Determination of genetic variation and taxonomy in lentil (Lens Miller) species by chloroplast DNA polymorphism

Summary Chloroplast DNA restriction fragment lenght polymorphisms (RFLP) were used to examine the taxonomic relationships of cultivated and wild lentil (Lens Miller) species and identify the extent of genetic variation in this genus. Twelve accessions representing all Lens subspecies were digested with four hexanucleotide-recognizing restriction endonucleases. These digests randomly surveyed 540 base pairs, or 0.4% of the approximately 125 kilobase lentil chloroplast genome. A high degree of gragment length conservation was seen among members of crossability group I, i.e., L. c. ssp. culinaris, L. c. ssp. orientalis and L. c. ssp. odemensis. Accessions of the two subspecies comprising crossability group II, i.e., L. n. ssp. nigricans and L. n. ssp. ervoides, showed the greatest amount of variation when compared to the cultivated lentil, L. c. ssp. culinaris. Limited variation was observed within subspecies except for L. n. ssp. nigricans, where accessions of the normal cytotype were highly polymorphic to those of the differentiated cytotype. Chloroplast DNA RFLPs reaffirm hypotheses that propose L. c. ssp. orientalis as the progenitor to the cultivated lentil. The implications of this study on taxonomy and genetic resources is also discussed.     

The breeding of the cultivated potato species solanum x juzepczukii and S. x curtilobum II. The resynthesis of S. x juzepczukii and S. x curtilobum

Summary The possibility of combining anew the genomes of wild and cultivated progenitors of triploid S. x juzepczukii and pentaploid S. x curtilobum by following the known evolutionary pathway of these species was investigated.The resynthesis of S. x juzepczukii was easy, and a wide range of synthetic forms was bred. Among these were forms with higher frost resistance (-5°C) than has been found in natural S. x juzepczukii. The total tuber glycoalkaloid content of several synthetic hybrids was lower than or as low as that of natural clones. Most synthetic hybrids were more vigorous than natural S. x juzepczukii and produced about the same types of tubers as are found in the natural range of variation. The best diploid parents were found in the species S. goniocalyx.The attempt to resynthesize pentaploid S. x curtilobum has not been successful but tetraploid plants were obtained in the process. An explanation for the occurrence of tetraploids resulting from triploid x tetraploid and/or diploid crosses is offered.The newly bred tetraploids contain at least one genome from S. acaule (possibly two) and hybridize easily with ssp. andigena. They thus provide a means for the transfer of S. acaule germ plasm into the tetraploid cultivated gene pool which would profit from the frost resistance of S. acaule.     

Immunity to Clavibacter michiganensis subsp. sepedonicus: Screening of exotic Solanum species

Summary Accessions from exotic Solanum species, including diploid and tetraploid species, were screened for immunity to Clavibacter michiganensis subsp. sepedonicus, the causal agent of potato ring rot. The diploid species included S. infundibuliforme, S. lesteri, S. megistacrolobum, S. tuberosum Group Phureja, S. polyadenium, S. pinnatisectum, S. raphanifolium, S. sparsipilum, S. sanctae-rosae, S. tuberosum Group Stenotomum, S. toralapanum, and S. verrucosum. The tetraploid species included S. tuberosum Group Andigena, S. acaule, S. fendleri, S. hjertingii, S. oplocense, S. polytrichon, and S. stoloniferum. Apparent immunity was initially found in several diploid species, but was not present during subsequent retesting. Immunity was found in nine accessions of tetraploid S. acaule. These accessions maintained their immunity during testing over an eight-month period. S. acaule appears to be a good source of immunity for introgression studies.     

