Interspecific Backcross Progeny Research Articles

Characterization of Ogura CMS fertility-restored interspecific hybrids and backcross progenies from crosses between broccoli and rapeseed

Interspecific F1 hybrids were obtained from crosses between two Ogura cytoplasmic male sterility (Ogura CMS) broccoli lines (B14 and 137) and a rapeseed restorer line to introgress the Ogura CMS’s fertility-restoration gene Rfo from rapeseed into broccoli. Ten Rfo-positive interspecific, triploid plant progenies, phenotypically between rapeseed and broccoli, were obtained but still contained multiple rapeseed genes, with reduced fertility and seed setting potential under natural pollination. For fertility and seed setting improvement, successive backcrosses with broccoli were carried out to yield BC2 progenies. Screening revealed six plants with Rfo among 25 BC2 progenies, which were investigated for agronomic traits, ploidy determination, pollen viability and SSR (simple sequence repeat) markers to evaluate for ideal individuals closer to broccoli. Pollen viability tests were carried out at different stages for various hybrid parents. The results were as follows: 137-F1 (68.54%) > B14-F1 (65.32%) > 137-BC1 (48.15%) > B14-BC1 (32.41%). Although there were significant differences among the BC1 and BC2 individuals, the genetic background of BC2 was closer to the parent broccoli compared with that of BC1 plants. The existence of the Rfo gene in broccoli not only facilitates broccoli germplasm innovation, combination of interspecific hybridization and backcrossing, but may also shed light on trait segregation in different generations.

GWAS analysis using interspecific backcross progenies reveals superior blue catfish alleles responsible for strong resistance against enteric septicemia of catfish.

Infectious diseases pose significant threats to the catfish industry. Enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri is the most devastating disease for catfish aquaculture, causing huge economic losses annually. Channel catfish and blue catfish exhibit great contrast in resistance against ESC, with channel catfish being highly susceptible and blue catfish being highly resistant. As such, the interspecific backcross progenies provide an ideal system for the identification of quantitative trait locus (QTL). We previously reported one significant QTL on linkage group (LG) 1 using the third-generation backcrosses, but the number of founders used to make the second- and third-generation backcross progenies was very small. Although the third-generation backcross progenies provided a greater power for fine mapping than the first-generation backcrosses, some major QTL for disease resistance may have been missing due to the small numbers of founders used to produce the higher generation backcrosses. In this study, we performed a genome-wide association study using first-generation backcrosses with the catfish 690K SNP arrays to identify additional ESC disease resistance QTL, especially those at the species level. Two genomic regions on LG1 and LG23 were determined to be significantly associated with ESC resistance as revealed by a mixed linear model and family-based association test. Examination of the resistance alleles indicated their origin from blue catfish, indicating that at least two major disease resistance loci exist among blue catfish populations. Upon further validation, markers linked with major ESC disease resistance QTL should be useful for marker-assisted introgression, allowing development of highly ESC resistant breeds of catfish.

A genome-wide association study of heat stress-associated SNPs in catfish.

Heat tolerance is a complex and economically important trait for catfish genetic breeding programs. With global climate change, it is becoming an increasingly important trait. To better understand the molecular basis of heat stress, a genome-wide association study (GWAS) was carried out using the 250K catfish SNP array with interspecific backcross progenies, which derived from crossing female channel catfish with male F1 hybrid catfish (female channel catfish×male blue catfish). Three significant associated SNPs were detected by performing an EMMAX approach for GWAS. The SNP located on linkage group 14 explained 12.1% of phenotypical variation. The other two SNPs, located on linkage group 16, explained 11.3 and 11.5% of phenotypical variation respectively. A total of 14 genes with heat stress related functions were detected within the significant associated regions. Among them, five genes-TRAF2, FBXW5, ANAPC2, UBR1 and KLHL29- have known functions in the protein degradation process through the ubiquitination pathway. Other genes related to heat stress include genes involved in protein biosynthesis (PRPF4 and SYNCRIP), protein folding (DNAJC25), molecule and iron transport (SLC25A46 and CLIC5), cytoskeletal reorganization (COL12A1) and energy metabolism (COX7A2, PLCB1 and PLCB4) processes. The results provide fundamental information about genes and pathways that is useful for further investigation into the molecular mechanisms of heat stress. The associated SNPs could be promising candidates for selecting heat-tolerant catfish lines after validating their effects on larger and various catfish populations.

