Microsatellite-anchored Fragment Length Polymorphism Research Articles

Identification of MFLP fingerprint for higher seed zinc accumulation in barley DH population

Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley breeding. To develop a specific molecular markers, 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara-3771 (high-Zn-accumulator) were screened under field and glasshouse conditions. Microsatellite-anchored fragment length polymorphism (MFLP) fingerprint generated by SSR-anchor primer MF128 in combination with AFLP primer MseI-AGA (5?-GATGAGTCCTGAGTAAAGA-3?) was identified as a candidate marker for tagging seed Zn accumulation gene. The sequencing of the band showed a marker of 369 bp with the sequence of SSR anchor primer MF128 and MseI-AGA at the two ends as expected. The MFLP marker associated with higher seed Zn accumulation has potential to be converted to a simple, sequence-specific, PCR-based, low-cost marker amenable to large populations, making it potentially viable for marker-assisted selection in barley breeding. This marker might be useful in the improvement of barley productivity and nutritional quality in Zn-deficient environments.

Development of a DNA marker tightly linked to low-alkaloid gene iucundus in narrow-leafed lupin (Lupinus angustifolius L.) for marker-assisted selection

Narrow-leafed lupin (Lupinus angustilolius L.) is a grain legume of exceptionally high nutritive value and much versatile food and animal feed around the world. The development of lupin as a modern crop was limited by its high concentration of alkaloids. Progress in breeding necessitates a better understanding of the genetics underlying the trait – low-alkaloid level (sweet). Marker-assisted selection would allow a better targeting of the desired genes. The microsatellite-anchored fragment length polymorphism (MFLP) fingerprinting technology was applied to an F8 recombination inbred line (RIL) population to identify and select candidate markers linked to the low-alkaloid gene iucundus. Four MFLP markers were identified as candidate markers based on their banding patterns in the F8 RIL population. One of these candidate markers showing the best correlation with phenotypes in the representative germplasm was selected and successfully converted into a simple PCR-based co-dominant marker, named IucLi. This established marker IucLi is 0.9 cM away from the sweet (low-alkaloid) gene iucundus. The accuracy between marker genotype and phenotype is 100% in the common 25 cultivars and 86.4% among the 125 accessions of narrow-leafed lupin core collection. Marker IucLi is being used in narrow-leafed lupin breeding for selection of ‘sweet’ individuals. The marker is also used to develop near-isogenic lines to further characterise and fine mapping the iucundus locus.

Characterization of the multiple resistance traits of somatic hybrids between Solanum cardiophyllum Lindl. and two commercial potato cultivars

Interspecific somatic hybrids between commercial cultivars of potato Solanum tuberosum L. Agave and Delikat and the wild diploid species Solanum cardiophyllum Lindl. (cph) were produced by protoplast electrofusion. The hybrid nature of the regenerated plants was confirmed by flow cytometry, simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphism (MFLP) markers and morphological analysis. Somatic hybrids were assessed for their resistance to Colorado potato beetle (CPB) using a laboratory bioassay, to Potato virus Y (PVY) by mechanical inoculation and field trials, and foliage blight in a greenhouse and by field trials. Twenty-four and 26 somatic hybrids of cph+cv. Agave or cph+cv. Delikat, respectively, showed no symptoms of infection with PVY, of which 3 and 12, respectively, were also resistant to foliage blight. One hybrid of cph+Agave performed best in CPB and PVY resistance tests. Of the somatic hybrids that were evaluated for their morphology and tuber yield in the field for 3years, four did not differ significantly in tuber yield from the parental and standard cultivars. Progeny of hybrids was obtained by pollinating them with pollen from a cultivar, selfing or cross-pollination. The results confirm that protoplast electrofusion can be used to transfer the CPB, PVY and late blight resistance of cph into somatic hybrids. These resistant somatic hybrids can be used in pre-breeding studies, molecular characterization and for increasing the genetic diversity available for potato breeding by marker-assisted combinatorial introgression into the potato gene pool.

Development of a co-dominant DNA marker tightly linked to gene tardus conferring reduced pod shattering in narrow-leafed lupin (Lupinus angustifolius L.)

The reduced pod shattering gene tardus is one of the most important domestication genes in narrow-leafed lupin (Lupinus angustifolius L.). In development of a molecular marker linked to the tardus gene, we incorporated the concept of marker validation during the initial candidate marker identification stage. Four dominant microsatellite-anchored fragment length polymorphism (MFLP) markers were identified as candidate markers based on their banding patterns in an F8 recombination inbred line (RIL) population. One specific marker best correlating with phenotypes in the representative germplasm was selected and converted to a simple PCR-based marker. This established marker, designated as “TaLi”, is located at a distance of 1.4 cM from the tardus gene. DNA sequencing revealed six insertion/deletion sites between the non-shattering marker allele and the shattering marker allele. Validation of marker TaLi on 25 domesticated commercial cultivars and 125 accessions of the lupin core collection found a 94% marker and tardus phenotype match. Marker TaLi is the first simple PCR-based marker that can be widely used for non-shattering pod selection in narrow-leafed lupin breeding program.

