A comparative analysis of genetic polymorphism in wild and cultivated barley from Tibet using isozyme and ribosomal DNA markers.

This study was conducted to address some of the issues concerning the possible significance of Tibet in the origin and evolution of cultivated barley. A total of 1757 barley accessions from Tibet, including 1496 entries of Hordeum vulgare ssp. vulgare (HV), 229 entries of the six-rowed wild barley H. vulgare ssp. agriocrithon (HA), and 32 entries of the two-rowed wild barley H. vulgare ssp. spontaneum (HS), were assayed for allozymes at four esterase loci. A subsample of 491 accessions was surveyed for spacer-length polymorphism at two ribosomal DNA loci. Genetic variation is extensive in these barley groups, and the amount of genetic diversity in cultivated barley of this region is comparable with that of cultivated barley worldwide. The level of genetic variation of HA is significantly lower than the other two barley groups, and there is also substantial heterogeneity in the level of polymorphism among different agrigeographical subregions. However, little genetic differentiation was detected among the three barley groups (HV, HA, and HS), as well as among different agrigeographical subregions. Comparison of the results from this and previous studies indicated a strong differentiation between Oriental and Occidental barley, thus favoring the hypothesis of a diphyletic origin of cultivated barley.     

A DNA marker closely linked to the <Emphasis Type="Italic">vrs1</Emphasis> locus (row-type gene) indicates multiple origins of six-rowed cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The origin of six-rowed cultivated barley was studied using a DNA marker cMWG699 closely linked to the vrs1 locus. Restriction patterns of the PCR-amplified product of the cMWG699 locus were examined in 280 cultivated (Hordeum vulgare ssp. vulgare) and 183 wild (H. vulgare ssp. spontaneum) barleys. Nucleotide sequences of the PCR products were also examined in selected accessions. Six-rowed cultivated barleys were divided into two distinct groups, types I and II. Type I six-rowed cultivated barley was distributed widely while type II six-rowed cultivated barley was found only in the Mediterranean region. The type I sequence was also found in a wild barley accession from Turkmenistan whereas the type II sequence was also found in a two-rowed cultivated barley from North Africa and a wild barley from Morocco. These results suggested that the six-rowed type I and II barleys were derived from two-rowed type I and II barleys, respectively, by independent mutations at the vrs1 locus. Received: 3 November 2000 / Accepted: 17 April 2001     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

应用微卫星标记研究西藏野生大麦的遗传多样性

以西藏不同地区的106份野生大麦为材料,其中包括50份野生二棱大麦（HS）,27份野生瓶形大麦（HL）和29份野生六棱大麦（HA）,用Liu等（1996）发表的SSR连锁图的每个连锁群的两个臂的不同位置上选取3～5个共30个SSR标记,研究了西藏3类野生大麦的遗传多样性。结果表明,这3类野生大麦在遗传组成及等位变异频率分布上存在着明显的遗传分化。在总样本中,共检测到229个等位变异,平均每个SSR位点检测到7．6个等位变异,其中70个为这3类野生大麦间共同的等位变异,等位变异数在这3类野生大麦间有明显的差异,亚种问的遗传多样性明显高于亚种内的遗传多样性。其遗传多样性大小顺序为HS〉HL〉HA。聚类分析表明,野生二棱大麦、野生六棱大麦分别聚在不同的两类,而野生瓶形大麦中各有约50％的材料分别聚在这两类。根据本研究及前人研究结果,我们认为中国栽培大麦是从野生二棱大麦经野生瓶形大麦向野生六棱大麦进化的。该结果支持了栽培大麦起源的“野生二棱大麦单系起源论”的观点。     

Comparison of restriction fragment length polymorphisms in wild and cultivated barley.