QTL analysis of growth and wood chemical content traits in an interspecific backcross family of white poplar (Populus tomentosa×P. bolleana) ×P. tomentosa

The genetic control of tree growth and wood chemical content traits was studied using interspecific backcross progeny between clone TB01 (Populus tomentosa × Populus bolleana) and clone LM50 (P. tomentosa). In total, 247 and 146 amplified fragment length polymorphism (AFLP) markers from genetic maps previously constructed for backcross parents LM50 and TB01 were used for the analyses of quantitative trait loci (QTL). These markers were distributed among 19 linkage groups and covered 3265 and 1992 cM in the backcross parents, respectively. A total of 32 putative QTLs, associated with five growth and chemical content traits, sylleptic branch number, sylleptic branch angle, stem volume, wood cellulose content, and wood lignin content, were detected. These QTLs were dispersed among 16 linkage groups in parent LM50 and 10 groups in parent TB01. The phenotypic variance explained by each QTL ranged from 7.0% to 14.6%. QTLs controlling sylleptic branch number and stem volume were colocalized in two linkage groups, TLG6 and TLG8, respectively. The favorable alleles were mostly from P. tomentosa, which is phenotypically superior to P. bolleana for sylleptic branch angle, stem volume, and wood chemical content traits. The favorable alleles for sylleptic branch number were from P.bolleana. These AFLP markers that were associated with the QTLs have potential use in P. tomentosa breeding programs.

Genetic Architecture of Transcript-Level Variation in Differentiating Xylem of a Eucalyptus Hybrid

Species diversity may have evolved by differential regulation of a similar set of genes. To analyze and compare the genetic architecture of transcript regulation in different genetic backgrounds of Eucalyptus, microarrays were used to examine variation in mRNA abundance in the differentiating xylem of a E. grandis pseudobackcross population [E. grandis x F(1) hybrid (E. grandis x E. globulus)]. Least-squares mean estimates of transcript levels were generated for 2608 genes in 91 interspecific backcross progeny. The quantitative measurements of variation in transcript abundance for specific genes were mapped as expression QTL (eQTL) in two single-tree genetic linkage maps (F(1) hybrid paternal and E. grandis maternal). EQTL were identified for 1067 genes in the two maps, of which 811 were located in the F(1) hybrid paternal map, and 451 in the E. grandis maternal map. EQTL for 195 genes mapped to both parental maps, the majority of which localized to nonhomologous linkage groups, suggesting trans-regulation by different loci in the two genetic backgrounds. For 821 genes, a single eQTL that explained up to 70% of the transcript-level variation was identified. Hotspots with colocalized eQTL were identified in both maps and typically contained genes associated with specific metabolic and regulatory pathways, suggesting coordinated genetic regulation.

Male accessory gland secretory proteins in a few members of the Drosophila nasuta subgroup.

Male accessory gland secretory proteins in seven members of the Drosophila nasuta subgroup have been analyzed by SDS-PAGE. The study revealed remarkable simplicity in the patterns. The protein fractions, which migrate in three groups, could be categorized as "major" and "minor." The number of major fractions varies from a maximum of eight to a minimum of four. Group I consists of high molecular weight fractions, and group III, low molecular weight fractions. Among different members analyzed, the variation with respect to pattern and the number of fractions are confined largely to group III protein fractions, while group I and II fractions are found to be conserved to a greater extent. These proteins are PAS positive and group III fractions are not sensitive to silver staining. Analysis of these tissue specific proteins in the F1 and F2 of interspecific crosses and backcross progeny as well as volume analysis revealed that a 26-kD fraction in D. n. nasuta follows an autosomal pattern of inheritance, while a 55-kD and a 25-kD fraction in D. n. albomicans and a 24-kD fraction in D. n. kepulauana follow an X-linked pattern of inheritance.