Molecular marker linked to a chromosome region regulating seed Zn accumulation in barley

Zinc deficiency is a critical nutritional problem in soils, restricting yield and nutritional quality of barley (Hordeum vulgare L.). Some genotypes (Zn-efficient) can produce greater yield and accumulate more Zn in seed under Zn deficiency than standard (Zn-inefficient) genotypes. However, there is little information regarding the genetics of Zn uptake/accumulation and location of genes conferring Zn efficiency in barley. Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley. With the aim of developing molecular markers for increased accumulation of Zn in seed, a population of 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara 3771 (high-Zn-accumulator) was screened in the field and glasshouse for seed Zn concentration and content. One dominant DNA polymorphism was detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. The candidate MFLP marker was isolated from the MFLP gel, re-amplified by PCR, cloned, sequenced, and converted into simple sequence-specific and PCR-based marker. This marker, located on the short arm of chromosome 2H, might be useful for the improvement of barley nutritional quality and productivity programs in Zn-deficient environments. However, high seed Zn alone can not replace the need for Zn fertilization.

Development of a sequence-specific PCR marker linked to the gene “pauper” conferring low-alkaloids in white lupin (Lupinus albus L.) for marker assisted selection

Seeds and plants of wild type Lupinus albus are bitter and contain high level of alkaloids. During domestication, at least three genes conferring low-alkaloid content were identified and incorporated into commercial varieties. Australian lupin breeders exclusively utilize one of these sweetness genes, “pauper”, in all varieties to prevent possible bitterness contamination via out-crossing. A cross was made between a sweet variety Kiev Mutant (containing pauper gene) and a bitter type landrace P27174, and the population was advanced into F8 recombinant inbred lines (RILs). Twenty-four plants representing sweetness and bitterness were subjected to DNA fingerprinting by the microsatellite-anchored fragment length polymorphism (MFLP) technique. A dominant polymorphism was discovered in an MFLP fingerprint. The MFLP marker was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the software program MapManager with marker score data and alkaloid phenotyping data from a segregating population containing 190 F8 RILs indicated that the marker is linked to the pauper gene at the genetic distance of 1.4 centiMorgans (cM). This marker, which is designated as “PauperM1”, is capable of distinguishing the pauper gene from the other two low-alkaloid genes exiguus and nutricius. Validation on germplasm from the Australian lupin breeding program showed that the banding pattern of the marker PauperM1 is consistent with the alkaloid genotyping on a wide range of domesticated varieties and breeding lines. The PauperM1 marker is now being implemented for marker assisted selection in the Australian albus lupin breeding program.

Characterisation of genetic diversity and DNA fingerprinting of Australian chickpea (Cicer arietinum L.) cultivars using MFLP markers

Chickpea (Cicer arietinum L.) is one of the major grain legume crops in the world. In this study, the genetic diversity of 24 Australian chickpea cultivars released between 1987 and 2005 was investigated with microsatellite-anchored fragment length polymorphism (MFLP) DNA markers. Among the cultivars examined, 30 cultivar-specific markers were identified and all were unequivocally identified using the DNA fingerprints developed in this study. Most of the cultivars were grouped into two major clusters; cv. Flipper was separated from the rest based on total character differences of DNA polymorphism. The MFLP approach proved suitable in the analysis of genetic diversity among the chickpea cultivars studied and the genetic relationship identified will be useful for chickpea breeding programs in selecting parent materials.

A strategy to develop molecular markers applicable to a wide range of crosses for marker assisted selection in plant breeding: a case study on anthracnose disease resistance in lupin (Lupinus angustifolius L.)

A key challenge in marker-assisted selection (MAS) for molecular plant breeding is to develop markers linked to genes of interest which are applicable to multiple breeding populations. In this study representative F2 plants from a cross Mandalup (resistant to anthracnose disease) × Quilinock (susceptible) of Lupinus angustifolius were used in DNA fingerprinting by Microsatellite-anchored Fragment Length Polymorphism (MFLP). Nine candidate MFLP markers linked to anthracnose resistance were identified, then ‘validated’ on 17 commercial cultivars. The number of “false positives” (showing resistant-allele band but lack of the R gene) for each of the nine candidate MFLP markers on the 17 cultivars ranged from 1 to 9. The candidate marker with least number of false positive was selected, sequenced, and was converted into a co-dominant, sequence-specific, simple PCR based marker suitable for routine implementation. Testing on 180 F2 plants confirmed that the converted marker was linked to the R gene at 5.1 centiMorgan. The banding pattern of the converted marker was consistent with the disease phenotype on 23 out of the 24 cultivars. This marker, designated “AnManM1”, is now being used for MAS in the Australian lupin breeding program. We conclude that generation of multiple candidate markers, followed by a validation step to select the best marker before conversion to an implementable form is an efficient strategy to ensure wide applicability for MAS.

Development of two sequence-specific PCR markers linked to the le gene that reduces pod shattering in narrow-leafed Lupin (Lupinus angustifolius L.)

Wild types of narrow-leaf lupin (Lupinus angustifolius L.) have seed pods that shatter upon maturity, leading to the loss of their seeds before or during the harvest process. Two recessive genes have been incorporated into domesticated cultivars of this species to maximize harvest-ability of the produce. One of these genes is called lentus (le). Two microsatellite - anchored fragment length polymorphism (MFLP) candidate markers were identified as closely linked to the le gene in a recombinant inbred line (RIL) population derived from a domesticated x wild type cross. The candidate MFLP markers were isolated from the gel, re-amplified by PCR, cloned and sequenced. The MFLP polymorphisms were converted into sequence-specific PCR-based markers. Linkage analysis by MapManager indicated that one of the markers, LeM1, was 2.6 centiMorgans (cM) and the other, LeM2, was 1.3 cM from the gene, with both being on the same side. The correlation between the marker genotype and the plant phenotype for the le gene is 95% for the Australian cultivars, and approximately 36% on wild types tested. These markers may be useful in marker assisted selection for the le gene when introgressing wild material into lupin breeding programs.

Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin (Lupinus angustifolius L.)

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.