Broadening the genetic base of lentil cultivars through inter‐sub‐specific and interspecific crosses of Lens taxa

Wild Lens taxa are invaluable sources of useful traits for broadening genetic base of cultivated lentil. Nine inter‐sub‐specific and interspecific crosses were made successfully between cultivated (Lens culinaris ssp. culinaris) and wild lentils (L. culinaris ssp. orientalis, odemensis, lamottei and ervoides). The effect of species groups, day length and temperature on crossability in lentils was evident under normal winter sowing in New Delhi and in summer Himalayan nursery at Sangla in Himachal Pradesh, India, although pollen fertility assessed in all the cross‐combinations showed no significant variation. True hybridity of nine inter‐sub‐specific and interspecific crosses was confirmed through morphological and molecular (ISSR) markers, in which three of 120 primers could confirm the hybridity of all the crosses. All cross‐combinations were also studied for important quantitative traits related to yield. The range, mean and coefficient of variation were estimated in parental lines, F₁ and F₂ generations to determine the extent of variability generated in cultivated lentils through the introgression of genes from wild L. taxa. A high level of heterosis was observed in F₁ crosses for important traits studied. Substantially higher variations for seed yield and its attributing traits were exhibited in F₂ generations indicating transgressive segregation. The results of the present investigation revealed that wild L. taxa can be successfully exploited for lentil improvement programmes, and the variations generated could be easily utilized for broadening the genetic base of cultivated lentil gene pool for improving the yield as well as wider adaptation.     

Introgression of phenoxy herbicide resistance from Raphanus raphanistrum into Raphanus sativus

Phenoxy herbicides such as 2,4‐dichlorophenoxy acetic acid (2,4‐D) and 4‐chloro‐2‐methylphenoxy acetic acid (MCPA) are selective herbicides used extensively in agriculture for weed control. Wild radish (Raphanus raphanistrum) is a problem weed across the globe and heavily infests crop fields in Australia. Phenoxy herbicides are used to selectively control dicot weeds, including wild radish. As a result of selection, phenoxy‐resistant wild radish populations evolved in Western Australia. In this research, introgression of phenoxy resistance from wild radish to cultivated radish (Raphanus sativus) was investigated following classical breeding procedures. F₁ progeny were generated by crossing MCPA‐resistant R. raphanistrum and MCPA‐susceptible R. sativus. F₁ hybrids were screened for MCPA resistance. The MCPA‐resistant F₁ hybrids were used to produce three generations of backcross progeny. Genetic analyses of F₁ and backcross progeny demonstrated introgression of the MCPA‐resistant trait from wild radish to cultivated radish. Implications of phenoxy resistance introgression into cultivated radish include potential development of herbicide‐tolerant radish cultivars or other members of the Brassicaceae family.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

Exploitation of wild annual Cicer species for widening the gene pool of chickpea cultivars

Introgression of unadapted genes from the wild Cicer species could contribute to the widening of genetic base of important traits such as yield, yield attributes and resistance to major biotic and abiotic stresses. An attempt was made successfully to intercross two wild annual Cicer species with three cultivated chickpea cultivars. Four interspecific cross‐combinations were made, and their true hybridity was ascertained through morphological and molecular markers. These cross‐combinations were also studied for some important quantitative traits under real field conditions. The range, mean and coefficient of variation of agro‐morphological traits were assessed in the parental lines, their F₁ and F₂ generations to determine the extent of variability generated in cultivated chickpea varieties. A high level of heterosis was recorded for number of pods/plant and seed yield/plant in F₁ generation. Three cross‐combinations of ‘Pusa 1103’ × ILWC 46, ‘Pusa 256’ × ILWC 46 and ‘Pusa 256’ × ILWC 239 exhibited substantially higher variability for important yield‐related traits. The present research findings indicate that these wild annual Cicer species can be easily exploited to broaden the genetic base of cultivated gene pool for improving seed yield as well as adaptation.     

Molecular evaluation of genetic variability in wild populations of mulberry (Morus serrata Roxb.)

Sixteen populations of the wild mulberry, Morus serrata Roxb., were analysed for their genetic diversity with the aim of using them in introgressive breeding programmes with cultivated relatives. Five genets from each population were collected from different natural populations of M. serrata present in Uttaranchal and Himachal Pradesh in India, and diversity of morpho‐anatomical traits and inter‐simple sequence repeat (ISSR) markers were studied. Significant amounts of genetic diversity were observed among these populations for morpho‐anatomical as well as DNA markers. The 17 ISSR primers generated a total of 95 DNA markers, 51 of which were polymorphic, revealing 67% polymorphism among the populations. The pair‐wise genetic distance, estimated from these DNA markers varied from 0.091 between Urgam‐3 and Kathpuria to 0.258 between Dakrakao‐1 and Dunda with an average genetic distance of 0.165. Clustering analysis grouped these 16 populations into three broad groups. The grouping showed a moderate correlation with the geographical distances. Based on the morphological traits and molecular genetic variability, plants of Urgam‐1, Bhowali farm, Nainitikar, Dunda or Korwa‐2 can be selected for breeding and conservation programmes.     

