Molecular Mapping and Character Tagging in Brassica juncea—I. Degree,Nature and Linkage Relationship of RFLPs and Their Association With Quantitative Traits

For molecular mapping and character tagging in Indian mustard (Brassica juncea), cultivar Varuna and exotic collection BEC 144 were chosen on the basis of morphological and molecular differences. High degree of RFLP was detected between them using genomic DNA clones of Pst! subgenomic library as probes. Of the 48 probe — enzyme combinations, 89.5% were polymorphic. Majority of the probes revealed duplicate loci and a high frequency of null alleles (71.4%). Segregation analysis in the F² population revealed significant deviation from expected 1:2:1 ratio for 32% of the markers. Using the computer package MAPMAKER, 15 markers could be placed in six linkage groups which covered a total length of 173.9 eM. Based on single factor analysis of variance, three significant marker-quantitative trait associations viz. BJG 59a primary branches/plant, BJG 42Gb - secondary branches/plant and BJG 433-days to f1owering could be identified. The BEC 144 alleles at the quantitative trait loci (QTL) in the marked genomic regions enhanced the trait expression. The putative gene action at these loci was found to be non-additive.     

Molecular Markers for Seed Oil Content in Indian Mustard

With the objective of identifying molecular markers for the seed oil content in mustard (Brassica juncea), which is known to be quantitatively inherited, 94 F₆ generation recombinant inbred lines (RILs) developed from an intervarietal cross (Varuna × BEC144), were characterised. Oil content, determined by pulsed nuclear magnetic resonance technique, varied from 25.7% to 52.3% among the RILs. The frequency distribution of the trait was normal with mean 39.24 and standard deviation 4.52. Bulked segregant analysis of the RILs enabled fast and efficient screening of 219 random decamer primers for identification of informative primers. Single factor analysis of variance revealed significant association of oil content with three RAPD markers, viz., OPH11₁₃₅₀, OPJ06₁₂₀₀ and OPL05₆₀₀ which individually explained 6% of the trait variance. Molecular data indicated dispersion of desirable quantitative trait loci in the two parent varieties.     

Development and validation of CAPS and AFLP markers for white rust resistance gene in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

White rust, caused by Albugo candida, is a very serious disease in crucifers. In Indian mustard (Brassica juncea), it can cause a yield loss to the extent of 89.9%. The locus Ac2(t) controlling resistance to white rust in BEC-144, an exotic accession of mustard, was mapped using RAPD markers. In the present study, we developed: (1) a more tightly linked marker for the white rust resistance gene, using AFLP in conjunction with bulk segregant analysis, and (2) a PCR-based cleaved amplified polymorphic sequence (CAPS) marker for the closely linked RAPD marker, OPB06₁₀₀₀. The data obtained on 94 RILs revealed that the CAPS marker for OPB06₁₀₀₀ and the AFLP marker E-ACC/M-CAA₃₅₀ flank the Ac2(t) gene at 3.8 cM and 6.7 cM, respectively. Validation of the CAPS marker in two different F₂ populations of crosses Varuna × BEC-144 and Varuna × BEC-286 was also undertaken, which established its utility in marker-assisted selection (MAS) for white rust resistance. The use of both flanking markers in MAS would allow only 0.25% misclassification and thus provide greater efficiency to selection.Communicated by C. Möllers     

Mapping of yield influencing QTL in <Emphasis Type="Italic">Brassica juncea</Emphasis>: implications for breeding of a major oilseed crop of dryland areas