An Ancient Conserved Gene Expressed in the Human Inner Ear: Identification, Expression Analysis, and Chromosomal Mapping of Human and Mouse Antiquitin (ATQ1)

We constructed and screened a human fetal cochlear cDNA library to identify genes involved in hearing and deafness. From this library we isolated a cDNA corresponding to the highly conserved ancient gene antiquitin (ATQ1). The plant homolog ofATQ1is thought to be involved in regulating turgor pressure, a function that also would be essential for cells of the mammalian cochlea. Northern blots of 13 human fetal tissues show antiquitin to be highly expressed in cochlea, ovary, eye, heart, and kidney. Using RT-PCR of rat cochlear hair cell-specific cDNA libraries, we detect antiquitin expression in outer hair cells, but not in inner or vestibular type 1 hair cells, suggesting that antiquitin is not expressed ubiquitously in the cochlea. HumanATQ1was mapped to human chromosome region 5q31 using fluorescencein situhybridization, and mouseATQ1was mapped to mouse chromosome 18 by single-strand conformation polymorphism mapping of interspecific backcross progeny DNAs. Four human antiquitin-like sequences, possibly pseudogenes, were also identified and mapped.

The gene for murine megakaryocyte growth and development factor (thrombopoietin, Thpo) is located on mouse chromosome 16

The canine MGDF genomic clone was isolated using a degenerative oligonucleotide probe based on the amino terminal sequences of the MGDF protein purified from aplastic canine serum. A mouse genomic library (1 x 10{sup 6} pfu) was then screened with a mixture of six canine MGDF oligomers. Phage DNA was isolated from the positive clone and sequenced. Comparison of the murine MGDF and the human MGDF gene showed that their genomic structures are similar in sizes of exons and introns. To determine chromosomal location of the murine MGDF gene (locus designation thrombopoietin, Thpo), interspecific backcross progeny were generated by mating (C57BL/6J X Mus spretus) F{sub 1} females and C57BL/6J males as described. This interspecific backcross mapping panel has been typed for over 1700 loci that are well distributed among all of the autosomes as well as the X chromosome. A total of 205 N{sub 2} mice were used to map the Thpo locus. The probe used in these studies was an {approximately}440-bp fragment of mouse genomic DNA consisting of exon 6, intron 6, and part of exon 7. C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphismsmore » (RFLPs) using a mouse genomic probe. A fragment of 6.5 kb was detected in EcoRI-digested C57BL/6J DNA, and a fragment of 4.6 kb was detected in EcoRI-digested M. spretus DNA. The 4.6-kb M. spretus-specific EcoRI fragment was used to follow the segregation of the Thpo locus in backcross mice. 5 refs., 1 fig.« less

A single gel analysis of 575 dominant and codominant restriction landmark genomic scanning loci in mice interspecific backcross progeny.

We identified 575 new NotI landmarks of C57BL/6(B)- and M. spretus (S)-specific, dominant and codominant loci which were segregated in B x S interspecific backcrosses (BSS), using the restriction landmark genomic scanning (RLGS) spot mapping method. All of these loci were visualized on a single RLGS profile which was produced with NotI-PvuII-PstI. These landmarks include 250 newly identified S-specific spots in addition to the previously reported 325 B-specific spots. The S-specific spots were identified by reading full or half intensity, based on the property that the spot intensity of the autoradiographic signal reflected the copy number of an end-labeled restriction landmark. The cumulative map is 1341 cM and it is based upon 985 meiotic events in 72 backcross progeny. This map covers 90% of the total estimated length of the mouse genetic map. This map provides a good tool for the high-speed genome scanning assay in the mouse genome by a single RLGS gel analysis.

Fine genetic mapping defines the genetic order of Pax9, Tcf3a, and Acrodysplasia (Adp).

We present here the fine genetic mapping of the proximal part of mouse Chromosome (Chr) 12 between D12Mit54 and D12Mit4. This chromosomal region contains three loci, Pax9, Tcf3a, and Acrodysplasia (Adp), which seem to play an important role in pattern formation during mouse embryogenesis. The Adp mutation, which was created by transgene integration, causes skull, paw, and tail deformities. Pax9, which is expressed in the face, paws, and tail, once qualified as a possible candidate for the Adp locus. We analyzed 997 interspecific backcross progeny for recombination between the markers D12Mit54 and D12Mit4; we recovered 117 recombinants, which were further typed for Pax9, Tcf3a, Adp, D12Mit88, D12Nds1, D12Mit36, and D12Mit34. This study represents the first instance in which all the above loci have been included in a single analysis, thereby allowing unambiguous determination of the genetic order and distance between D12Mit54 and D12Mit4. From our results, we conclude that the Adp locus is distinct from either Pax9 or Tcf3a.