S‐locus diversity and cross‐compatibility of wild Prunus avium for timber breeding

Prunus avium is primarily cultivated for its fruit, sweet cherries. However, it is also used to produce high‐quality timber. In a P. avium seed orchard, gametophytic self‐incompatibility is a restriction for free pollen flow and should be considered when establishing basic forest materials. In this study, S‐locus diversity and cross‐incompatibility of wild cherry individuals in clonal banks established for breeding for timber production were investigated. Wild cherry trees (140) with outstanding forest growth habit, collected in northern Spain, grafted and planted in two clonal banks, were genotyped at the S‐locus. The self‐incompatibility S‐locus genes, S‐RNase and SFB, were analysed by PCR. Twenty‐two S‐haplotypes, resulting in 72 different S‐genotypes, were identified. The genotypes were grouped into 33 incompatibility groups and 39 unique genotypes. This initial S‐locus analysis revealed large genetic diversity of wild cherry trees from the Spanish northern deciduous forest, and provides useful information for seed orchard design. Wild P. avium displays significantly more genetic diversity than what is detected in local cultivars, revealing a narrowing of genetic diversity during local domestication.     

Chromosome pairing and fertility of interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative with the potential to improve the drought and salt tolerances of white clover (Trifolium repens). Previous work has shown that it is possible to efficiently produce large T. repens × T. occidentale breeding populations using colchicine‐doubled (4×) T. occidentale. For effective introgression (backcross) breeding, it is also essential that interspecific chromosome pairing and recombination occur. In this study, it was apparent that chromosome pairing was occurring not only between T. occidentale and T. repens subgenomes, but also between the ancestral subgenomes of T. repens. Thus, interspecific hybridization has the potential for major genome recombination and opens the way for introgression of traits from T. occidentale into white clover.     

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.     

Characterizing morphological traits and estimating genetic relationship for intermediate soybean collected from South Korea

Twenty‐two wild, 16 intermediate and 20 cultivated soybean varieties were used to analyse relationships among the species of subgenus Soja, genus Glycine using 11 morphological traits and simple sequence repeat (SSR) markers. These genotypes using eleven agronomic characteristics were divided into three clusters: cluster I included 20 G. max, cluster II included 12 intermediate, and cluster III included 22 G. soja and four intermediate lines. Genetic relationships among the species of subgenus Soja showed three distinct clusters. Cluster I consisted of 20 G. max cultivars and two intermediate type lines, and cluster II consisted of 13 intermediate type and two G. soja lines; however, cluster III consisted of 20 G. soja and one intermediate type lines. These phenotypic and genetic data suggest that the intermediate type lines could be distinguished between G. max and G. soja lines. However, the intermediate type could not be classified as a new species.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Assessment of cytoplasmic polymorphisms by PCR-RFLP of the mitochondrial orfB region in wild and cultivated radishes (Raphanus)

In order to determine the genetic relationship between wild and cultivated radish species, and those among the cultivated species, structural and sequence variations in the mitochondrial orfB gene region were studied in one cultivated and two wild species of Raphanus. Using PCR amplification patterns and RFLP of a PCR product of the region, 232 wild and 420 cultivated radish plants were classified into one of three types of orfB variation. The wild radish (especially the Japanese one) showed large polymorphism in each population with eight of 13 Japanese populations studied containing all three types, whereas cultivars were generally monomorphic. Although type 1 having Ogura male sterile cytoplasm was present with the highest frequency in Japanese wild radish, most cultivars were divided into type 2 or 3 with normal cytoplasm. Type 2 was widely distributed in European, Chinese and major Japanese varieties, while some Chinese varieties and several Japanese local radishes had type 3 cytoplasm. The comparison provides valuable information about the origin and differentiation of cultivated radishes and the relationship between cultivated and wild radishes.     