Quantitative trait loci (QTL) analysis of yield influencing traits was carried out in Brassica juncea (AABB) using a doubled haploid (DH) mapping population of 123 lines derived from a cross between Varuna (a line representing the Indian gene pool) and Heera (representing the east European gene pool) to identify potentially useful alleles from both the parents. The existing AFLP based map of B. juncea was further saturated with RFLP and SSR markers which led to the identification of the linkage groups belonging to the A (B. rapa) and B (B. nigra) genome components of B. juncea. For QTL dissection, the DH lines were evaluated at three different environments and phenotyped for 12 quantitative traits. A total of 65 QTL spread over 13 linkage groups (LG) were identified from the three environments. QTL analysis showed that the A genome has contributed more than the B genome to productivity (68% of the total QTL detected) suggesting a more prominent role of the A genome towards domestication of this crop. The east European line, Heera, carried favorable alleles for 42% of the detected QTL and the remaining 58% were in the Indian gene pool line, Varuna. We observed clustering of major QTL in a few linkage groups, particularly in J7 and J10 of the A genome, with QTL of different traits having agronomically antagonistic allelic effects co-mapping to the same genetic interval. QTL analysis also identified some well-separated QTL which could be readily transferred between the two pools. Based on the QTL analysis, we propose that improvement in yield could be achieved more readily by heterosis breeding rather than by pure line breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.     

Mapping of QTLs for oil content and fatty acid composition in Indian mustard [Brassica juncea (L.) Czern. and Coss.]

An AFLP linkage map of Brassica juncea (L.) Czern and Coss was constructed using 88 recombinant inbred lines (RILs) from a cross between an Indian cultivar ‘Varuna’ and an accession from Poland ‘BEC-144’. The map included 91 AFLP markers organized on 19 linkage groups covering a total map distance of 1679.1 cM. A total of 14 QTLs were detected for oil content (2 QTLs), erucic acid (2 QTLs), eicosenoic acid (2 QTLs), linolenic acid (3 QTLs), linoleic acid (3 QTLs) and palmitic acid (2 QTLs). A specific genomic region on LG2 was associated with contents of three fatty acids: erucic acid, eicosenoic acid and linoleic acid. Some of the markers showed absolute linkage with the QTLs associated with the levels of linolenic acid, linoleic acid and oil content. These markers may be used for improvement of fatty acid profile of B. juncea.     

Molecular mapping reveals two independent loci conferring resistance to Albugo candida in the east European germplasm of oilseed mustard Brassica juncea

White rust caused by Albugo candida (Pers.) Kuntze is a major disease of the oilseed mustard Brassica juncea. Almost all the released varieties of B. juncea in India are highly susceptible to the disease. This causes major yield losses. Hence, there is an urgent need to identify genes for resistance to white rust and transfer these to the existing commercial varieties through marker-assisted breeding. While the germplasm belonging to the Indian gene pool is highly susceptible to the disease, the east European germplasm of B. juncea is highly resistant. In the present study, we have tagged two independent loci governing resistance to A. candida race 2V in two east European lines, Heera and Donskaja-IV. Two doubled haploid populations were used; the first population was derived from a cross between Varuna (susceptible Indian type) and Heera (partially resistant east European line) and the second from a cross between TM-4 (susceptible Indian type) and Donskaja-IV (fully resistant east European line). In both the resistant lines, a single major locus was identified to confer resistance to white rust. In Heera, the resistance locus AcB1-A4.1 was mapped to linkage group A4, while in Donskaja-IV, the resistant locus AcB1-A5.1 was mapped to linkage group A5. In both the cases, closely linked flanking markers were developed based on synteny between Arabidopsis and B. juncea. These flanking markers will assist introgression of resistance-conferring loci in the susceptible varieties.     

Detection and Mapping of Duplicate Loci in Brassica juncea

Restriction fragment length polymorphism (RFLP) analysis of Brassica juncea genome using 39 random homologous genomic DNA clones and chlorophyll a, b binding polypeptide (cab-3c) cDNA of tomato as probes revealed high degree of sequence duplication. The average number of hybridizing fragments per probe (8) was much higher than that earlier reported using cDNA probes in B. juncea. Null alleles observed for majority (56.2%) of the polymorphic duplicate loci suggested a significant role of insertion/deletion events in evolution of mustard genome. Distortion in segregation was evident in respect of only 9.6% of the segregating loci indicating that the mapping population used was relatively unbiased and thus can be used efficiently for genome mapping as well as for location of genes. Forty-nine polymorphic duplicate loci could be mapped to 15 linkage groups. Arrangement of these loci on different linkage groups revealed intra and inter-chromosomal duplications as well as duplication of chromosome blocks.Three of the eight cab loci could be mapped on three different linkage groups. Null allelic situation for seven of the cab loci suggested the role of DNA rearrangement in evolution of this multigene family in B. juncea.     