Ribosomal DNA repeat unit polymorphism and heritability in peanut (Arachis hypogaea L.) accessions and related wild species

Repeat unit length and restriction site variation in ribosomal RNA geneclusters (rDNA) was surveyed in 77 Arachis accessions, includingsamples from 39 accessions of cultivated Arachis hypogaea(2n=4x=40), 36 accessions representing 15 related tetraploid and diploidwild species, and two synthetic amphidiploids. Total genomic DNA wasdigested with five restriction enzymes, and probed with three heterologousribosomal clones of wheat and broad bean. Four rDNA repeat unit lengthclasses were recognized in the Arachis species. Restriction site analysisshowed that some SacI, BamHI and TaqI cleavage sites in rDNA unit werehighly conserved. With few exceptions, the variable BamHI and EcoRV siteswere able to differentiate the taxonomic sections and species, respectively.Arachis hypogaea and A. duranensis accessions produced fourrDNA length classes. Among these, three were identical with those of otherArachis species. A SacI restriction site (s) from probe (Ver6-5) cangenerally distinguish the two subspecies A. hypogaea ssp. hypogaea and A. hypogaea ssp. fastigiata. Forty nine per centof bands were polymorphic across the A. hypogaea accessionsanalysed. This study does not support A. batizocoi to be a progenitorof A. hypogaea. For the gene array, the contribution from eachparental genome can be detected in the two synthetic amphidiploids.     

Nuclear RFLP between pepper cultivars (Capsicum annuum L.)

Summary Forty one nuclear probes, distributed across the different linkage groups of the previously published map, were used to examine restrition fragment length polymorphism between cultivated peppers. Total DNA from thirteen accessions of Capsicum annuum var. annuum and one accession of C. baccatum var. pendulum was separately cut with ten restriction enzymes. The analyses were restricted to only one enzyme per probe to reduce the polymorphism redundancy. Nei & Li's genetic distances between accessions were calculated from the 141 resultant nuclear DNA restriction fragments. The genetic variation was larger between C. annuum and C. baccatum than between C. annuum cultivars. Large fruited related accessions closely clustered together. Distances between the small fruited cultivars were larger than within the bell pepper group. A correspondence analysis performed on differences between the global RFLP patterns of each accessions of C. annuum revealed the particular genomic structure of four small fruited cultivars: Criollo de Morelos 334, H3, Perennial and Doux Long des Landes. The percentage of probes revealing at least one RFLP with at least one enzyme ranged from zero to fifty percent for all the pairwise comparisons of C. annuum accessions. 82 presumptive loci were detected with a mean number of 1.46 alleles per locus within C. annuum and 1.83 within all the accessions. This result indicates that molecular markers will be more usable in intraspecific study of C. annuum than isozyme markers.     

PCR-RFLP analysis of chloroplast gene regions in Cajanus (Leguminosae) and allied genera

Twenty-eight species belonging to five genera of the sub tribe Cajaninae; viz., Cajanus(15 species), Rhynchosia (10 species), Dunbaria, Flemingia and Paracalyx were analysed for variations in four chloroplast generegions, rbcL, trnS-psbC, 16S and trnL-UAA. The four chloroplast gene regions were amplified with specific primers and subsequently digested with 15 restriction enzymes. Rhynchosia did not show any inter-specific differences in the four gene regions for any of the enzymes used. In Cajanus, the inter-specific PCR-RFLP profile of the four gene regions for all the enzymes were similar except for the Pst I digests of trnS-psbC gene region. At inter-generic level, rbcL gene region did not show polymorphism with any of the enzymes used; while in trnS-psbC gene region polymorphism was observed only with Pst I and Hae III digestions. Inter-generic PCR-RFLP of 16S did not reveal any variation. The trnL-UAA gene region had restriction site only forEcoR I in which Cajanus and Rhynchosia showed similar profiles while Dunbaria showed a different profile. The trnL-UAA gene region in Paracalyx and Flemingia did not have restriction site for this enzyme. Despite the analyses of four gene regions using 15 restriction enzymes, differentiation at genus level could not be obtained. These observations indicated limited divergence of the chloroplast genome among the four genera of the sub-tribe Cajaninae suggesting close relationships of the taxa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Natural hybridization between Solanum acaule Bitt. and S. megistacrolobum Bitt. in the province of jujuy,Argentina

