Detection and mapping of SSRs in rye ESTs from aluminium-stressed roots

Aluminium toxicity is a major problem for crop production on acid soils. Rye (Secale cereale L.) has one of the most efficient group of genes for aluminium tolerance, at least, four independent and dominant loci, Alt1, Alt2, Alt3 and Alt4, located on chromosome arms 6RS, 3RS, 4RL and 7RS, have been described. The increasing availability of expressed sequence tags in rye and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In order to obtain simple sequence repeat (SSR) markers related with Al tolerance more than 1,199 public accessible rye cDNA sequences from Al-stressed roots were exploited as a resource for SSR markers development. From a total of 21 S. cereale microsatellite (SCM) loci analysed, 12 were located on chromosomes 1R, 2R, 3R, 4R and 5R, using wheat–rye addition lines or mapped using a F₂ population segregating for Al tolerance. Seven SCM loci were included in a rye map with other SCIM and RAPD markers. Moreover, 14 SCM loci could be associated to proteins with known or unknown function. The possible implications of these sequences in aluminium tolerance mechanisms are discussed.     

AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.)

Rye (Secale cereale L.) is considered to be the most aluminum (Al)-tolerant species among the Triticeae. It has been suggested that aluminum tolerance in rye is controlled by three major genes (Alt genes) located on rye chromosome arms 3RL, 4RL, and 6RS, respectively. Screening of an F₆ rye recombinant inbred line (RIL) population derived from the cross between an Al-tolerant rye (M39A-1–6) and an Al-sensitive rye (M77A-1) showed that a single gene controls aluminum tolerance in the population analyzed. In order to identify molecular markers tightly linked to the gene, we used a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis techniques to evaluate the F₆ rye RIL population. We analyzed approximately 22,500 selectively amplified DNA fragments using 204 primer combinations and identified three AFLP markers tightly linked to the Alt gene. Two of these markers flanked the Alt locus at distance of 0.4 and 0.7 cM. Chromosomal localization using cloned AFLP and a restriction fragment length polymorphism (RFLP) marker indicated that the gene was on the long arm of rye chromosome 4R. The RFLP marker (BCD1230) co-segregated with the Alt gene. Since the gene is on chromosome 4R, the gene was designated as Alt3. These markers are being used as a starting point in the construction of a high resolution map of the Alt3 region in rye. Received: 29 March 2000 / Accepted: 9 July 2001     

From the rye Alt3 and Alt4 aluminum tolerance loci to orthologous genes in other cereals

The major limit to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which limit growth by inhibiting root elongation. Aluminum tolerance in rye is controlled by (at least) four independent loci (Alt1, Alt2, Alt3 and Alt4) located on chromosome arms 6RS, 3RS, 4RL and 7RS, respectively. In this work, we analyzed several F₂ populations in which two different Alt loci were segregating. We constructed a map of chromosome 7R, which contains the Alt4 locus and microsatellite and PCR-markers (B1, B4, B11, B26 and BCD1230). These markers were mapped to the S arm of 7R using wheat-rye addition lines. Our results show that all these markers are linked to the Alt4 locus already known to be on 7RS. In addition, the OPS14 ₇₀₅ RAPD marker was linked to the Alt3 locus using bulked segregant analysis. This RAPD marker was transformed into a SCAR (ScOPS14 ₇₀₅ ) and was localized to arm 4RL using wheat-rye addition lines. Finally, this SCAR was linked to the Alt3 locus at a genetic distance of 23.4 cM. In light of the current findings, and taking into account the synteny relationships in cereals, we propose candidate Alt3 and Alt4 orthologues in other cereals.     

Development of PCR-based codominant markers flanking the Alt3 gene in rye.

