Identification of RAPD markers linked to the Ns locus in potato

Using the RAPD method and bulked segregant analysis we identified four RAPD markers linked to a dominant gene Ns, responsible for a hypersensitive reaction of potato (Solanum tuberosum L.) to potato virus S (PVS) infection. The markers OPE15⁵⁵⁰, OPJ13⁵⁰⁰, OPG17⁴⁵⁰ and OPH19⁹⁰⁰ were found to be closely linked to the Ns gene in diploid potato clones. They are situated at 2.6, 3.3, 4.6 and 6.6 cM from Ns, respectively. As a source of the gene, clone G-LKS 678147/60, which is known to carry Ns transferred from S. tuberosum ssp. andigena was used. These RAPD markers were not amplified in resistant tetraploid clones containing Ns derived from the clone MPl65 118/3, also having an andigenum origin. This suggests that there may be two separate loci of Ns in the sources identified, or different alleles with the same specificity at a single locus, or that the genetic background of tetraploids tested results in different RAPD amphlification patterns.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

Rapid and convenient gel‐free screening of SCAR markers in wheat using SYBR green‐based melt‐profiling

Sequence characterized amplified region (SCAR) markers that are highly desirable in crop breeding for marker‐assisted selection (MAS) are routinely analysed by gel‐based methods that are low‐throughput, time‐consuming and laborious. In this study, we showed a rapid and convenient method for analysis of SCAR markers in a gel‐free manner. Seven SCAR markers, linked to rust resistance genes (Sr24, Sr26 and Sr31) and seed quality traits (Pina, Pinb and Glu‐D1) in wheat (Triticum aestivum), were amplified on a real‐time PCR machine using custom reaction mixture. Subsequently, melting curve analysis was performed, to assess the specificity of amplicons. Using the amplicon‐specific melt‐profiles, the presence/absence of SCAR markers was analysed in fifteen genotypes and five F₂ populations. Unlike the fluorescence‐based in‐tube detection methods, the present method used the amplicon‐specific melt‐profiles to evaluate the status of the SCAR markers, thus eliminating the need for gel‐based analysis. Results also showed feasibility of multiplex analysis of two markers with well‐separated melting profiles. Overall, the approach is a rapid, convenient and cost‐effective method for high‐throughput screening of SCAR markers.     

Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheat

The stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non‐carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP‐PCR). AP‐PCR markers viz. SS30.2_580(H) associated with the Sr31 gene and SS26.1₁₁₀₀ associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F₂ population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP‐PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2₅₇₆ and SCSS26.1₁₁₀₀ respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F₂ generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker‐assisted selection.     

Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene <Emphasis Type="Italic">mdm1 (t)</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Maize dwarf mosaic is one of the devastating and wide spread viral diseases in the world. The present investigation was carried out to develop DNA markers closely linked to the resistance gene mdm1 (t). Linkage between the markers and phenotypes was confirmed by analyzing an F₂ population obtained from a cross between a resistant parent ‘Huangzaosi’ and a susceptible parent ‘Mo17(478)’. Four AFLP markers were found in the maize dwarf mosaic resistant plants. By using (BSA) bulked segregant analysis, two of the four AFLP markers were transformed into Sequence-characterized amplified regions markers (SCARs), nominated Rsun-1 and Rsun-2. The two amplified fragment length polymorphism (AFLP) markers, RHC-1and RHC-2, from the amplification products of primer combination E-AGC/M-CAA and E-AGC/M-GAA, showed linkage with the mdm1 (t) gene in a genetic distance 1.6 and 2.0 cM, respectively. The results indicate that the new SCAR markers will be valuable to distinguish resistant plants from susceptible plants in plantlets growing in seedbeds. The markers developed in this study are suitable for marker-assisted selection for maize dwarf mosaic resistance.     