Summary Evidence is provided that hybridization of the tetraploid (2n=4x=48), self-fertile tuber-bearing species Solanum acaule Bitt. with the diploid (2n=2x=24), self-incompatible, tuber-bearing S. megistacrolobum Bitt. takes place in several localities of the province of Jujuy in the high, cold plateaux of the Argentine Puna. The triploid hybrids (2n=3x=36) closely resemble S. acaule in growth habit, leaf morphology and floral structures and for this reason they can be easily overlooked for that species in the field. Experimental data show that S. acaule can be crossed with S. megistacrolobum though the crossability is rather low and variable according to the particular cross considered. The artificial hybrid obtained compares well with the natural hybrid in morphology and chromosome number. The hybrids, though almost completely male sterile, are successful colonizers of disturbed areas around farmers' dwellings, cattle enclosures and other areas where the soil is rich in organic matter.There is some evidence that the Tilcara material of S. acaule subsp. aemulans and the hybrids of S. acaule x S. megistacrolobum have some characters in common, which can be interpreted as having a similar origin.It is postulated that S. acaule subsp. aemulans, in Jujuy at any rate, is not a primitive form of S. acaule as thought by Hawkes and Hjerting, but rather a fertile hybrid derivative of S. acaule x S. megistacrolobum through the functioning of 2n gametes.We also provide evidence that S. bruecheri Correll should not be considered a hybrid of S. acaulle x S. megistacrolobum but a synonym for S. gourlayi Hawkes. The new name, S. x indunii Okada et Clausen, is proposed to designate this hybrid.     

Identification of Resistance Genes Against Powdery Mildew in Four Accessions of Hordeum Vulgare SSP. Spontaneum

Summary Four newly detected accessions of wild barley (Hordeum vulgare ssp. spontaneum) resistant to powdery mildew caused by Blumeria graminis f. sp. hordei were studied with the aim of finding the number of genes/loci conferring the resistance of individual accessions, the type of inheritance of the genes and their relationships to the Mla locus. F₂ populations after crosses between the winter variety ‘Tiffany’ and four wild barley accessions and use of microsatellite DNA markers were focused on the identification of individual resistance genes/loci by means of their chromosomal locations. In PI466495, one locus conferring powdery mildew resistance was identified in highly significant linkage with the marker Bmac0213. This location is consistent with the known locus Mla on chromosome 1HS. In the other three accessions the resistance was determined by two independent loci. In PI466197, PI466297 and PI466461, one locus was identified on chromosome 1HS and three new loci were revealed on chromosomes 2HS (highly significant linkage with Bmac0134), 7HS (highly significant linkage with Bmag0021) and 7HL (significant linkage with EBmac0755). Our prospective aim is identification of further linked DNA markers and the exact location of the resistance genes on the barley chromosomes.     

Diversity in selected wild and cultivated species of pigeonpea using RFLP of mtDNA

Diversity in 28 accessions representing 12 species of the genus, Cajanus arranged in 6 sections including 5 accessions of the cultivated species, C. cajan, and 4 species of the genus Rhyncosia available in the germplasm collection at ICRISAT was assessed using RFLP with maize mtDNA probes. Cluster analysis of the Southern blot hybridization data with 3 restriction enzymes – 3 probe combinations placed the genus Rhyncosia in a major group well separated from all the species belonging to the genus Cajanus. Within the genus Cajanus, the 4 accessions of C. platycarpus belonging to section Rhynchosoides formed a separate group in contrast to those in other sections of pigeonpea. In the section, Cajanus all the 5 accessions of C. cajan were grouped together and C. cajanifolius belonging to the same section was in a subgroup by itself closer to the main group. The four accessions of C. scarabaeoides, were together and the other species belonging to section Cantharospermum were in different subgroups. The intra-specific variation was seen even within accessions of certain pigeonpea wild species such as C. scarabaeoides, C. platycarpus, C. acutifolius, and even the cultivated species of C. cajan. This study suggests that RFLP of mtDNA can be used for the diversity analysis of pigeonpea and it gives some indications on the maternal lineage among the species. The variations in the mitochondrial DNA hybridization patterns also suggest the extensive rearrangement of the organelle genome among the Cajanus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resistance to late blight (Phytophthora infestans) in ten wild Solanum species