Effect of Indian mustard on feeding,larval survival and development of Plutella xylostella at constant temperatures

Feeding behavior of Plutella xylostella under optional to non-optional conditions was studied at 10°C, 15°C, 20°C, and 25°C on Indian mustard, Brassica juncea. The study reveals that the variety Pusa Bahar was significantly less preferred by the larvae as compared with Pusa Bold and Varuna under optional to non-optional conditions. Larvae of P. xylostella consumed more food at 25°C than 20°C, 15°C and 10°C. Larval survival was found to be highest on cabbage (control) as compared with Indian mustard and was found to vary with host plants and temperature. The larval survival decreased to 11.29% on Pusa Bahar at 10°C. Increasing the temperature from 10°C to 20°C, larval mortality resulted more on Varuna than Pusa Bahar and Pusa Bold. Developmental period was prolonged on Pusa Bold at 10°C while it was shortest on cabbage at 25°C. A total of 536.47 degree days were required to complete the development by immature stages on Varuna at 25°C and 421.64 degree days on cabbage.     

Genetic Positioning of Centromeres through Half-Tetrad Analysis in Gynogenetic Diploid Families of the Zhikong Scallop (Chlamys farreri)

Centromere mapping is a powerful tool for improving linkage maps, investigating crossover events, and understanding chiasma interference during meiosis. Ninety microsatellite markers selected across all linkage groups (LGs) from a previous Chlamys farreri genetic map were studied in three artificially induced meiogynogenetic families for centromere mapping by half-tetrad analysis. Inheritance analyses showed that all 90 microsatellite loci conformed to Mendelian inheritance in the control crosses, while 4.4 % of the microsatellite loci showed segregation departures from an expected 1:1 ratio of two homozygote classes in meiogynogenetic progeny. The second division segregation frequency (y) of the microsatellites ranged from 0.033 to 0.778 with a mean of 0.332, confirming the occurrence of partial chiasma interference in this species. Heterogeneity of y is observed in one of 42 cases in which markers were typed in more than one family, suggesting variation in gene–centromere recombination among families. Centromere location was mostly in accordance with the C. farreri karyotype, but differences in marker order between linkage and centromere maps occurred. Overall, this study makes the genetic linkage map a more complete and informative tool for genomic studies and it will also facilitate future research of the structure and function of the scallop centromeres.     

Development and characterization of <Emphasis Type="Italic">Brassica juncea – fruticulosa</Emphasis> introgression lines exhibiting resistance to mustard aphid (<Emphasis Type="Italic">Lipaphis erysimi</Emphasis> Kalt)

Background

Mustard aphid is a major pest of Brassica oilseeds. No source for aphid resistance is presently available in Brassica juncea . A wild crucifer, Brassica fruticulosa is known to be resistant to mustard aphid. An artificially synthesized amphiploid, AD-4 (B. fruticulosa × B. rapa var. brown sarson) was developed for use as a bridge species to transfer fruticulosa resistance to B. juncea. Using the selfed backcross we could select a large number of lines with resistance to mustard aphid. This paper reports cytogenetic stability of introgression lines, molecular evidence for alien introgression and their reaction to mustard aphid infestation.

Results

Majority of introgression lines had expected euploid chromosome number(2n= 36), showed normal meiosis and high pollen grain fertility. Well-distributed and transferable simple-sequence repeats (SSR) markers for all the 18 B. juncea chromosomes helped to characterize introgression events. Average proportions of recipient and donor genome in the substitution lines were 49.72 and 35.06%, respectively. Minimum alien parent genome presence (27.29%) was observed in the introgression line, Ad3K-280 . Introgressed genotypes also varied for their resistance responses to mustard aphid infestations under artificial release conditions for two continuous seasons. Some of the test genotypes showed consistent resistant reaction.

