Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

Density enhancement of a faba bean genetic linkage map (Vicia faba) based on simple sequence repeats markers

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple

Apple Glomerella leaf spot (GLS) is a severe fungal disease that damages apple leaves during the summer in China. Breeding new apple varieties that are resistant to the disease is considered the best way of controlling GLS. Fine mapping and tightly linked marker are critically essential for the preselection of resistant seedlings. In this study, a population of 207 F₁ individuals derived from a cross between ‘Golden Delicious’ and ‘Fuji’ was used to construct a fine simple sequence repeat (SSR)‐based genetic linkage map. The position of R_gls, a locus responsible for resistance to GLS, was identified on apple linkage group (LG) 15 using SSR markers CH05g05 and CH01d08, which was adapted from a published set of 300 SSR markers that were developed using the bulked segregant analysis (BSA) method. These two SSR markers flanked the gene, and its recombination rate was 8.7% and 23.2%, respectively. A total of 276 newly developed SSR markers around the target region and designed from the genome apple assembly contig of LG15 were screened. Only nine of these were determined to be linked to the R_gls locus. Thus, a total of 11 SSR markers were in linkage with R_gls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the R_gls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene R_gls was constructed, and the locations of the 11 effective primers in the ‘Golden Delicious’ apple genome sequence were anchored. This result facilitates better understanding of the molecular mechanisms underlying the trait of resistance to GLS and could be used in improving the breeding efficiency of GLS‐resistant apple varieties.     

Generation and analysis of expressed sequence tag sequences from a soft‐seeded pomegranate cDNA library

The cultivation of soft‐seeded pomegranate is an important direction in pomegranate breeding. To comprehensively understand the molecular mechanisms involved in the formation of soft‐seeded pomegranate (Punica granatum. var. Hongmanaozi), we established an expressed sequence tag (EST) resource. Two thousand valid sequences were generated, from which 907 unigenes were obtained after initial assembly using the clustalx program. Among these unigenes, 51 showed no similarity to any protein in the public databases, 433 matched with proteins of unknown function, and 423 matched with proteins of known or putative functions. The 423 unigenes were further classified into 13 categories. Among these categories, protein synthesis, cell structure, protein destination and storage, secondary metabolism, signal transduction and transporters accounted for 8%, 8%, 4%, 7%, 6% and 17%, respectively. We also successfully developed 10 highly polymorphic expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for pomegranate. The results provide a new tool for future activities in pomegranate breeding.     

Development of a PCR marker tightly linked to mollis, the gene that controls seed dormancy in Lupinus angustifolius L.

Wild plants of Lupinus angustifolius avoid extinction in a drought year by production of seeds with coats that are impermeable to water, preventing germination of a large percentage of the seed in any given year. Domesticated cultivars of this species carry the recessive gene mollis, making the seed coat permeable to water and, in turn promoting good crop establishment in the year of sowing. A dominant microsatellite‐anchored fragment length polymorphism candidate marker was identified as being tightly linked to mollis in a population of recombinant inbred lines derived from domesticated and wild‐type parents. The candidate marker was excised from the gel, amplified by PCR, sequenced and extended beyond the SSR end of the original MseI‐SSR fragment. Two single nucleotide polymorphisms were found within this extended sequence. Specific primers were designed to create a marker 209 bp long. PCR products of these primers run on a single strand conformation polymorphism gel resolved in a co‐dominant fashion. This marker will be used in marker‐assisted selection for mollis when introgressing wild material into lupin breeding programmes.     

Efficient development of Haynaldia villosa chromosome 6VS‐specific DNA markers using a CISP‐IS strategy

Development of effective molecular markers linked to Pm21 deriving from Haynaldia villosa is critical for wheat breeding of powdery mildew resistance. In this study, we designed 12 pairs of conserved‐intron scanning primers (CISPs), using intron‐containing conserved genes located on the short arm of Brachypodium distachyon chromosome 3 (3BdS) aligned with cDNA or expressed sequence tags (ESTs) of Triticeae crops. Of 12 CISP primer pairs, 11 amplified DNA both in H. villosa and in wheat, and four displayed H. villosa chromosome 6VS‐specific polymorphisms. Six non‐polymorphic DNAs were further sequenced for designing internal primers, and five additional 6VS‐specific markers were obtained. Of the total nine 6VS‐specific co‐dominant markers, six could effectively trace Pm21 in F₂ population derived from the hybrid between the T6AL.6VS line and ‘Yangmai 158’. This study demonstrated that Brachypodium genomic information could be powerfully utilized to develop molecular markers in H. villosa or other Triticeae species.     