This study was undertaken to assess the relative level of molecular diversity between cultivated barley, Hordeum vulgare ssp. vulgare (HV), and one of its wild relatives, H. vulgare ssp. spontaneum (HS), and to identify possible restriction fragment length polymorphism (RFLP) patterns that may provide information concerning the phylogenetic relationship between these two barley groups. A total of 363 barley accessions were assayed, including 95 entries of HV collected from 36 major barley growing countries of the world and 268 entries of HS from 25 natural populations in Israel and Iran. The 26 RFLP marker loci used in the survey represent single-copy, low-copy, and repetitive DNA sequences and mark all of the chromosome arms. A randomization test, on the basis of equal sample sizes, showed that HS is more polymorphic than HV, as evaluated by the number of alleles and diversity indices. The analysis also indicated extensive RFLP differentiation between these two barley groups; highly significant differences of allele frequencies were detected at the majority of the loci. The HV sample can be subdivided according to winter or spring growth habits, and two- or six-rowed spikes. Analysis of genetic polymorphisms in these subgroups showed that levels of diversity were about equal in spring and winter groups and also in the groups with two- and six-rowed spikes. However, significant differences of allelic frequencies were detected between subgroups of the two divisions.     

Genetic and morphological characterization of the barley <Emphasis Type="Italic">uniculm2</Emphasis> (<Emphasis Type="Italic">cul2</Emphasis>) mutant

Axillary meristem growth and development help define plant architecture in barley (Hordeum vulgare L). Plants carrying the recessive uniculm2 (cul2) mutation initiate vegetative axillary meristem development but fail to develop tillers. In addition, inflorescence axillary meristems develop into spikelets, but the spikelets at the distal end of the inflorescence have an altered phyllotaxy and are sometimes absent. Double mutant combinations of cul2 and nine other recessive mutations that exhibit low to high tiller number phenotypes resulted in a uniculm vegetative phenotype. One exception was the occasional multiple shoots produced in combination with granum-a; a high tillering mutant that occasionally produces two shoot apical meristems. These results show that the CUL2 gene product plays a role in the development of axillary meristems into tillers but does not regulate the development of vegetative apical meristems. Moreover, novel double-mutant inflorescence phenotypes were observed with cul2 in combination with the other mutants. These data show that the wild-type CUL2 gene product is involved in controlling proper inflorescence development and that it functions in combination with some of the other genes that affect branching. Our genetic analysis indicates that there are genetically separate but not distinct regulatory controls on vegetative and inflorescence axillary development. Finally, to facilitate future positionally cloning of cul2, we positioned cul2 on chromosome 6(6H) of the barley RFLP map.     

AB-QTL analysis in spring barley: III. Identification of exotic alleles for the improvement of malting quality in spring barley (<Emphasis Type="Italic">H. vulgare</Emphasis> ssp. <Emphasis Type="Italic">spontaneum</Emphasis>)

Malting quality is genetically determined by the complex interaction of numerous traits which are expressed prior to and, in particular, during the malting process. Here, we applied the advanced backcross quantitative trait locus (AB-QTL) strategy (Tanksley and Nelson, Theor Appl Genet 92:191–203, 1996), to detect QTLs for malting quality traits and, in addition, to identify favourable exotic alleles for the improvement of malting quality. For this, the BC₂DH population S42 was generated from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). A QTL analysis in S42 for seven malting parameters measured in two different environments yielded 48 QTLs. The exotic genotype improved the trait performance at 18 (37.5%) of 48 QTLs. These favourable exotic alleles were detected, in particular, on the chromosome arms 3HL, 4HS, 4HL and 6HL. The exotic allele on 4HL, for example, improved α-amylase activity by 16.3%, fermentability by 0.8% and reduced raw protein by 2.4%. On chromosome 6HL, the exotic allele increased α-amylase by 16.0%, fermentability by 1.3%, friability by 7.3% and reduced viscosity by 2.9%. Favourable transgressive segregation, i.e. S42 lines exhibiting significantly better performance than the recurrent parent Scarlett, was recorded for four traits. For α-amylase, fermentability, fine-grind extract and VZ45 20, 16, 2 and 26 S42 lines, respectively, surpassed the recurrent parent Scarlett. The present study hence demonstrates that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve malting quality traits.     