Reproductive compatibility studies between wild and cultivated strawberries (Fragaria × ananassa) to obtain “bridge species” for breeding programmes

The Fragaria genus has become relevant due to worldwide economic importance of the octoploid hybrid F. × ananassa. Interspecific hybridizations are widely employed in breeding programmes to introduce useful characteristics from wild species to cultivated varieties, although many reproductive barriers prevent gene flow between species. Hybrid genotypes that can act as a bridge between related wild species and cultivated strawberry could be useful to overcome those barriers. The aim of this work was to obtain interspecific hybrids of Fragaria and to analyse possible reproductive barriers. In the Banco de Germoplasma de Frutilla‐UNT, crossings were carried out among wild species of Fragaria with different ploidy levels and cultivated strawberry. To confirm hybrid condition of genotypes obtained, histological techniques and microsatellite markers were used. When wild species were crossed with cultivated strawberry in both directions, there was low production of achenes, while in crosses between wild species of Fragaria genus, achene production was very high. The percentage of germination of achenes was high when crossed species had the same ploidy level, and very low or null when they presented different ploidy levels. Although pre‐zygotic incompatibility associated to the degree of domestication and postzygotic barriers related to different ploidy levels of the progenitors were detected, new hybrid genotypes of Fragaria were obtained. These new hybrids could be used as “bridge species” in breeding programmes, since preliminary results showed no incompatibility barriers when they were crossed with Duchesnea indica.     

Sexual hybridization between Capsella bursa‐pastoris (L.) Medik (♀) and Camelina sativa (L.) Crantz (♂) (Brassicaceae)

The development of transgenic oilseed Camelina sativa (2n = 40) and the potential for hybridization with its weedy relative Capsella bursa‐pastoris (2n = 36) necessitates a careful evaluation of the reproductive compatibility between the species. Here, we conducted over 1800 crosses (emasculation and manual pollination) to examine the ability of 10 Canadian C. bursa‐pastoris (♀) accessions to hybridize with five accessions of C. sativa (♂). Seven hybrids were confirmed among 586 putative hybrids screened with species‐specific markers, indicating a hybridization rate of 1.5 hybrids per 10 000 ovules pollinated. All seven hybrids had intermediate DNA content compared to their parents, were morphologically distinct, had low (1.9%) pollen fertility and failed to produce selfed or backcrossed seed. Given the abundance of C. bursa‐pastoris along field margins, hybrids will likely be generated in the wild, but they will be unable to establish lineages unless fertility is restored. The large number of crosses and the diversity captured by the use of multiple accessions resulted in strong statistical power and a high degree of confidence in the estimated hybridization rate.     

Gene flow between cultivated sunflower and <Emphasis Type="Italic">Helianthus petiolaris</Emphasis> (Asteraceae)

Helianthus petiolaris (Asteraceae) native to North America has naturalized in Argentina. The extensive overlapping with sunflower crop regions, the coincidence of life cycles and the common pollinators facilitate interspecific crosses with sunflower, H. annuus var. macrocarpus. To estimate the occurrence of crop-to-wild and wild-to-crop gene flow, off-type plants in progenies of pure stands of both species flowering in coincidence and the presence of crop alleles in H. petiolaris populations were investigated in 26 wild populations and nine crop fields. Morphological traits and RAPD markers were used to attempt hybrid characterization. Off-type individuals were found in frequencies of 0.5 and 0.3% among crop progenies and wild populations, respectively. Off-type plants showed intermediate values for metric morphological traits and low fertility traits. Some off-type plants proved to carry crop alleles based on molecular analysis. The average frequency of cultivar-marker introgression across the wild populations was very low (0.02). Although observed hybridization rates seem to be low, the extension of crop-wild overlapping in Argentina make hybrid formation a noticeable process. Therefore, the new sunflower varieties and eventually GM varieties would transfer their traits through pollen flow and they would persist in H. petiolaris populations.     

Construction of a crop—wild hybrid population for broadening genetic diversity in cultivated sunflower and first evaluation of its combining ability: the concept of neodomestication

The genetic base of sunflower elite lines is very narrow, due to many years of selection and breeding. To broaden the genetic diversity of the cultivated sunflower, in 1995 73 wild sunflower populations were crossed with 3 cultivated lines (Testers), and 219 hybrid offspring’s were evaluated in the field. GCA and SCA effects were computed suggesting for all traits a genetic potential for improvement through selection. Study of the hybrids revealed that the wild accessions bear different genetic abilities to combine with the testers for traits of morphological architecture, phenology and yield (seed weight and seed oil). The variance due to GCA and SCA showed that gene action was additive for days to flowering, branching and plant height. Genotypes derived from the same geographic origin may have either good or poor general combing ability. The correlation between GCA and per se genotype performance was positive for all traits except for seed oil content. This was the first attempt to evaluate wild-cultivated hybrids in sunflower on a large scale and will be the starting point for the management of hybrid Helianthus annuus populations for breeding. GCA and SCA estimations will facilitate the definition of strategies to manage and exploit the natural diversity for this crop.     