Nineteen accessions of the tuber-bearing species Solanum berthaultii, S. chacoense, S. leptophyes, S. microdontum, S. sparsipilum, S. sucrense, S. venturii, S. vernei and S. verrucosum were tested for their resistance to late blight in two years of field experiments. Plants were artifically inoculated with zoospores of race 1.2.3.4.5.7.10.11 and the development of the disease was followed. Resistance ratings, calculated as the areas under the disease progress curves (ADPC), demonstrated a high resistance in all accessions except in S. sparsipilum, S. leptophyes and their interspecific hybrid. Segregations suggest that major genes for resistance are present in S. sucrense and S. venturii, and may also play a role in S. verrucosum. It is not yet certain wether the resistance of the other accessions is comparable to the partial and durable resistance of S. tuberosum cultivars like Pimpernel, as inheritance and mechanism have yet to be established. However, segregations suggesting the presence of single major genes with complete dominance were not found in these other accessions. Tuber initiation in the field occurred in only one accession, S. tuberosum ssp. andigena, and maturity of the clones was not related to their resistance. In the other accessions maturity types could not be assessed, as the clones require short day conditions for tuber initiation.     

Reaction of wild species of the geneus Lens to drought

Summary Susceptibility to drought stress is a key factor in dry land lentil (Lens culinaris Medikus) production in the Mediterranean region of West Asia and North Africa. This study examined the response to drought stress of 121 accessions representing all subspecies of the genus Lens; cultivated, and the wild L. culinaris ssp. orientalis (Boiss.) Ponert, L. culinaris ssp. odemensis (Ladiz.), L. nigricans M.B. Godr. ssp. nigricans Godr. and L. nigricans ssp. ervoides (Brign.) Ladiz. for their potential use in breeding for dry land conditions. Accessions were grown under two moisture regimes (dry land and dry land plus supplemental irrigation) at Breda, Syria during the 1990–91 and 1991–92 seasons. The cultivated lentil had markedly superior seed and straw production than did the wild Lens species. Time to flowering accounted for less than 10% of the variation in yield of wild accessions under rainfed conditions in the two seasons, showing that, in contrast to the cultivated germplasm, drought escape was relatively unimportant in wild lentil. Performance under drought in wild lentil, measured in terms of dry land seed yield or drought susceptibility index (S), was randomly distributed among collection locations with little relation to collection site aridity. Direct selection of wild lentil germplasm for biomass yield under dry conditions is of little value and an evaluation of wild accessions in hybrid combination is needed.     

Maternal inheritance of cytoplasmic organelles in intergeneric hybrids of Carica papaya L. and Vasconcellea spp. (Caricaceae Dumort., Brassicales)

The mode of inheritance of chloroplast and mitochondrial DNA has been determined in intergeneric hybrids between C. papaya and four different Vasconcellea species by employing a restriction fragment length polymorphism analysis of a PCR-amplified chloroplast and mitochondrial DNA region. Artificial F₁ hybrids were produced between a female specimen of C. papaya and male specimens of either V. parviflora, V. goudotiana, V. cundinamarcensis or V. quercifolia. The hybridization patterns of all hybrids correspond in all cases with that of the C. papaya mother, and are different from that of the paternal Vasconcellea species, thus indicating the maternal inheritance of cpDNA and mtDNA in intergeneric hybrids between C. papaya and wild relatives of the Vasconcellea genus.     

Analysis of bulked and redundant accessions of Brassica germplasm using assignment tests of microsatellite markers