Identification of molecular markers associated with resistance to squash silverleaf disorder in summer squash (<Emphasis Type="Italic">Cucurbita</Emphasis> <Emphasis Type="Italic">pepo</Emphasis>)

Squash silverleaf (SSL), caused by the silverleaf whitefly [Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain)], is an important physiological disorder that affects squash (Cucurbita spp.) by reducing yield potential. Breeding squash with resistance to SSL disorder can be facilitated by using marker-assisted selection (MAS). Resistance to SSL disorder, in Cucurbita pepo, is conferred by a single recessive gene (sl). The objective of this study was to identify molecular markers associated with resistance. A zucchini squash, SSL disorder resistant breeding line, ‘Zuc76’ (sl/sl) and a SSL disorder susceptible zucchini cultivar ‘Black Beauty’ (Sl/Sl) were screened with 1,152 randomly amplified polymorphic DNA (RAPD) primers and 432 simple sequence repeat (SSR) markers to identify polymorphisms. Using F₂ and BC₁ progeny segregating for SSL disorder resistance, three RAPD (OPC07, OPL07 and OPBC16) primers and one SSR (M121) marker were found associated with sl. Fragments amplified by RAPD primer OPC07 was linked in coupling phase to sl, whereas RAPD primer OPL07 was linked in repulsion phase. RAPD primer OPBC16 and SSR marker M121 were co-dominant. The allelic order of these loci was found to be M121–sl–OPC07–OPL07–OPBC16. The closest marker to sl is M121 with an estimated genetic distance of 3.3 cM. The markers identified in this study will be useful for breeding summer squash (C. pepo) for SSL disorder resistance derived from zucchini squash breeding line ‘Zuc76’.     

Fine‐mapping a fibre strength QTL QFS‐D11‐1 on cotton chromosome 21 using introgressed lines

An initial F₂ mapping population of 223 plants of the cross between TM‐1 (Gossypium hirsutum L.) × H102 (Gossypium barbadense L.) was used to map QTLs controlling fibre strength in cotton. A genetic linkage map with 408 SSR markers was constructed with a total length of 3872.6 cM. Multiple‐QTL model of the software MapQTL version 5.0 was used to map QTLs related to fibre strength of the F_2 : 3 population. QTL QFS‐D11‐1 conferring fibre strength was mapped between NAU2950 and NAU4855 on chromosome 21 (Chr. 21) which explained 23.4% of phenotypic variation. Introgressed lines (ILs), that is, IL‐D11‐1, IL‐D11‐2 and IL‐D11‐3 were obtained through marker‐assisted backcrossing in TM‐1 background. An F₂ population of 758 plants derived from cross IL‐D11‐2 × TM‐1 was used for fine‐mapping QTL QFS‐D11‐1. QFS‐D11‐1 was mapped between markers NAU2110 and NAU2950, adjacent to its initial interval NAU2950–NAU4855 with phenotypic variation explaining 35.8%. QFS‐D11‐1 was further mapped to 0.6 cM from the flanking marker NAU2950. The results will give a basis for marker‐assisted selection of QFS‐D11‐1 in cotton breeding and to lay the foundation for cloning QFS‐D11‐1.     

Validation of a set of informative simple sequence repeats markers for variety identification in Pak‐choi (Brassica rapa L. ssp. chinensis var. communis)

A core set of 21 simple sequence repeats (SSR) markers was developed for Pak‐choi (Brassica rapa ssp. chinensis var. communis) variety identification. We initially selected 74 SSR markers which exhibited high polymorphism and reproducibility in SSR detection from 2129 SSRs. Using the 74 SSR‐based dendrogram for 45 inbred lines as calibration, 21 core SSRs were selected out. The utility of this core set SSRs was firstly tested in 45 inbred lines and finally verified in 102 commercial varieties. We also constructed a molecular ladder for each core SSR as a reference standard. Diversity analysis of this core SSR panel in 102 varieties demonstrated that each marker generates 2–3 alleles (averaged 2.33), with polymorphism information content values ranging from 0.01 to 0.56 (averaged 0.31). The averaged values of Shannon information index, observed heterozygosity, expected heterozygosity and Wright's fixation index were 0.59, 0.43, 0.38 and −0.09, respectively. Furthermore, the 21 SSR‐based classifications for 102 varieties were consistent with traditional classification based on morphology. This core SSR panel represents an effective tool for genetic variation analysis in Pak‐choi.     