Genetic diversity of feral alfalfa (<Emphasis Type="Italic">Medicago</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.) populations occurring in Manitoba,Canada and comparison with alfalfa cultivars: an analysis using SSR markers and phenotypic traits

Feral populations of cultivated crops may act as reservoirs for novel traits and aid in trait movement across the landscape. Knowledge on the genetic diversity of feral populations may provide new insights into their origin and evolution and may help in the design of efficient novel trait confinement protocols. In this study, the genetic diversity of 12 feral alfalfa (Medicago sativa) populations originating from southern Manitoba (Canada) and 10 alfalfa cultivars and a M. falcata germplasm were investigated using eight SSR markers (i.e., microsatellites) and 14 phenotypic traits. We found that the genetic diversity observed in feral populations was similar to the diversity detected among the 10 cultivars. Analysis of molecular variance revealed that there was great genetic variation within (99.8%) rather than between different feral populations. Cluster analysis (unweighted pair-group method using arithmetic average) revealed no differentiation between feral populations and cultivars for neutral loci. High levels of population differentiation for phenotypic traits (and not for neutral markers) suggest the occurrence of heterogeneous selection for adaptive traits. The phenotypic traits we studied did not distinctly separate feral populations from cultivars but there was evidence of natural selection in feral populations for traits including winter survivability, rhizome production, and prostrate growth habit. Our results suggest that feral alfalfa populations need to be considered in the risk assessment of alfalfa containing novel genetically modified (GM) traits. Further, feral alfalfa populations may be regarded as a source of new germplasm for plant improvement.     

Genetic dissection of seed vigour under artificial ageing conditions using two joined maize recombinant inbred line populations

Seed vigour plays an important role in agricultural production, and seeds with high sowing quality are necessary for improving agriculture production. In our study, two connected maize recombinant inbred line (RIL) populations derived from Yu82 × Shen137 and Yu537A × Shen137 crosses were evaluated for the mean germination time (MGT) and other related traits under three artificial ageing treatments. We used meta‐analysis to integrate genetic maps and identify quantitative trait loci (QTLs) across the two populations. In total, 74 QTLs and 20 meta‐QTLs (mQTLs) were identified. Four key mQTLs, mQTL2‐2, mQTL5‐4, mQTL6 and mQTL8, which contained initial QTLs with R² values >10% and included 5–9 initial QTLs, may be hot spots of important QTLs for the associated traits. Twenty‐two key candidate genes associated with four seed vigour‐related traits mapped to 14 mQTLs. In particular, the GRMZM2G163749, GRMZM2G122172/GRMZM2G554885/GRMZM2G122871 and GRMZM2G150367 genes mapped within the important mQTL5–4, mQTL6 and mQTL8 regions, respectively. Fine mapping for the genetic regions of these three mQTLs merits further study and could be utilized for marker‐assisted breeding.     

Identification and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

Morphology and SSR fingerprinting of newly developed <Emphasis Type="Italic">Cynara cardunculus</Emphasis> genotypes exploitable as ornamentals

The species Cynara cardunculus includes the globe artichoke (var. scolymus), the cultivated cardoon (var. altilis) and the wild cardoon (var. sylvestris). The three taxa are sexually compatible and originate fertile F ₁ progenies, which, given the high heterozygosity of the species, are highly segregating. We report the characterization of two F ₁ populations, one bred from a cross between globe artichoke and cultivated cardoon, and the other between globe artichoke and wild cardoon. Both populations featured a wide array of phenotypes in relation to several traits, and some of the newly developed genotypes are of interest for the ornamental market. The two populations were genotyped at 50 microsatellite (SSR) loci: in the globe artichoke × wild cardoon and globe artichoke × cultivated cardoon progenies 116 and 97 alleles were respectively detected. SSR pattern scores were used to produce an UPGMA dendrogram and a PCoA plot. A set of nine SSR loci, evenly dispersed across the genome, was shown to be sufficient to unambiguously identify each segregant. The molecular fingerprinting is useful for establishing the true to type correspondence of propagative materials in nurseries and ensures the effective correspondence between the real and the declared identity of a clone.     

Independent selection for seed free tryptophan content and vernalization response in chickpea domestication

Chickpea shows a distinct domestication trajectory vis‐a‐vis pod dehiscence and growth cycle mediated by vernalization insensitivity compared with its companion Near Eastern legumes. Our objectives were: (i) to map the quantitative trait loci (QTLs) associated with vernalization response and seed free tryptophan in domesticated × wild chickpea progeny and (ii) estimate the genetic correlation between vernalization response and free tryptophan content. A domesticated × wild chickpea cross was used to document phenotypic segregation in both traits and to construct a skeletal genetic map for QTL detection. A number of vernalization response and seed free tryptophan content QTLs were documented in both F₂ and F₃ generations. No significant genetic correlation between these two traits was observed. Epistatic relationship between two free tryptophan loci was documented. It is evident that selection for high seed tryptophan is easier to accomplish relative to selection for vernalization insensitivity. This suggests that the two traits were selected independently in antiquity, thereby corroborating earlier claims for conscious selection processes associated with chickpea domestication.