Comparative Mapping of Lipocalin Genes in Human and Mouse: The Four Genes for Complement C8 γ Chain, Prostaglandin-d-Synthase, Oncogene-24P3, and Progestagen-Associated Endometrial Protein Map to HSA9 and MMU2

The lipocalin superfamily encompasses a large set of quite distantly related proteins that act as carriers for small, lipophilic molecules. The lipocalin genes coding for orosomucoid, the alpha 1-microglobulin/bikunin precursor, and the major urinary protein map to MMU4, while their human counterparts map to the homologous HSA9q34 area where three other lipocalin genes for complement C8 gamma chain (C8G), progestagen-associated endometrial protein (PAEP), and prostaglandin D synthase (PTGDS) are also located. By linkage analyses in an interspecific backcross progeny in mouse, the Lcn2 gene coding for the oncogenic lipocalin 24p3, as well as the 3 lipocalin genes for C8G, PTGDS, and PAEP, have now been assigned to MMU2. The first three genes map to proximal MMU2, which is known to be homologous to HSA9q34. Paep is more distally located, which extends the number of regions with conserved syntenies between HSA9q34 and MMU2. By in situ hybridization, the human LCN2 gene maps to HSA9q34. Our data indicate that the lipocalin locus arrangement in the human/mouse ancestor is closer to that found at HSA9q than to that in the MMU genome.

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7.

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development until their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (mCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in interspecific backcross progeny revealed linkage of mCd19 with hemoglobin beta (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere--Hbb--mCd19--H19--Int-2--telomere. The genetic distances between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C beta polypeptide.

The gene for murine CTP: Phosphocholine cytidylyltransferase (Ctpct) is located on mouse chromosome 16

CTP:phosphocholine cytidylyltransferase is the rate-controlling enzyme in phosphatidylcholine biosynthesis and is essential for the survival of eukaryotic cells. The murine cDNA for the cytidylyltransferase was cloned and sequenced. A genomic clone was isolated and the chromosomal location of the Ctpct locus determined by Southern blot hybridization of DNAs from a panel of interspecific backcross progeny derived from matings of [(C57BL/6J × Mus spretus)F1 × C57BL/6J] mice. These data place the Ctpct gene on mouse chromosome 16 between the Smst and Stf-1 genes.

Mapping of the phenol sulfotransferase gene (STP) to human chromosome 16p12.1-p11.2 and to mouse chromosome 7.

We have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotransferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5' end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, beta 1 polypeptide (PRKCB1), in the region from the distal portion of 16p11.2 to p12.1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, we have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers.

Extension of the physical map in the region of the mouse X chromosome homologous to human Xq28 and identification of an exception to conserved linkage

We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to human Xq28 to include the loci Gdx (DXS254Eh), P3 (DXS253Eh), G6pd, Cf-8, and F8a. Gdx, P3, and G6pd are demonstrated to be physically linked to the X-linked visual pigment locus ( Rsvp) within a maximal distance of 340 kb, while G6pd and Cf-8 are approximately 900 kb apart. These studies favor a gene order of cen- Rsvp-Gdx-P3-G6pd-(Cf-8)-tel and extend the physical map of this region to 5 million bp. In conjunction with previous physical mapping studies in both mouse and human, the results suggest conserved linkage for loci in this region of the mouse X chromosome and human Xq28. However, employing pulsed-field gel electrophoresis and genetic pedigree analysis of interspecific backcross progeny, we have found close linkage of a clone encoding a mouse homolog for human factor VIII-associated gene A (F8A) to DXPas8, thus revealing the first exception to conserved gene order between murine and human loci in the region.

Analysis of sex-chromosome aneuploidy in interspecific backcross progeny between the laboratory mouse strain C57BL/6 and Mus spretus.

