A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

Chloroplast substitution overcomes leaf chlorosis in a Moricandia arvensis-based cytoplasmic male sterile Brassica juncea

 A male sterile Brassica juncea line based on Moricandia arvensis cytoplasm was developed previously by backcrossing the somatic hybrid M. arvensis+B. juncea, and the gene for restoring fertility was introgressed. The CMS line is very severely chlorotic because of the presence of alien chloroplasts and flowering is delayed by 30–40 days, making it unsuitable for the exploitation of heterosis. We have resorted to another cycle of protoplast fusion between green fertile B. juncea and chlorotic male sterile B. juncea, and developed green male-sterile plants. Molecular analysis revealed that in green male-sterile plants chloroplasts of M. arvensis origin were substituted by those from B. juncea, giving rise to intergeneric cytoplasmic hybrids with mitochondria of M. arvensis origin. With the development of dark-green male-sterile plants, the CMS fertility restoration system is suitable for the production of hybrid mustard. Received: 23 February 1998 / Accepted: 12 May 1998     

Classification of rice germplasm: III. High-resolution fingerprinting of cytoplasmic genetic male-sterile (CMS) lines with AFLP

 The cytoplasmic genetic male-sterile (CMS) lines developed at the International Rice Research Institute are valuable in producing tropical rice hybrids. Efficient use of CMS lines in hybrid rice production will depend on their level of genetic diversity. Aside from morphological characterization, molecular analysis based on DNA markers can provide information on the genetic diversity of the germplasm. The Amplified Fragment Length Polymorphism (AFLP) technique was used to fingerprint 71 CMS lines and four rice cultivars, ‘IR64’, ‘Azucena’, ‘IR74’, and ‘FR13A’. Eleven primer pair combinations specific to the enzymes PstI and MseI were used to generate 530 AFLP markers, 176 of which were polymorphic. Each CMS line revealed a distinct fingerprint. The AFLP marker-based dendrogram depicted genetic variation among the CMS lines. The CMS lines developed in japonica background grouped with ‘Azucena’, a japonica cultivar. None of the CMS lines clustered with ‘FR13A’, a flood-tolerant traditional indica variety. ‘IR64’ was found to be distinct from the other indica CMS lines and clustered with lines developed in its background. The grouping of CMS lines into a few groups is useful for breeders in selecting genetically diverse CMS lines for hybrid rice production and in avoiding test crossing every CMS line empirically. This study demonstrated that AFLP is a powerful and reliable tool in determining the genetic relationships and in producing distinct fingerprints of rice cultivars. Received: 20 December 1996 / Accepted: 9 October 1997     

Somatic cell hybridization of ’oxy’ CMS Brassica juncea (AABB) with B. oleracea (CC) for correction of chlorosis and transfer of novel organelle combinations to allotetraploid brassicas

Alloplasmic lines of cultivated Brassica species with B. oxyrrhina cytoplasm are male-sterile and suffer from severe chlorosis. We developed male-sterile lines corrected for chlorosis by fusing protoplasts of CMS B. juncea (AABB) with ’oxy’ cytoplasm and normal B. oleracea (CC). A large number of male-sterile AABBCC somatic hybrids with desirable organelle combinations, i.e. chloroplasts of B. oleracea and mitochondria with recombinant genomes, were recovered. While no recombination was observed in the chloroplast genome, the mitochondrial genome showed extensive recombination that resulted in the appearance of totally novel banding patterns in some of the hybrids. Hybrids with a parental-type mitochondrial genome as well as recombinant patterns close to either of the parental types were also obtained. Using AABBCC somatic hybrids as bridging material, we transferred the desirable organelle combinations to B. juncea (AABB), B. napus (AACC), and B. carinata (BBCC). Many of these lines are now at advanced stages of backcrossing and show stable inheritance of the CMS character and do not suffer from chlorosis. Received: 9 August 1999 / Accepted: 14 September 1999     

Large double-stranded RNA molecules in Phaseolus vulgaris L. are not associated with cytoplasmic male sterility

Summary Two large double-stranded RNA molecules, 15 and 16 kilobases, were detected in cytoplasmic male sterile (CMS) Phaseolus vulgaris by agarose gel electrophoresis. A number of smaller RNA molecules were observed in Sprite, a maintainer line, and recurrent backcrossing of CMS P. vulgarisxSprite resulted in a combined electrophoretic pattern of the two large and numerous small RNA molecules. The large RNA molecules were seed and pollen-transmissible, but were not transmitted by grafting. The RNAs were present in revertant and restored lines derived from CMS-Sprite and therefore were not associated with the cytoplasmic male sterile trait.Florida Agr. Exp. Stn. Journal Series No. 8405     