ISSR and SCAR markers linked to the mungbean yellow mosaic virus (MYMV) resistance gene in blackgram [Vigna mungo (L.) Hepper]

A recombinant inbred line (RIL) mapping population (F₈) was generated by crossing Vigna mungo (cv. TU 94‐2) with Vigna mungo var. silvestris and screened for mungbean yellow mosaic virus (MYMV) resistance. The inter simple sequence repeat (ISSR) marker technique was employed to identify markers linked to the MYMV resistance gene. Of the 100 primers screened, 54 showed amplification of which 36 exhibited polymorphism between the parents TU 94‐2 (resistant) and V. mungo var. silvestris (susceptible). Individual plants from 53 RIL populations were analysed and one marker (ISSR811₁₃₅₇) was identified as tightly linked to the MYMV resistant gene at 6.8 cM. Both the phenotype as well as the ISSR811₁₃₅₇ marker segregated in a 1 : 1 ratio. The ISSR811₁₃₅₇ marker was sequenced and sequence characterized amplified region (SCAR) primers were designed (YMV1‐F and YMV1‐R) to amplify the marker. Screening for the SCAR marker in the RIL population distinguished the MYMV resistant and susceptible plants, agreeing well with the phenotypic data. The ISSR811₁₃₅₇ marker was validated using diverse blackgram genotypes differing in their MYMV reaction. The marker will be useful for the development of MYMV‐resistant genotypes in blackgram.     

Identification of molecular markers associated with sweet potato resistance to sweet potato virus disease in Kenya

Sweet potato virus disease (SPVD), a result of the co-infection of whitefly transmitted Sweet potato chlorotic stunt virus (genus Crinivirus, family Closteroviridae) and the aphid transmitted Sweet potato feathery mottle virus (genus Potyvirus, family Potyviridae), is the most destructive disease of sweet potato in East Africa. A study was conducted to establish if genotypes identified as resistant or susceptible to SPVD in Kenya could be distinguished using molecular markers. A total of 47 unrelated sweet potato genotypes were selected from germplasm collections and classified into two phenotypic groups as resistant or susceptible to SPVD. Genotype selection was based on disease severity or days to symptom development in plants following graft inoculation. Amplified fragment length polymorphism (AFLP) marker profiles were generated for each individual and used in association studies to identify markers suitable for classifying the two pre-defined phenotypic groups. Analysis of molecular variance showed significant (P < 0.002) variation between the two groups using 206 polymorphic AFLP markers. Discriminant analysis and logistic regression statistical methods were used to select informative markers, and to develop models that would classify the two phenotypic groups. A training set of 30 genotypes consisting of 15 resistant and 15 susceptible were used to develop classification models. The remaining 17 genotypes were used as a test set. Four markers, which gave 100% correct classification of the training set and 94% correct classification of the test set, were selected by both statistical methods.     

多重PCR技术鉴定番茄Ty-1和Mi基因

利用同一PCR反应体系,对分别与番茄(Lycopersion esculentum)抗黄化曲叶病毒(Tomato YellowLeaf Curl virus)病的Ty-1基因和番茄抗根结线虫(root-knot nematode)的Mi基因紧密连锁的SCAR标记进行同时扩增筛选,扩增的特异性片段与单引物扩增片段完全吻合.与Ty-1基因紧密连锁的SCAR1标记为共显性标记,抗感材料均产生398 bp的特异片段,纯合和杂合抗病基因型存在TaqⅠ酶切位点,酶切后分别产生了303 bp和95 bp以及398 bp、303 bp和95 bp的特异性片段,而感病基因型无此酶切位点.与Mi基因紧密连锁的SCAR2标记也为共显性标记,抗感材料均产生750 bp的特异片段,纯合和杂合抗病基因型存在TaqⅠ酶切位点,酶切后分别产生了570bp和180bp以及750bp、570bp和180bp的特异性片段,而感病基因型无此酶切位点.酶切结果仍为750 bp的产物.经反复验证,结果准确可靠,可以用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定.     

RAPD and SCAR markers for powdery mildew resistance gene er in pea

In pea, a single recessive gene (er) on linkage group 6 confers resistance to powdery mildew caused by Erysiphe pisi. The present study aims to identify molecular markers linked to the er gene. Screening of the powdery mildew‐resistant cultivar ‘DMR11’ and its susceptible nearisogenic line for polymorphism revealed linkage of two RAPD primers (OPO‐02 and OPU‐17) to the er gene and a sequence characterized polymorphic region (SCAR) primer, ScOPD‐10₆₅₀ with er in a population of 83 F₂ plants in the order: OPU‐17 ‐ er ‐ ScOPD‐10₆₅₀ ‐ OPO‐02. The markers ScOPD‐10₆₅₀ and OPU‐17 being coupled with the allele causing resistance would substantially increase the efficiency of marker‐assisted selection in peabreeding for powdery mildew.     