Sex-chromosomal aneuploidy was identified in four female progeny of 200 interspecific backcrosses between laboratory mice (C57BL/6Ros) and Mus spretus. The progeny included two 39,XO monosomy mice resulting from a backcross with M. spretus, as well as a 41,XXX trisomic mouse and a 40,XX/41,XXX mosaic mouse resulting from two separate backcrosses with C57BL/6 mice. The parental origin and meiotic stage of the aneuploidies was determined for each of the mice using a series of markers that identified allelic differences in the parental X-chromosome genes present in the hybrid female. Two of the probes identified differences in repeated elements between the M. spretus and laboratory mouse X chromosomes, whereas the remaining sites involved restriction fragment length differences of single-copy genes detectable by Southern analysis. These markers indicated that the aneuploidies were most likely of maternal origin and that the trisomy resulted from a nondisjunction at the second meiotic division. In contrast, the mosaic female could have originated either from a trisomic embryo that had lost a single X in a portion of its cells or from a mitotic nondisjunction during early embryogenesis that resulted in XXX and XO daughter cells, with subsequent loss of the XO cells.

Polymorphisms revealed by PCR with single, short-sized, arbitrary primers are reliable markers for mouse and rat gene mapping.

Ten single, arbitrarily designed oligodeoxynucleotide primers, with 50-70% (G+C) content, were used to amplify by polymerase chain reaction (PCR) sequences with DNA templates from several mouse species (Mus spretus, Mus musculus musculus, and Mus musculus domesticus), as well as DNA from the laboratory rat (Rattus norvegicus). Eight of these ten primers, used either individually or associated in pairs, generated a total of 13 polymorphic products which were used as genetic markers. All of these polymorphic sequences but one were mapped to a particular mouse chromosome, by use of DNA panels prepared either from interspecific backcross progeny of the type (C57BL/6 x Mus spretus)F1 x C57BL/6 or DNA samples prepared from two sets of recombinant inbred (RI) strains (AKXL and BXD). Six rat-specific DNA segments were also assigned to a particular chromosome with DNA panels prepared from 18 rat/mouse somatic cell hybrids segregating rat chromosomes. From these experiments we conclude that, under precisely standardized PCR conditions, the DNA molecules amplified with these arbitrarily designed primers are useful and reliable markers for genetic mapping in both mouse and rat.

Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.

Molecular genetic analysis of the Ta 25H deletion: Evidence for additional deleted loci

Seventeen linking clones sublocalized to the central region of the mouse X Chromosome (Chr) were screened against genomic DNA from male mice carrying the tabby-25H (Ta25H) deletion. Two of these linking clones, lambda EM131 and lambda EM169, were found to be deleted in Ta25H/Y animals. Genetic mapping through Mus musculus domesticus/Mus spretus interspecific backcross progeny, segregating for the original tabby (Ta) gene mutation, was utilized to order these markers and to define nearest flanking markers to the Ta25H deletion (lambda EM140 and lambda EM171). The size of the Ta25H deletion was thus estimated as up to 4.5 centiMorgans (cM). The order of markers, proximal to distal, was found to be lambda EM140/lambda EM131, mouse androgen receptor gene (Ar)/lambda EM169, Ta/lambda EM171. A putative CpG-rich island and a highly evolutionarily conserved DNA probe were isolated from the DXCrc169 locus which co-segregates with the Ta locus in this study.

Close linkage of the murine locus bare patches to the X-linked visual pigment gene: Implications for mapping human X-linked dominant chondrodysplasia punctata

The murine X-linked dominant mutation bare patches ( Bpa) has a phenotype similar to and is likely homologous to human X-linked dominant chondrodysplasia punctata (CDPX2). Classic two-point linkage analysis in the mouse with distant markers suggested that Bpa maps near glucose-6-phosphate dehydrogenase ( G6pd). We have confirmed the regional localization using interspecific matings with Mus spretus. We have also detected a restriction fragment length polymorphism (RFLP) at the murine X-linked visual pigment ( Rsvp) locus in inbred Bpa females using the restriction enzyme PstI. Cumulative data from segregation of alleles using the PstI RFLP and analysis of interspecific backcross progeny at the Rsvp locus suggest that Bpa is tightly linked to Rsvp. Thus, the human CDPX2 gene probably maps within Xq27–Xq28 and not within Xp22.3-Xpter, where deletions associated with X-linked recessive chondrodysplasia punctata (CDPX) have been noted. This strategy should be applicable to the fine mapping of other dominant murine mutations.