二角山羊草细胞质小麦雄性不育系的育性特异性分析

利用大量小麦亲本材料和优良品种(系)与具有粘果、易变、偏凸和二角山羊草细胞质的小麦雄性不育系杂交,并对其杂交F1过氧化物同工酶进行了分析,结果表明：(1)二角山羊草细胞质与小麦核内的遗传物质组成两个不同的核质互作不育系统,粘、易、偏型不育系育性基本表现一致,而二角型不育系除了与前三种不育系具有相同的1BL／1RS保持系以外,对某些小麦近缘植物的杂交后代材料还表现出育性特异性。(2)粘、易、偏和二角型同核异质不育系5-1及其与V9125杂交F1过氧化物同工酶分析表明,粘、易、偏和二角型不育系5-1过氧化物同工酶带型基本表现一致,粘、易、偏不育系5-1与V9125杂交F1过氧化物同工酶带型基本表现一致,而二角型不育系5-1杂交F1过氧化物同工酶则表现出酶带减少变弱。     

Genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">hau</Emphasis>) in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">juncea</Emphasis> and its transfer to <Emphasis Type="Italic">B. napus</Emphasis>

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC₁F₁ population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.     

Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions

 A collection of 200 wheat (Triticum aestivum L. cv ‘Chinese Spring’) cytogenetic stocks (nullisomic, tetrasomic, nulli-tetrasomic, ditelosomic and deletion lines, addition and substitution stocks from intra- and inter-specific crosses) was utilized to determine the proteins encoded by some of the wheat and barley dehydrin genes, using a western blot procedure. Proteins extracted from seeds were reacted with antibodies that recognize dehydrins in a wide range of plants, including wheat and barley. Proteins encoded by dehydrin loci in chromosome arms 4DS, 5BL and 6AL of ‘Chinese Spring’ wheat were assigned by this method. There was also evidence of a regulatory factor on 5B in the vicinity of the dhn genes, and on 5H in wheat-barley addition lines, that is required for a normal level of expression of seed dehydrins in hexaploid wheat. Further understanding of this putative regulatory factor would be helpful for the interpretation of linkage studies that may relate dehydrin gene expression to phenotypes such as dehydration, salinity or low-temperature tolerance. Received: 27 August 1997 / Accepted: 4 February 1998     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Delimitation of a QTL region controlling cold tolerance at booting stage of a cultivar, ‘Lijiangxintuanheigu’, in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Low temperature at the booting stage of rice causes male sterility resulting in severe yield loss. Cold tolerance has long been an important objective in rice breeding. We identified a quantitative trait locus (QTL) for cold tolerance on the long arm of chromosome 3 from the cold-tolerant breeding line ‘Ukei 840’ by using F₂ and BC₁F₂ populations from crosses between ‘Ukei 840’ and ‘Hitomebore’. The cold tolerance of ‘Ukei 840’ is derived from the Chinese cultivar ‘Lijiangxintuanheigu’. The effect of this QTL on cold tolerance was confirmed by developing ‘Hitomebore’ chromosome segment substitution lines having ‘Lijiangxintuanheigu’ alleles on chromosome 3. By producing recombinants in chromosome 3, the QTL region for cold tolerance was delimited to the region of about 1.2-Mb region between RM3719 and RM7000. All lines heterozygous for the QTL showed seed fertilities as low as that of ‘Hitomebore’, suggesting that the ‘Lijiangxintuanheigu’ allele for cold tolerance in the QTL region is recessive. Determination of a 1.2-Mb nucleotide sequence of ‘Ukei 840’ and comparison with the published genomic sequence of ‘Nipponbare’ showed 254 SNPs, of which 11 were in coding regions of genes, seven in five genes being non-synonymous. SNPs were detected in the 5-kb upstream regions of 89 genes, but no differences of gene expression levels were detected between alleles of these genes. Although further delimitation is required to identify the gene responsible for cold tolerance of ‘Lijiangxintuanheigu’, SNP markers developed here will be useful for marker-assisted selection in a breeding program using ‘Lijiangxintuanheigu’ as a donor of cold tolerance.     