Fine mapping of a QTL for the number of spikelets per panicle by using near‐isogenic lines derived from an interspecific cross between Oryza sativa and Oryza minuta

We constructed a high‐resolution physical map for the qSPP7 QTL for spikelets per panicle (SPP) on rice chromosome 7 across a 28.6‐kb region containing four predicted genes. Using a series of BC₇F₄ near‐isogenic lines (NILs) derived from a cross between the Korean japonica cultivar ‘Hwaseongbyeo’ and Oryza minuta (IRGC Acc. No. 101144), three QTLs for the number of SPP, grains per panicle and primary branches were identified in the cluster (P ≤ 0.01). All three QTLs were additive, and alleles from the O. minuta parent were beneficial in the ‘Hwaseongbyeo’ background. qSPP7 was mapped to a 28.6‐kb region between the two simple sequence repeat (SSR) markers RM4952 and RM21605. The additive effect of the O. minuta allele at qSPP7 was 23 SPP, and 43.6% of the phenotypic variance was explained by the segregation of the SSR marker RM4952. Colocalization of the three QTLs suggested that this locus was associated with panicle structure and had pleiotropic effects. The NIL populations and molecular markers are useful for cloning qspp7.     

Mapping of male sterility gene ms10 in chilli pepper (Capsicum annuum L.)

Most of the hybrid seed in chilli are produced manually, but the use of male sterility (MS) can reduce the cost of hybrid seed production. MS‐12, a nuclear male‐sterile (NMS) line developed at Punjab Agricultural University, Ludhiana (India), has been utilized to develop commercial F₁ hybrids. A recessive gene, designated as ms10, governs MS in MS‐12. Due to recessive gene control, development of new NMS lines incorporating ms10 gene is tedious and time‐consuming. We identified SSR markers AVRDC‐PP12 and AVRDC_MD997* linked to the ms10 gene. A total of 558 primer pairs were screened following bulked segregant analysis (BSA). Linkage analysis in 210 F₂ plants indicated that the two SSR markers were linked to the ms10 gene and the marker AVRDC‐PP12 was closest to the gene at 7.2 cM distance. The marker was mapped to chromosome 1 at genome position 175 694 513 to 175 694 644. Until more closely linked markers are developed, the marker AVRDC‐PP12 would facilitate transfer of ms10 gene through marker‐assisted selection (MAS). Fine mapping would lead to cloning of the ms10 gene.     

A novel aphid resistance locus in cowpea identified by combining SSR and SNP markers

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1‐57‐2 and for introgressing the resistance into elite cultivars by marker‐assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F₂ population from ‘Apagbaala’ x SARC 1‐57‐2. A SNP‐genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514 Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP‐SNP‐based marker panel for background selection in MABC, the resistance from SARC 1‐57‐2 was introduced into elite susceptible cultivar ‘Zaayura’. Five BC₄F₃ lines of improved ‘Zaayura’ that were isogenic except for the resistance locus region had phenotypes similar to SARC 1‐57‐2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker‐assisted breeding.     

Fine mapping of Rf2, a major locus controlling pollen fertility restoration in sorghum A1 cytoplasm,encodes a PPR gene and its validation through expression analysis

Sorghum is one of the pioneering cereal crops where cytoplasmic male sterility (CMS) was successfully exploited for mass production of F₁ hybrid seed. Mapping genes for fertility restoration (Rf) is an important aspect of understanding the molecular basis of fertility restoration in crop plants. In this study, we fine‐mapped a fertility restoration locus, Rf2 of sorghum reported earlier (Jordan, Mace, Henzell, Klein, & Klein, 2010 ), involving two F₂ populations (296A × RS29 and 296A × DSV1) and newly developed SSR markers delimited Rf2 locus to 10.32‐kb region on chromosome 2. The Rf2 locus was tightly linked with two new SSRs, MS‐SB02‐3460 (0.14 cM) and MS‐SB02‐3466 (0.75 cM) on both sides, and hosted only one gene (Sobic.002G057050) of PPR gene family. Another new SSR marker developed in the study, MS‐SB02‐37912, forms the part of PPR gene and could act as a perfect marker in marker‐assisted breeding for fertility restoration involving Rf2 in sorghum breeding. The strong involvement of Sobic.002G057050 gene in fertility restoration was supported through RNA expression analysis.     