Genomic amplification of the <Emphasis Type="Italic">Gret1</Emphasis> retroelement in white-fruited accessions of wild <Emphasis Type="Italic">Vitis</Emphasis> and interspecific hybrids

Retrotransposons are retrovirus-related mobile sequences that have the potential to replicate via RNA intermediates and increase the genome size by insertion into new sites. The retroelement, Gret1, has been identified as playing a key role in generating fruit color variation in cultivated grape (Vitis vinifera L.) due to its insertion into the promoter of VvMybA1. Fruit color variation is an important distinguishing feature of cultivated grapes and virtually no fruit color variation is observed in wild grape species. The presence and relative copy number of Gret1 was assessed using quantitative PCR on 22 different Vitis species, only four of which (plus interspecific hybrids) are known to contain white accessions. Gret1 copy number was observed to vary by species as well as by color within species and was significantly higher in white-fruited accessions across all taxa tested. Additionally, genomic regions surrounding Gret1 insertion were sequenced in white V. vinifera, hybrid, V. labrusca, V. aestivalis, and V. riparia accessions.     

Microsatellite variation in <Emphasis Type="Italic">Avena sterilis</Emphasis> oat germplasm

The Avena sterilis L. collection in the Plant Gene Resources of Canada (PGRC) consists of 11,235 accessions originating from 27 countries and is an invaluable source of genetic variation for genetic improvement of oats, but it has been inadequately characterized, particularly using molecular techniques. More than 35 accessions have been identified with genes for resistance to oat crown and stem rusts, but little is known about their comparative genetic diversity. This study attempted to characterize a structured sample of 369 accessions representing 26 countries and two specific groups with Puccinia coronata avenae (Pc) and Puccinia graminis avenae (Pg) resistance genes using microsatellite (SSR) markers. Screening of 230 SSR primer pairs developed from other major crop species yielded 26 informative primer pairs for this characterization. These 26 primer pairs were applied to screen all the samples and 125 detected alleles were scored for each accession. Analyses of the SSR data showed the effectiveness of the stratified sampling applied in capturing country-wise SSR variation. The frequencies of polymorphic alleles ranged from 0.01 to 0.99 and averaged 0.28. More than 90% of the SSR variation resided within accessions of a country. Accessions from Greece, Liberia, and Italy were genetically most diverse, while accessions from Egypt, Georgia, Ethiopia, Gibraltar, and Kenya were most distinct. Seven major clusters were identified, each consisting of accessions from multiple countries and specific groups, and these clusters were not well congruent with geographic origins. Accessions with Pc and Pg genes had similar levels of SSR variation, did not appear to cluster together, and were not associated with the other representative accessions. These SSR patterns are significant for understanding the progenitor species of cultivated oat, managing A. sterilis germplasm, and exploring new sources of genes for oat improvement.     

Genetic marking of glume color in <Emphasis Type="Italic">Triticum spelta</Emphasis> L. var. <Emphasis Type="Italic">caeruleum</Emphasis> using gliadins

Using gliadins as genetic markers, Triticum spelta L. var. caeruleum accessions were analyzed to identify genetic control of the dark color of glumes. The research material was F₂ and BC₁ plants from crosses between spelt accessions and white-glumed common wheat varieties. The segregation for glume color fitted the monogenic control of the trait. The electrophoretic analysis of gliadins in grains from the hybrid plants has shown that the Gli-Alj* allele in the T. spelta var. caeruleum accessions is linked to the allele for the dark (black) color of glumes at the Rg-A1 locus.     

Large-scale DNA polymorphism study of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> reveals the origin and divergence of Asian rice

Polymorphism over ∼26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (π = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (π = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to ∼50 kb in O. sativa ssp. indica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.     

Development of a SCAR marker associated with salt tolerance in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) from a semi-arid region

Durum wheat (Triticum turgidum ssp. durum) is one of the main species of cultivated wheat. In arid and semi-arid areas, salinity stress reduces durum wheat productivity. This study used 26 durum wheat accessions from semi-arid regions in Tunisia to analyze plant tolerance to salt stress. Salt stress was experimentally applied by regularly submerging pots in NaCl solution. The salt tolerance trait index (STTI) and salt tolerance index (STI) of various growth parameters were used as criteria to select for salt tolerance. Analysis of genetic relationships was carried out to determine the genetic distance between durum wheat accessions. Based on simple sequence repeats analysis, a molecular marker for salt stress resistance in durum wheat was developed. Salt-treated plants had reduced morphological traits compared to control plants. Most STTIs in all genotypes were below 100 %. Based on STI, 8 accessions were found to be salt-resistant, 16 were salt-moderate, two were salt-susceptible. Analysis of the genetic relationships among 28 Tunisian durum wheat accessions revealed that landraces of the same nominal type are closely related. Of the 94 SSR primers investigated, three were selected and used to design sequence characterized amplified region (SCAR) primers. One SCAR primer pair, KUCMB_Xgwm403_2, produced a 207 bp band that was present in salt-resistant durum wheat lines but absent in salt-susceptible lines. The results suggest that KUCMB_Xgwm403_2 could be a potential genetic tag for salt-tolerant durum wheats.     

