germplasm exchange in Niger. Next-steps for conserving rice and crop wild relatives in Niger could be taken using the findings of this research.     

云南杂交玉米SSR标记核心引物的筛选及多样性分析

为降低合成引物成本,加快杂交玉米品种鉴定进程,筛选适用于云南玉米杂交种SSR标记鉴定的核心引物。本研究用玉米SSR标记鉴定规程中推荐的40对SSR引物对12个杂交玉米品种DNA进行扩增,筛选出多态性高、扩增效果好、稳定性好的20对引物,推荐其作为云南玉米杂交种特异性鉴定的核心引物。并用筛选的核心引物对云南220个杂交玉米材料进行多样性分析及评价。筛选的20对SSR引物在12个杂交玉米品种中共检测到216个等位变异,平均每对引物检测到10.80个。20对SSR核心引物在220个杂交玉米材料中共检测到236个等位变异,平均每对引物检测到11.80个;平均有效等位变异为3.158 8;平均Shannon-Weaver多样性信息指数为3.028 2。聚类分析表明,220个杂交玉米材料间的相似系数为0.62~0.95。以0.62为阈值可将220个杂交玉米材料划分为两大类群,第Ⅰ类群包括10个玉米材料,占4.5%;第Ⅱ类群包括210个玉米材料,占95.5%。本研究筛选的20对SSR引物多态性高、扩增效果好、稳定性好,可作为云南玉米杂交种特异性鉴定的核心引物。     

Dual role of the pigmentation gene B in affecting carotenoid and vitamin E content in squash (Cucurbita pepo) mesocarp

The yellow and orange colorations of the mesocarp of pumpkins and squash of Cucurbita pepo are due to the presence of carotenoid pigments that also greatly contribute to their nutritional value. Carotenoids, as well as tocopherols (vitamin E), are isoprenoid compounds formed by branches of a common biosynthetic pathway. Photodiode array HPLC analysis was used to simultaneously determine the content and composition of fruit-flesh carotenoids and tocopherols in five pairs of near-isogenic lines differing in the allelic state of genes previously identified as having profound effects on fruit color. The dominant B allele promoted carotenoid accumulation up to 5-fold and prevented tocopherol accumulation in all genetic backgrounds. The dominant L-2 allele doubled carotenoid content and, in combination with the dominant B allele, increased carotenoid content by 10-15-fold as compared to the recessive l-2 allele. The genes D and L-1 had no significant effects on mesocarp tocopherols or carotenoids content. These results indicate that the B gene, which affects both carotenoids and tocopherols, may play a regulatory role in the flux of the isoprenoid pathway products.     

Varietal and interspecific influence on micronutrient contents in citrus from the Mediterranean area

To specify the genotypic variation of Mediterranean Citrus juices, the contents of carotenoids, flavonoids, and vitamin C were determined by high-performance liquid chromatography. A selection of orange varieties and Mandarin species from the Mediterranean area (Citrus sinensis, Citrus deliciosa Ten, and Citrus clementina Hort. ex Tan) was evaluated using carotenoid profiles and flavanones contents. Among the eight varieties of orange (Salustiana, Hamlin, Shamouti, Pera, Valencia, Maltaise, Sanguinelli, and Cara-cara) and two Mandarin species, only three cultivars (Pera, Sanguinelli, and Shamouti) and the two Mandarin species displayed a high content of vitamin A (374, 381, and 272 ER L(-1) for the three orange cultivars and 1156 and 960 retinol equivalent (RE) L(-1) for the Mandarins) due to a high content of beta-cryptoxanthin. These same Citrus were also rich in hesperidin (502, 537, 552, 767, and 754 mg L(-1), respectively). Principal component analysis allowed the Mediterranean orange varieties and Mandarin species to be differentiated on the basis of nutritional criteria. Strong correlations were observed between beta-cryptoxanthin and hesperidin (r = 0.92) and between beta-cryptoxanthin and beta-carotene (r = 0.98). In contrast, vitamin C content was not correlated with carotenoids and flavanone glycosides. The Mandarin and orange group was quite distinct. The orange varieties could be divided in two groups. In addition, a diversity tree allowed a genetic approach to differentiating Citrus cultivars on the basis of Euclidian distances. This representation showed that the hybrid Clementine was nearer to its parent Mandarin than to its parent orange, suggesting that beta-cryptoxanthin was a dominant genetic factor. With regard to vitamin A, Mandarin and its hybrid Clementine appeared to be the best Citrus species.     

Analysis of genetic diversity in the tuber mustard (Brassica juncea var. tumida Tsen et Lee) in the Yangtze river basin of China