This study was conducted to determine if Brassica germplasm bulks created and maintained by the USDA-ARS North Central Plant Introduction Station (NCRPIS) were made with genetically indistinguishable component accessions and to examine newly identified putative duplicate accessions to determine if they can be bulked. Using ten microsatellite primer pairs, we genotyped two bulks of B. rapa L. ssp. dichotoma (Roxb.) Hanelt comprising four accessions and three bulks of B. rapa L. ssp. trilocularis (Roxb.) Hanelt comprising fourteen accessions, as well as four pairs of putatively duplicate accessions of B.␣napus L. Assignment tests on ten individual plants per accession were conducted using a model-based clustering method to arrive at probabilities of likelihood of accession assignment. The assignment tests indicated that one of the two bulks of B. rapa ssp. dichotoma involves genetically heterogeneous accessions. It was observed in the B. rapa ssp. trilocularis bulks that the component accessions could be differentiated into groups, with misassignments observed most frequent within groups. In B. napus, only one of the four pairs of putative duplicates showed significant genetic differentiation. The other three pairs of putative duplicates lack differences and support the creation of bulks. The results of the assignment tests were in agreement with cluster analyses and tests of population differentiation. Implications of these results in terms of germplasm management include the maintenance and/or re-creation of some Brassica germplasm bulks by excluding those accessions identified as being unique in this study.     

Variation in the ribosomal RNA units of the generaLens andCicer

Summary Investigation of the organization of the tandemly repeated 5S and 18–25S ribosomal RNA (rRNA) genes was carried out on members of the generaLens andCicer using restriction endonuclease digestion and Southern hybridization. The 5S rRNA unit (gene+spacer) inLens (0.38 to 0.50 kb) andCicer 0.50 to 0.44 kb) is the largest among the legumes. InL. nigricans andL. culinaris ssp.odemensis a second repeat size was detected. Restriction maps for the 18–25S units ofLens andCicer species were developed. The 18–25S rRNA unit varied from 8.3 to 9.8 kb inLens and 10.5 to 11.4 kb inCicer. The only detectable variability in theLens andCicer 18–25S rRNA unit was in the length of the intergenic spacer (IGS) region, except for a HindIII site in the IGS region ofL. nigricans ssp.nigricans and in allCicer species.Similarities in the size of both 5S and 18–25S rRNA units were noted betweenL. culinaris ssp.culinaris andL. culinaris ssp.orientalis, which supports previous evidence thatL. culinaris ssp.orientalis is the progenitor of the cultivated lentil. Differences in the arrangement of both 5S and 18–25S rRNA units were noted betweenL. nigricans ssp.nigricans andL. nigricans ssp.ervoides, indicating either divergent evolution of these subspecies or alternatively incorrect taxonomy.On the basis of the size of the 5S rRNA unit,C. bijugum, C. chorassanicum andC. echinospermum formed a group with a slightly smaller unit than the otherCicer species. Similarities in the length of both 5S and 18–25S rRNA units ofC. arietinum andC. reticulatum possibly confirmC. reticulatum as the progenitor ofC. arietinum. Cicer cuneatum has a smaller 18–25S rRNA unit than the otherCicer species as a result of a smaller intergenic spacer.     

Agronomical performance and breeding value of selected strains of diploid wheat,Triticum monococcum

Summary Twenty-one germplasm accessions and breeding lines of einkorn wheat (Triticum monococcum ssp. monococcum and ssp. sinskajae) were grown at two sites in Italy and evaluated for various field and seed characteristics. Grain yields of germplasm accessions were relatively high (317–3238 kg/ha), but distinctly lower than those of four modern cultivars of tetraploid (T. turgidum ssp. durum) and hexaploid wheat (T. aestivum ssp. Aestivum) included in the experiments as controls. As expected, all Einkorns-including some substantially higher yielding crossbred lines (3415–4362 kg/ha)-were defective for one or more agronomically relevant features. However, a few of the accessions examined were found to contain, as a group, practically all the genes needed to breed monococcums having the main field attributes of a modern wheat cultivar: high yielding capacity, good threshability, large kernel size, earliness, short stature and adequate lodging resistance. Still higher yielding diploid wheats, more responsive to improved growing conditions and of better seed quality, could probably be obtained from crosses with wild monococcums bearing mostly two-seeded florets and with accessions producing less slender-shaped kernels. Some of the Einkorns examined were found to carry minor genes for easy threshing which might enhance the efficacy of the major gene for soft glumes carried by T. monococcum ssp. sinskajae, a partially free-threshing diploid wheat taxon. Seed protein percentage of monococcums was markedly higher than that of durum and bread wheat cultivars even in those cases where their grain yields surpassed those of the polyploid checks. The possibilities offered by diploid wheat in the exploitation of novel endosperm mutants and F₁ hybrid vigour, as well as in the fields of celiac disease, crop diversification and resistance to agro-biological stresses are discussed. Breeding priorities and strategies are also proposed.     