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) genotypes

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.     

AFLP based assessment of genetic relationships among shiitake (<Emphasis Type="Italic">Lentinula</Emphasis> ssp.) mushrooms

Despite the economical importance of shiitake (Lentinula ssp.) mushrooms, until the present date little information exists on cultivated and wild species in correlation with geographic origin applying molecular techniques. Use of a high resolution molecular tool like AFLP for assessing genetic similarity and geographical diversity would be an important step towards understanding of different Lentinula species. Thirteen wild and 17 cultivated accessions of 3 Lentinula species were analysed with 64 EcoRI–MseI primer combinations and finally 32 reproducible and polymorphic primer combinations were considered for the analysis. A total of 816 informative AFLP markers were generated and scored as binary data. These data were analysed using various method packages for cluster analysis, genetic diversity and genetic differentiation. Percentage polymorphism was high (62.99%) among the species studied. Different clustering analysis segregated the wild and the cultivated species into two major branches, with the wild samples being further grouped according to their geographic location. Overall polymorphisms among cultivated strains in the USA were higher than that of the cultivated strains in Japan (58.9%).     

Development of candidate introgression lines using an exotic barley accession (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> ssp.<Emphasis Type="Italic"> spontaneum</Emphasis>) as donor

In the present paper, we report on the selection of two sets of candidate introgression lines (pre-ILs) in spring barley. Two BC₂DH populations, S42 and T42, were generated by introgressing an accession of Hordeum vulgare ssp. spontaneum (ISR42-8, from Israel) into two different spring barley cultivars, Scarlett (S) and Thuringia (T). From these BC₂DH populations two sets with 49 (S42) and 43 (T42) pre-ILs were selected, and their genomic architecture as revealed by SSR marker analysis was characterised. The selected pre-ILs cover at least 98.1% (S42) and 93.0% (T42) of the exotic genome in overlapping introgressions and contain on average 2 (S42) and 1.5 (T42) additional non-target introgressions. In order to illustrate a potential application and validation of these pre-ILs, the phenotypic effect of the exotic introgression at the locus of the major photoperiod response gene Ppd-H1 was analysed. Pre-ILs carrying the introgression at the Ppd-H1 locus flowered significantly earlier than the elite parents, and the introgression maintained its effect across the two genetic backgrounds and across four tested environments. The selected pre-ILs represent a first promising step towards the assessment and utilization of genetic variation present in exotic barley. They may promote the breeding progress, serve for the verification of QTL effects and provide a valuable resource for the unravelling of gene function, e.g. by expression profiling or map-based cloning.     

Patterns of DNA Variation Among Three Centromere Satellite Families in <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">A</Emphasis>. <Emphasis Type="Italic">lyrata</Emphasis>

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Brian Morton]     

Analysis of genetic variability in two diploid <Emphasis Type="Italic">Musa</Emphasis> cultivars using RAPD

Twenty accessions of the sparsely cultivated diploid Musa cultivars Matti (AA) and Rasakadali (AB) were subjected to random amplified polymorphic DNA (RAPD) assay. A total of 14 random primers were used for the estimation of interand intracultivar variations. Out of 86 bands generated, 64 were polymorphic (74.4 % polymorphism). The cluster analysis grouped the cultivars into two major clusters: cluster I with 10 accessions of Matti and 2 of Rasakadali and cluster II comprising the remaining 8 accessions of Rasakadali. The coefficient of genetic similarity (GS) was from 0.73 to 0.99, suggesting low level of intercultivar variation. The accessions of Rasakadali with mean GS of 0.89 were genetically more diverse than those of Matti (GS = 0.93).