Mapping and validation of QTLs for resistance to an Indian isolate of Ascochyta blight pathogen in chickpea

Ascochyta blight (AB) caused by Ascochyta rabiei, is globally the most important foliar disease that limits the productivity of chickpea (Cicer arietinum L.). An intraspecific linkage map of cultivated chickpea was constructed using an F₂ population derived from a cross between an AB susceptible parent ICC 4991 (Pb 7) and an AB resistant parent ICCV 04516. The resultant map consisted of 82 simple sequence repeat (SSR) markers and 2 expressed sequence tag (EST) markers covering 10 linkage groups, spanning a distance of 724.4 cM with an average marker density of 1 marker per 8.6 cM. Three quantitative trait loci (QTLs) were identified that contributed to resistance to an Indian isolate of AB, based on the seedling and adult plant reaction. QTL1 was mapped to LG3 linked to marker TR58 and explained 18.6% of the phenotypic variance (R ²) for AB resistance at the adult plant stage. QTL2 and QTL3 were both mapped to LG4 close to four SSR markers and accounted for 7.7% and 9.3%, respectively, of the total phenotypic variance for AB resistance at seedling stage. The SSR markers which flanked the AB QTLs were validated in a half-sib population derived from the same resistant parent ICCV 04516. Markers TA146 and TR20, linked to QTL2 were shown to be significantly associated with AB resistance at the seedling stage in this half-sib population. The markers linked to these QTLs can be utilized in marker-assisted breeding for AB resistance in chickpea.     

Analysis of QTL for yield-related traits in cassava using an F<Subscript>1</Subscript> population from non-inbred parents

A cassava F₁ population raised from the cross SC6 × Mianbao was used to construct a genetic linkage map. The map incorporated 200 polymorphic amplified fragment length polymorphism, sequence-related amplified polymorphism, simple sequence repeat (SSR), and expressed sequence tag (EST)–SSR markers which fit a 1:1 segregation ratio. It comprised 20 linkage groups (LGs) and spanned a genetic distance of 1645.1 cM with an average marker interval of 8.2 cM. Fifty-seven repeatedly detected QTLs (rd-QTLs) for three phenotypic traits (fresh root yield, root dry matter content, and root starch content) were identified in the F₁ population in four trials of year 2003, 2004, 2005, and 2008 by inclusive composite interval mapping. Among the 57 rd-QTLs, 25 rd-QTLs were linked to SSR/EST–SSR markers, which will help to facilitate marker-assisted selective breeding in cassava, and 15 marker intervals on ten LGs showed pleiotropic effects.     

Identification of microsatellite polymorphisms in an expressed portion of the rye genome

For the purposes of genetics and application the number of simple sequence repeat (SSR) markers in rye has to be increased significantly to cover the entire genome. To this end, more than 8000 publicly accessible rye cDNA sequences from anthers, cold‐stressed leaves, and aluminium‐stressed and unstressed roots were exploited as a resource for SSR marker development. A total of 157 Secale cereale micro‐satellite (SCM) loci out of 528 SSRs comprising di‐, tri‐ and tetra‐nucleotide motifs could be assayed on automated sequencers. One‐hundred expressed sequence tag (EST)‐derived SCM loci displayed a length polymorphism among a sample of 15 rye accessions. Of the SCM, 45% could be associated with proteins of known or unknown function. Recently published ESTs from different rye tissues proved to be a valuable resource for SSR marker development in rye.     

Identification of SSR markers linked to the Phytophthora resistance gene Rps1-d in soybean

To identify markers for the Phytophthora resistance gene, Rps1‐d, 123 F_{2 : 3} families were produced from a cross between Glycine max (L.) Merr. ‘Tanbakuro’ (a Japanese traditional black soybean) and PI103091 (Rps1‐d) as an experimental population. The results of virulence tests produced 33 homozygous resistant, 61 segregating and 29 homozygous susceptible F_{2 : 3} families. The chi‐squared test gave a goodness‐of‐fit for the expected ratio of 1 : 2 : 1 for resistant, segregating and susceptible traits, suggesting that the inheritance of Rps1‐d is controlled by a monogenic dominant gene. Simple sequence repeat (SSR) analyses of this trait were carried out using the cultivars ‘Tanbakuro’ and PI103091. Sixteen SSR primers, which produced 19 polymorphic fragments between the two parents, were identified from 41 SSR primers in MLG N. Eight SSR markers were related to Rps1‐d, based on 32 of the 123 F_{2 : 3} families, consisting of 16 homozygous resistant and 16 homozygous susceptible lines. The remaining 91 families were analysed for these eight markers, and a linkage map was constructed using all 123 F_{2 : 3} families. The length of this linkage group is 44.0 cM. The closest markers, Sat_186 and Satt152, are mapped at 5.7 cM and 11.5 cM, respectively, on either side of the Rps1‐d gene. Three‐way contingency table analysis indicates that dual‐marker‐assisted selection using these two flanking markers would be efficient.