Conclusions

B.juncea-fruticulosa introgression set may prove to be a very powerful breeding tool for aphid resistance related QTL/gene discovery and fine mapping of the desired genes/QTLs to facilitate marker assisted transfer of identified gene(s) for mustard aphid resistance in the background of commercial mustard genotypes.

     

Eliminating expression of erucic acid-encoding loci allows the identification of “hidden” QTL contributing to oil quality fractions and oil content in <Emphasis Type="Italic">Brassica juncea</Emphasis> (Indian mustard)

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2), fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.     

Development of InDel markers for Brassica rapa based on whole-genome re-sequencing

Genome-wide detection of short insertion/deletion length polymorphisms (InDels, <5 bp) in Brassica rapa (named the A genome) was performed by comparing whole-genome re-sequencing data from two B. rapa accessions, L144 and Z16, to the reference genome sequence of Chiifu-401-42. In total, we identified 108,558 InDel polymorphisms between Chiifu-401-42 and L144, 26,795 InDels between Z16 and Chiifu-401-42, and 26,693 InDels between L144 and Z16. From these, 639 InDel polymorphisms of 3–5 bp in length between L144 and Z16 were selected for experimental validation; 491 (77 %) yielded single PCR fragments and showed polymorphisms, 7 (1 %) did not amplify a product, and 141 (22 %) showed no polymorphism. For further validation of these intra-specific InDel polymorphisms, 503 candidates, randomly selected from the 639 InDels, were screened across seven accessions representing different B. rapa cultivar groups. Of these assayed markers, 387 (77 %) were polymorphic, 111 (22 %) were not polymorphic and 5 (1 %) did not amplify a PCR product. Furthermore, we randomly selected 518 InDel markers to validate their polymorphism in B. napus (the AC genome) and B. juncea (the AB genome), of which more than 90 % amplified a PCR product; 132 (25 %) showed polymorphism between the two B. napus accessions and 41 (8 %) between the two B. juncea accessions. This set of novel PCR-based InDel markers will be a valuable resource for genetic studies and breeding programs in B. rapa.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.     

Segregation and Linkage Analysis of 75 Novel Microsatellite DNA Markers in Pair Crosses of Japanese Abalone (Haliotis discus hannai) Using the 5′-Tailed Primer Method

We present novel microsatellite markers of the Japanese abalone (Haliotis discus hannai) for general mapping studies in this species. A total of 75 microsatellite markers were developed, and the allele-transmission patterns of these markers were studied in three families generated by pair crosses. For allele scoring, we employed the 5′-tailed primer polymerase chain reaction (PCR) technique, which substantially reduces the cost for fluorescent labeling of primers. Of the 225 possible marker-family combinations (75 markers × 3 families), 18 cases of informative null-allele segregation were inferred. When such null-allele segregations were allowed, more than 70% of the 75 markers in the families turned out to be markers with an expected segregation ratio of 1:1:1:1, allowing maximal exploitation of the codominant nature of microsatellite markers. There were 16 instances of segregation distortion at the 5% significance level. The test for independence of segregation assigned the 75 markers into 17 linkage groups, which is in close agreement with the haploid chromosome number of H. discus hannai (n = 18). Six markers could not be placed into any linkage group. We suggest that these markers could help construct a H. discus hannai linkage map.     

Effect of K application on leaf carbonic anhydrase and nitrate reductase activities, photosynthetic characteristics, NPK and NO3 contents, growth, and yield of mustard

In a sand culture experiment on mustard (Brassica juncea L. Czern. & Coss) cv. Varuna, all tested characteristics at 60 d stage and yield characteristics at harvest were enhanced by K application as its levels increased from 5 to 10, 15, 20, 25, and 30 mM K, with 20 mM K proving best.