Tetrahaploids from colchicine-induced octaploid Solanum acaule subsp. Aemulans

Summary Crosses between colchicine-induced octaploid Solanum acaule ssp. aemulans (2n=8x =96) with diploid S. tuberosum, Group Phureja, carrying a homozygous embryo marker, produced a high percentage of S. acaule tetrahaploids besides pentaploid and hexaploid F₁ hybrids. These results are discussed in relation to the behaviour of autopolyploid and allopolyploid Solanum species, which basically differ in the occurrence of haploids and the ploidy level of F₁ hybrids after pollination of both types of poly-ploids with Phureja.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.     

The nature of the Bolivian weed potato species Solanum sucrense Hawkes

Summary Two theories for the origin of the Bolivian weed potato species Solanum sucrense Hawkes have been tested. The first was that it had an allopolyploid origin as a hybrid of the cultivated tetraploid S. tuberosum ssp. andigena (Juz. et Buk.) Hawkes with the wild/weed tetraploid cytotype of S. oplocense Hawkes. The second hypothesis postulated that it was a cross of the weed diploid species S. sparsipilum (Bitt.) Juz. et Buk. with S. oplocense.Synthetic hybrids of S. tuberosum ssp. andigena x S. oplocense and of S. sparsipilum x S. oplocense have been compared morphologically with S. sucrense accessions. Their crossability has also been investigated.The crossability and morphological studies strongly suggested that the former hypothesis was the more likely. This was borne out by observations made on the 1974 University of Birmingham Expedition when segregating populations of S. sucrense and S. oplocense were collected growing with feral S. tuberosum ssp. andigena.The evidence from this study indicates that the S. tuberosum ssp. andigena x S. oplocense hybrid has formed the basis of the S. sucrense gene pool. It is suggested that this hybridogenic taxon be maintained under the name Solanum x sucrense.The clarification of the taxonomic relationships of these three species will be of interest to Globodera resistance breeders, in view of the fact that these taxa are resistant to various pathotypes of the golden nematode.     

Breeding of the cultivated potato species Solanum x juzepczukii Buk. and Solanum x curtilobum Juz. etBuk.

Summary This study investigated the possibility of recombining anew the genomes of the wild and cultivated progenitors of triploid S. juzepczukii and pentaploid S. curtilobum by following the known evolutionary pathway of these two species. Before starting the actual breeding work, the natural variation of S. juzepczukii, S. curtilobum and their wild progenitor S. acaule was studied from the point of view of morphology, quantitative and qualitative tuber glycoalkaloid content and frost resistance. The morphological study was supplemented by a study of the soluble tuber proteins employing polyacrylamide slab-electrophoresis. From 137 accession of S. juzepczukii only 19 morphotypes were identified, 18 of which were also different in their protein spectra. The only red-tubered S. juzepczukii revealed a protein spectrum identical to that of the largest white-tubered group. On phylogenetic grounds, the occurrence of a red-tubered S. juzepczukii cannot be explained. It is concluded that this red clone is a somatic mutant for tuber colour which arose from a whitetubered clone. S. curtilobum was restricted in its variation to just two morphotypes differing only in tuber colour which are, however, identical chemotypes. This would be the case if one of the clones was a somatic mutant for tuber colour from the other one. The glycoalkaloids -solanine, -chaconine, tomatine, demissine and - and -solamarine are shown to be useful taxonomic characters which confirm earlier hypotheses on the origin of S. juzepczukii and S. curtilobum. Laboratory tests showed the two cultivated species to be resistant to about –3°C whereas S. acaule is resistant to temperatures sometimes below–5°C. The diploid progenitor of S. juzepczukii, S. stenotomum, also has forms resistant to –3°C. The results of this study demonstrate that the proposed breeding scheme is possible.