Quantitative trait loci for resistance to spot blotch caused by Bipolarissorokiniana in wheat (T.aestivum L.) lines ‘Ning 8201’ and ‘Chirya 3’

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat growing regions of the world. To identify quantitative trait loci (QTLs) for spot blotch resistance, two mapping populations were developed by making the crosses between common susceptible cultivar ‘Sonalika’ with the resistant breeding lines ‘Ning 8201’ and ‘Chirya 3’. Single seed descent derived F₆, F₇, F₈ lines of the first cross ‘Ning 8201’ × ‘Sonalika’ were evaluated for resistance to spot blotch in three blocks in each of the 3 years. After screening of 388 pairs of simple sequence repeat primers between the two parents, 119 polymorphic markers were used to genotype the mapping population. Four QTLs were identified on the chromosomes 2AS, 2BS, 5BL and 7DS and explained 62.9% of phenotypic variation in a simultaneous fit. The QTL on chromosome 2A was detected only in 1 year and explained 22.7% of phenotypic variation. In the second cross (‘Chirya 3’ × ‘Sonalika’), F₇ and F₈ population were evaluated in three blocks in each of the 2 years. In this population, five QTLs were identified on chromosomes 2BS, 2DS, 3BS, 7BS and 7DS. The QTLs identified in the ‘Chirya 3’ × ‘Sonalika’ population explained 43.4% of phenotypic variation in a simultaneous fit. The alleles for reduced disease severity in both the populations were derived from the respective resistant parent. The QTLs QSb.bhu-2B and QSb.bhu-7D from both populations were placed in the same deletion bins, 2BS1-0.53-0.75 and 7DS5-0.36-0.61, respectively. The closely linked markers Xgwm148 to the QTL on chromosome 2B and Xgwm111 to the QTL on chromosome 7D are potentially diagnostic markers for spot blotch resistance.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion

We document here the presence of a recombinant plastome in a cytoplasmic male sterile (CMS) line of Brassica juncea developed from the somatic hybrid Trachystoma ballii + B. juncea. Restriction endonuclease digestion of the chloroplast (cp) DNA has revealed that the recombinant plastome gives rise to novel fragments in addition to the parent-specific fragments. Analysis of the 16S rRNA region by Southern hybridization shows no variation between B. juncea, T. ballii and the CMS line. The rbcL gene region of the recombinant plastome is identical to that in T. ballii. Analysis with probes for psbA and psbD using single and double DNA digests indicates that the hybridization patterns of the recombinant plastome are identical to those of the parents in digests obtained with some restriction enzymes, while novel bands hybridize to probes in other digests. In the psbA region, a B. juncea-specific PstI site and a T. ballii-specific EcoRI site are found in the recombinant plastome. The psbD region of the recombinant plastome contains a B. juncea-specific HindIII site and T. ballii-specific BamHI and HpaII sites. These results indicate the occurrence of intergenomic recombination between the chloroplasts of T. ballii and B. juncea in the somatic hybrid from which the CMS line was developed. The recombined plastome appears to be a mosaic of fragments specific to both parents and the recombination event has occurred in the single-copy regions. These recombinational events have not caused any imbalance in the recombinant plastome in terms of chloroplast-related functions, which have remained stable over generations. Received: 3 March 1998 / Accepted: 5 August 1998     

Development of a CMS-specific marker based on chloroplast-derived mitochondrial sequence in pepper

Molecular markers developed from the flanking sequences of two cytoplasmic male sterility (CMS)-associated genes, orf456 and ψatp6-2, have been used for marker-assisted selection of CMS in pepper. However, in practice, the presence of orf456 and ψatp6-2 at substoichiometric levels even in maintainer lines hampers reliable selection of plants containing the CMS gene. In this study, we developed a novel CMS-specific molecular marker, accD-U, for reliable determination of CMS lines in pepper, and used the newly and previously developed markers to determine the cytoplasm types of pepper breeding lines and germplasms. This marker was developed from a deletion in a chloroplast-derived sequence in the mitochondrial genome of a CMS pepper line. CMS pepper lines could be unambiguously determined by presence or absence of the accD-U marker band. Application of orf456, ψatp6-2 and accD-U to various pepper breeding lines and germplasms revealed that accD-U is the most reliable CMS selection marker. A wide distribution of orf456, but not ψatp6-2, in germplasms suggests that the pepper cytoplasm containing both orf456 and ψatp6-2 has been selected as CMS cytoplasm from cytoplasm containing only orf456. Furthermore, factors other than orf456 may be required for the regulation of male sterility in pepper.     

Endogenous Hormones in Seeds, Germination Behaviour and Early Seedling Characteristics in a Normal and ogura Cytoplasmic Male Sterile Line of Rapeseed (Brassica napus L.)