Novel molecular marker associated with Tm2a gene conferring resistance to tomato mosaic virus in tomato

Tomato mosaic virus (ToMV) is an important Tobamovirus that causes significant crop losses. Resistance to the ToMV is conferred by the genes Tm1, Tm2 and Tm2^a. Among these three genes, Tm2^a confers resistance to most strains of the ToMV. Screening of genetic lines under field conditions based on phenotype is time‐consuming and challenging due to concerns associated with stability of the virus and its potential transmission to other plants. Tightly linked molecular markers associated with resistance genes can improve selection efficiency and avoid these problems. This study developed a PCR‐based marker based on restriction site differences from Tm2^a locus‐specific sequences, which was found to be useful in identifying the resistant and susceptible genotypes and was consistent with phenotypic data. The marker is a codominant cleaved amplified polymorphic sequence (CAPS) marker producing 270‐ and 600‐bp DNA fragments from resistant genotypes and an 870‐bp fragment from susceptible genotypes when digested with HaeIII restriction enzyme. This novel marker can be useful for tomato breeders to screen progeny from segregating populations for ToMV resistance.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Development of molecular markers linked to the Leptosphaeria maculans resistance gene Rlm6 and inheritance of SCAR and CAPS markers in Brassica napus × Brassica juncea interspecific hybrids

Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F₂ and F₃ generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F₂ and F₃ progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F₂ plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme.     

Identification of a SCAR marker associated with Bm, the beet mosaic virus resistance gene, on chromosome 1 of sugar beet

Beet mosaic virus (BtMV) is an aphid transmitted, viral disease of beet found worldwide. The Bm gene, a resistance gene effective against BtMV, was identified in the sugar beet line 8500 and backcrossed into a C37 background to produce line C719. Three populations were developed from the cross of line C719 with the susceptible line C37 with the intent of developing markers for use in marker‐assisted selection. The F₂ progeny of three crosses were scored for resistance. Two of the three populations conformed to a 3 : 1 ratio, indicating a single gene trait. Sequence characterized amplified region (SCAR) markers were developed by using bulked segregant analysis combined with random amplified polymorphic DNA type markers. The markers showed close association to the Bm resistance gene and were effective in all three populations. The A₁ allele for genetic male sterility also was found to be associated with Bm and the SCAR marker. Development of a single‐nucleotide polymorphism marker from the SCAR sequence was used to validate linkage to chromosome 1 using separate mapping populations. This marker will be useful for the introgression of the Bm gene into germplasm.     

Development of a coupling-phase SCAR marker linked to the powdery mildew resistance gene ‘er1’ in pea (Pisum sativum L.)

Pea powdery mildew is one of the major constraints in pea production worldwide, causing severe seed yield and quality loss. The resistance is governed by a single recessive gene er1 in majority of resistant cultivars, but er2 and Er3 have also been reported. The objective of the study was to find out tightly linked sequence characterized amplified regions (SCAR) markers to er1 gene using NILs. A total of 620 random amplified polymorphic DNA (RAPD) markers were screened for length polymorphism between seven sets of NILs. The 880 bp polymorphic band of the tightly linked RAPD marker OPX 04₈₈₀ was cloned, sequenced and a SCAR marker ScOPX 04₈₈₀ was developed. In a population of completely classified 208 F₂ plants (supported by phenotypic data from 208 F_2:3 and 4,390 F_3:4 families) ScOPX 04₈₈₀ was linked at 0.6 cM in coupling phase with er1 gene in the order ScOPX 04₈₈₀–er1–ScOPD 10₆₅₀. ScOPX 04₈₈₀ will correctly differentiate homozygous resistant plants from the susceptible accessions with more than 99 % accuracy. In combination with repulsion phase marker ScOPD 10₆₅₀, ScOPX 04₈₈₀ can help in an error free marker-assisted selection.     