Aluminum (Al) toxicity is considered to be a major problem for crop growth and production on acid soils. The ability of crops to overcome Al toxicity varies among crop species and cultivars. Rye (Secale cereale L.) is the most Al-tolerant species among the Triticeae. Our previous study showed that Al tolerance in a rye F6 recombinant inbred line (RIL) population was controlled by a single gene designated as the aluminum tolerance (Alt3) gene on chromosome 4RL. Based on the DNA sequence of a rice (Oryza sativa L.) BAC clone suspected to be syntenic to the Alt3 gene region, we developed two PCR-based codominant markers flanking the gene. These two markers, a sequence-tagged site (STS) marker and a cleaved amplified polymorphic sequence (CAPS) marker, each flanked the Alt3 gene at an approximate distance of 0.4 cM and can be used to facilitate high-resolution mapping of the gene. The markers might also be used for marker-assisted selection in rye or wheat (Triticum aestivum L.) breeding programs to obtain Al-tolerant lines and (or) cultivars.     

<Emphasis Type="Italic">Secale cereale</Emphasis> inter-microsatellites (SCIMs): chromosomal location and genetic inheritance

The polymerase chain reaction (PCR) was used to locate Secale cereale (inter-simple sequence repeat ISSR) or Secale cereale inter-microsatellite (SCIM) markers using wheat–rye addition lines in order to develop a set of molecular markers distributed on the seven rye chromosomes. The number of SCIM markers located on 1R, 2R, 3R, 4R, 5R, 6R and 7R chromosomes were 4, 3, 12, 3, 2, 9 and 8, respectively. Therefore, a total of 41 new SCIMs were located on the seven rye chromosomes. The segregation of the 63 different SCIM markers in three different F₂ was studied. The observed ISSR segregations were the 3:1 (50.7%), the 15:1 (12.7%) and the 1:1 (14.2%). The linkage analysis carried out indicated that seven of the segregating SCIMs were linked to chromosome 7R and two were linked to chromosome 4R. The use of the SCIM markers as a source of molecular markers that could be linked to interesting genes or other important agronomic traits is discussed.     

Molecular markers linked to the aluminium tolerance gene Alt1 in rye (Secale cereale L.)

 Rye has one of the most efficient group of genes for aluminium (Al) tolerance among cultivated species of Triticeae. This tolerance is controlled by at least two independent and dominant loci (Alt1 and Alt3) located on chromosomes 6RS and 4R. We used two pooled DNA samples, one of Al-tolerant individuals and another of Al-sensitive plants from one F₂ that segregated for the Alt1 locus. We also used two pooled DNA samples, one with genotypes 11 and another with genotypes 22 for the Lap1 locus (leucin aminopeptidase) from another F₂ progeny that segregated for this locus, located on the 6RS chromosome arm. We identified several RAPD markers associated with the pooled Al-tolerant plants and also with one of the bulks for the Lap1 locus. The RAPD fragments linked to Alt1 and Lap1 genes were transformed into SCAR markers to confirm their chromosomal location and linkage data. Two SCARs (ScR01 ₆₀₀ and ScB15 ₇₉₀₀ ) were closely linked to the Alt1 locus, ScR01 ₆₀₀ located 2.1 cM from Alt1 and ScB15 ₇₉₀ located 5.5 cM from Alt1, on the 6RS chromosome arm. These SCAR markers can aid in the transfer of Al tolerance genes into Al-sensitive germplasms. Received: 9 December 1997 / Accepted: 12 May 1998     

Candidate gene identification of an aluminum-activated organic acid transporter gene at the Alt4 locus for aluminum tolerance in rye (Secale cereale L.)