In the present study, analyses of SSR molecular markers were performed to investigate the genetic diversity of 133 tuber mustard cultivars. Eighty-one pairs of SSR primers from a total of 600 in Brassica produced stable amplified bands. In addition, 810 bands were detected among the cultivars, and 724 of those were polymorphic (89.38 %). The average number of bands per locus was 10.0 with a range from 5 to 16. Shannon’s information index for each SSR locus varied from 0.52 to 3.72, with an average of 2.74. The coefficients of genetic similarity in the SSR marker patterns among the 133 cultivars ranged from 0.77 to 0.91, with an average of 0.85. The cluster analysis showed that the cultivars could be classified into six clusters when the genetic similarity was 0.83, with 90.23 % of the cultivars included in Clusters 5 and 6. Principal component analysis was carried based on the SSR data. The results showed that the first three principal components could explain the genetic variation with 85.47, 0.67, and 0.61 %, and the 133 cultivars could be divided into six clusters according to the nearest phylogenetic relationship. It was indicated that the similarity was high and the genetic diversity was narrow among the 133 mustard tuber cultivars. 360 individuals from 24 cultivars were analyzed to reveal the genetic structure and genetic diversity within cultivars. A total of 925 alleles were detected in the 24 cultivars. Estimates of the mean number of alleles ‘A’, the effective allelic number ‘A_e’, the observed heterozygosity ‘H_o’, and expected heterozygosity ‘H_e’ were 6.0, 3.6, 0.64, and 0.37, respectively. An obvious genetic deviation from Hardy–Weinberg expectation was observed both among and within cultivars and a considerable genetic variation was revealed within rather than among cultivars. It is necessary to broaden the genetic basis of the breeding germplasm in tuber mustard. Based on their geographical distributions, the tuber mustard cultivars in this study can be divided into up-Yangtze river, mid-Yangtze river, and down-Yangtze river groups. Genetic diversity was highest in mid-Yangtze river group, followed by up-Yangtze river group, and then down-Yangtze river group. It was presumed that the origin center or genetic diversity center of tuber mustard was mid-Yangtze river, and the crop was transmitted along the Yangtze river in both directions.     

The potential of microsatellites for high throughput genetic diversity assessment in wheat and barley

Microsatellite (SSR) profiles from 65 wheats and 135 barleys have been generated, involving 14 and 22 loci, respectively. The wheat and barley varieties were chosen to represent the bulk of the area sown to these crops in the UK over the past 70 years. The profiling identified genotypic mixtures in some seed samples. Null alleles were common in wheat, but rare in barley. We describe attempts to increase the efficiency of data acquisition. High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants. Multiplex PCR was very dependent on the choice of primer combinations and seldom produced amplifications as consistently as when primer pairs were used individually. Background (non-specific) amplification was common to many primer pairs, and this hindered the use of both multiplex PCR and multiple sample loading. Sequential sample loading was the most effective strategy, although this was the least time-efficient of the measures used.     

Detection of genetic integrity of conserved maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) germplasm in genebanks using SNP markers

Twenty maize landrace accessions regenerated and conserved in five maize genebanks were investigated for genetic integrity using 1,150 Single Nucleotide Polymorphisms (SNPs) and 235 SNP haplotypes. The genetic diversity of three accessions changed significantly in terms of the average number of alleles per locus. Ten out of twenty accessions had significantly different SNP allelic frequencies, either after regeneration or in the same accession held in different genebanks. The proportion of loci with significant changes in SNP allelic frequency was very low (37/1,150). Changes in the major allelic frequency (MAF) for the majority of SNP loci (60.2–75.2%) were less than 0.05. For SNP haplotypes, the genetic diversity of four accessions changed significantly in terms of average number of haplotype alleles and polymorphic information content (PIC) per locus. The proportion of SNP haplotype alleles lost in the later generations ranged between 0 and 22.6%, and at the same time 0–19.9% of the SNP haplotype alleles appeared in later generations, however, these were absent in the earlier generations. Dynamic changes in genetic integrity, in terms of presence and absence of genes (alleles), by both SNP and SNP haplotype analysis were detected during regeneration. A suboptimum number of ears harvested in one generation can be combined with those from another, repeated regeneration to capture the diversity of the previous generation. Use of molecular markers during regeneration of accessions can help in understanding the extent of genetic integrity of the maize accessions in ex situ genebanks and in recommending the best practice for maintaining the original genetic diversity of the genebank accessions.     

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.     

黄淮麦区部分小麦种质资源的遗传差异分析

利用79对SSR引物对黄淮麦区129份种质资源进行了分析。结果表明,79对引物共检测到335个等位变异,每个引物检测到的等位变异数目2～8个,平均4.240个,变异最大的位点主要位于B组染色体上。79个SSR位点的遗传多态性信./010.015～0.820之间,平均0.498。种质资源间的遗传相似系数在0.253～0.909之间,平均0.492。聚类分析把这些种质45674大类和7个亚类。聚类结果与地域并不吻合,但能较好地反映出亲本的特性和其间的亲缘关系。     

Analysis of Genetic Diversity in the Brassica napus L. Gene Pool Using SSR Markers

Genetic diversity throughout the rapeseed (Brassica napus ssp. napus) primary gene pool was examined by obtaining detailed molecular genetic information at simple sequence repeat (SSR) loci for a broad range of winter and spring oilseed, fodder and leaf rape gene bank accessions. The plant material investigated was selected from a preliminary B. napus core collection developed from European gene bank material, and was intended to cover as broadly as possible the diversity present in the species, excluding swedes (B. napus ssp. napobrassica (L.) Hanelt). A set of 96 genotypes was characterised using publicly available mapped SSR markers spread over the B. napus genome. Allelic information from 30 SSR primer combinations amplifying 220 alleles at 51 polymorphic loci provided unique genetic fingerprints for all genotypes. UPGMA clustering enabled identification of four general groups with increasing genetic diversity as follows (1) spring oilseed and fodder; (2) winter oilseed; (3) winter fodder; (4) vegetable genotypes. The most extreme allelic variation was observed in a spring kale from the United Kingdom and a Japanese spring vegetable genotype, and two winter rape accessions from Korea and Japan, respectively. Unexpectedly the next most distinct genotypes were two old winter oilseed varieties from Germany and Ukraine, respectively. A number of other accessions were also found to be genetically distinct from the other material of the same type. The molecular genetic information gained enables the identification of untapped genetic variability for rapeseed breeding and is potentially interesting with respect to increasing heterosis in oilseed rape hybrids.