The Importance of the Bolivian Wild Potato Species in Breeding for Globodera pallida Resistance

Screening for resistance to the potato cyst nematode, Globodera pallida, in potatoes from. Bolivia, was carried out in 1983 and 1984, using a mixture of four nematode populations representing pathotypes Pa₁, Pa₂ and Pa₃ From the 66 accessions of 17 species and subspecies evaluated, highly resistant genotypes were identified in 21 accessions from seven species. All had Pf/Pi values of 2 or less, whereas the susceptible control, Solanum tuberosum cv. ‘Disiree’ had Pf/Pi values of more than 2G in both tests. Two diploid wild species, S. brevicaule and S. leptophyes, showed the best resistant. The geographical distributional of resistant populations and the evolution of resistance in wild potato populations are discussed.     

Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat

Powdery mildew is an important disease in most of the wheat production areas of the world. The resistance gene Pm21 (6AL/6VS trans-location) derived from Haynaldia villosa confers resistance to all available isolates of Erysiphe (Blumeria) graminis f. sp. tritici in China and Europe. The objective of this study was to develop fast and reliable sequence characterized amplified region (SCAR) markers linked to the Pm21 gene. A random amplified polymorphic DNA (RAPD) marker for Pm21, OPH17₁₄₀₀, was converted to SCAR markers after sequencing the two ends of the polymorphic DNA fragment. Two SCAR markers, SCAR₁₂₆₅ and SCAR₁₄₀₀, were developed to detect the Pm21 gene in different genetic backgrounds. The specific SCAR₁₂₆₅ marker enable large-scale accurate screening for the presence/absence of Pm21 allele.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Development of a SCAR marker linked with a MYMV resistance gene in mungbean (Vigna radiata L. Wilczek)

Yellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is the most important disease of mungbean, causing great yield loss. The present investigation was carried out to study the inheritance and identify molecular markers linked with MYMV resistance gene by using F₁, F₂ and 167 F_2 : 8 recombinant inbred lines (RILs) developed from the cross ‘TM‐99‐37’ (resistant) × Mulmarada (susceptible). The F₁ was susceptible, F₂ segregated in 3S:1R phenotypic ratio and RILs segregated in 1S:1R ratio in the field screening indicating that the MYMV resistance gene is governed by a single recessive gene. Of the 140 RAPD primers, 45 primers showing polymorphism in parents were screened using bulked segregant analysis. Three primers amplified specific polymorphic fragments viz. OPB‐07_600, OPC‐06₁₇₅₀ and OPB‐12₈₂₀. The marker OPB‐07₆₀₀ was more closely linked (6.8 cM) with a MYMV resistance gene as compared to OPC‐06₁₇₅₀ (22.8 cM) and OPB‐12₈₂₀ (25.2 cM). The resistance‐specific fragment OPB‐07₆₀₀ was cloned, sequenced and converted into a sequence‐characterized amplified region (SCAR) marker and validated in twenty genotypes with different genetic backgrounds.     

Development of RAPD and ISSR derived SCAR markers linked to <Emphasis Type="Italic">Xca1Bo</Emphasis> gene conferring resistance to black rot disease in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis> L.)

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) (Pam.) is the most devastating disease of cauliflower (Brassica oleracea var. botrytis L.; 2n = 2x = 18), taking a heavy toll of the crop. In this study, a random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) derived sequence characterized amplified region (SCAR) markers linked to the black rot resistance locus Xca1bo were developed and evaluated as a screening tool for resistance. The RAPD marker OPO-04₈₃₃ and ISSR marker ISSR-11₆₃₅ were identified as closely linked at 1.6 cM distance to the black rot resistance locus Xca1bo. Both the markers OPO-04₈₃₃ and ISSR-11₆₃₅ were cloned, sequenced and converted into SCAR markers and validated in 17 cauliflower breeding lines having different genetic backgrounds. These SCAR markers (ScOPO-04₈₃₃ and ScPKPS-11₆₃₅) amplified common locus and showed 100% accuracy in differentiating resistant and susceptible plants of cauliflower breeding lines. The SCAR markers ScOPO-04₈₃₃ and ScPKPS-11₆₃₅ are the first genetic markers found to be linked to the black rot resistance locus Xca1bo in cauliflower. These markers will be very useful in black rot resistance marker assisted breeding.