Endogenous hormones, namely cytokinins (CKs), indole-3-aceticacid (IAA) and abscisic acid (ABA) were quantified by specificenzyme-linked immunosorbent assay (ELISA) in the mature seedof normal (cv. Westar) and ogura (ogu) cytoplasmic male sterile(CMS) lines of rapeseed (Brassica napus L.). Dihydrozeatin (DZ)and dihydrozeatin riboside (DZR) were the major CK base andriboside, respectively, in seeds of both the normal and oguCMS lines. The normal seed had more than 4-fold DZ levels incomparison to that of ogu CMS. On the other hand, the ogu CMSseed had higher levels of CK o-glucosides and CK. nucleotidesthan normal seed. Seeds of the normal line contained 5-foldmore IAA but had one-quarter the level of ABA in comparisonto those of the ogu CMS line. The normal line also had greaterseed diameter and weight than the ogu CMS line and the normalseed germinated earlier than the male sterile seed. DZ (10^–6M) promoted the germination of ogu CMS seeds, but it was notcomparable to that of the normal line. ABA (10^–6 M) inhibitedseed germination of ogu CMS but had little effect on the normalline. The normal seedlings had shorter primary roots, more lateralroots, longer hypocotyls, greater cotyledon fresh weight andhigher chlorophyll levels in comparison to ogu CMS seedlings.Exogenously supplied DZ, IAA and ABA affected the various parametersof both the normal and ogu CMS seedlings, but in most casesdid not fully restore the differences in the two lines. The results presented show that in the ogura cytoplasmic malesterile line of B. napus (1) a number of seed and seedling characteristicsare affected, and (2) the altered seed morphology is accompaniedby changes in the levels of various hormones. Key words: Brassica napus, cytoplasmic male sterility, enzyme-linked immunosorbent assay, hormone, seed germination     

QTL Analysis for Root Protein in a Backcross Family of Cassava Derived from Manihot esculenta ssp flabellifolia

Root protein content of elite cassava is very low, largely due to breeder’s selection for other agronomic traits mainly fresh weight yield and disease resistance. Increased protein content in the root of cassava will improve its usefulness as a more complete food source in the developing world. An inter-specific F₁ hybrid CW 198 - 11 was earlier developed at International Center for Tropical Agriculture (CIAT), Cali, Colombia by genetic crosses of OW 230 - 1 (FLA 441 - 5) and CW 30–65 (an inter-specific hybrid between an improved cassava variety SG 427 - 87 and an accession of Manihot esculenta ssp flabellifolia (MESCFLAX – 80)). The inter-specific cross was ‘backcrossed’, in the sense of another cross to cassava (MTAI – 8) to generate a B₁P₂ family with 225 progenies in which major quantitative trait loci (QTL) for root protein in the backcross population of cassava were identified. A linkage map from the female parent of the backcross population was used for QTL detection. A total of three QTL (protg.7, protg.13 and protg.23) controlling protein were identified in three different environments. One QTL was expressed across all three environments. These results demonstrated high broad sense heritability of 61.6% for protein over 2 years, in two different locations. The individual effects of alleles at these QTL explained from 15% to 25% of the phenotypic variance. The consistency of QTL controlling protein across environments reveals their potential for use in marker-assisted recurrent selection.     

New CMS types in Plantago lanceolata and their relatedness

 Mitochondrial variation in Plantago lanceolata was used to detect new CMS types. Directional reciprocal crosses were made between plants which differed in mtDNA restriction patterns. Differential segregation of male steriles in reciprocal crosses indicated that the parents differed in CMS type. MtDNA variation revealed nine RFLP patterns, which could be categorised according to the sex phenotype of the plants as MS1 (brown-anther type), MS2 (petaloid flower type) and MS3 (more yellow anthers than MS1). A single mtDNA pattern was found within MS1, six mtDNA patterns were found within the MS2 group, and two mtDNA patterns were found within hermaphrodites which segregated MS3 in the crosses. MS1 and MS2 are known to represent different CMS types, CMSI and CMSII. In reciprocal crosses between plants with different mtDNA patterns within the MS2 group, different ratios of male steriles segregated in the crosses, indicating that the parents differed in CMS type. Within the MS2 group two CMS types were found, designated CMSIIa and b. Finally, the sex phenotype H/MS3 turned out to be a different type. From previous studies it was known that the MS3 phenotype can also occur in CMSI and CMSII types, hence it is unclear whether MS3 is diagnostic for CMSIII. Since the data in this study cannot distinguish between the new type being a fully restored new CMS type or a ‘Normal’ cytoplasm, it was denoted as CMSIII. In total, four CMS types were found in the material studied. CtDNA variation was screened and three chloroplast haplotypes were identified. Two haplotypes were associated with CMSI plants, and one haplotype with the other CMS types. The ctDNA variation indicated that the CMSI type is widespread within the species, due to migration rather than to recurrent mutation. This may lead to the conclusion that only a limited number of CMS types are successful. Received: 9 August 1996 / Accepted: 6 September 1996