Among cereal crops, rye is one of the most tolerant species to aluminum. A candidate gene approach was used to determine the likely molecular identity of an Al tolerance locus (Alt4). Using PCR primers designed from a wheat aluminum tolerance gene encoding an aluminum-activated malate transporter (TaALMT1), a rye gene (ScALMT1) was amplified, cloned and sequenced. Subsequently, the ScALMT1 gene of rye was found to be located on 7RS by PCR amplification using the wheat–rye addition lines. SNP polymorphisms for this gene were detected among the parents of three F₂ populations that segregate for the Alt4 locus. A map of the rye chromosome 7R, including the Alt4 locus ScALMT1 and several molecular markers, was constructed showing a complete co-segregation between Alt4 and ScALMT1. Furthermore, expression experiments were carried out to clarify the function of this candidate gene. Briefly, the ScALMT1 gene was found to be primarily expressed in the root apex and upregulated when aluminum was present in the medium. Five-fold differences in the expression were found between the Al tolerant and the Al non-tolerant genotypes. Additionally, much higher expression was detected in the rye genotypes than the moderately tolerant “Chinese Spring” wheat cultivar. These results suggest that the Alt4 locus encodes an aluminum-activated organic acid transporter gene that could be utilized to increase Al tolerance in Al sensitive plant species. Finally, TaALMT1 homologous sequences were identified in different grasses and in the dicotyledonous plant Phaseolus vulgaris. Our data support the hypothesis of the existence of a common mechanism of Al tolerance encoded by a gene located in the homoeologous group four of cereals. G. Fontecha and J. Silva-Navas contributed equally to this work.     

Linkage mapping of powdery mildew and greenbug resistance genes on recombinant 1RS from 'Amigo' and 'Kavkaz' wheat-rye translocations of chromosome 1RS.1AL.

Cultivated rye (Secale cereale L., 2n = 2x = 14, RR) is an important source of genes for insect and disease resistance in wheat (Triticum aestivum L., 2n = 6x = 42). Rye chromosome arm 1RS of S. cereale 'Kavkaz' originally found as a 1BL.1RS translocation, carries genes for disease resistance (e.g., Lr26, Sr31, Yr9, and Pm8), while 1RS of the S. cereale 'Amigo' translocation (1RSA) carries a single resistance gene for greenbug (Schizaphis graminum Rondani) biotypes B and C and also carries additional disease-resistance genes. The purpose of this research was to identify individual plants that were recombinant in the homologous region of.1AL.1RSV and 1AL.1RSA using both molecular and phenotypic markers. Secale cereale 'Nekota' (1AL.1RSA) and S. cereale 'Pavon 76' (1AL.1RSV) were mated and the F1 was backcrossed to 'Nekota' (1AL.1AS) to generate eighty BC1F2:3 families (i.e., ('Nekota' 1AL.1RSA x 'Pavon 76' 1AL.1RSV) x 'Nekota' 1AL.1AS). These families were genotyped using the secalin-gliadin grain storage protein banding pattern generated with polyacrylamide gel electrophoresis to discriminate 1AL.1AS/1AL.1RS heterozygotes from the 1AL.1RSA+V and 1AL.1AS homozygotes. Segregation of the secalin locus and PCR markers based on the R173 family of rye specific repeated DNA sequences demonstrated the presence of recombinant 1AL.1RSA+V families. Powdery mildew (Blumeria graminis) and greenbug resistance genes on the recombinant 1RSA+V arm were mapped in relation to the Sec-1 locus, 2 additional protein bands, 3 SSRs, and 13 RFLP markers. The resultant linkage map of 1RS spanned 82.4 cM with marker order and spacing showing reasonable agreement with previous maps of 1RS. Fifteen markers lie within a region of 29.7 cM next to the centromere, yet corresponded to just 36% of the overall map length. The map position of the RFLP marker probe mwg68 was 10.9 cM distal to the Sec-1 locus and 7.8 cM proximal to the powdery mildew resistance locus. The greenbug resistance gene was located 2.7 cM proximal to the Sec-1 locus.     

Identification of AFLP and microsatellite markers linked with an aluminium tolerance gene in barley ( Hordeum vulgare L.)

Barley is the most sensitive among the cereals to aluminium (Al) stress and breeding for more tolerant cultivars is a priority. To enhance selection efficiency for Al tolerance in barley, PCR-based AFLP and microsatellite markers linked to a locus conferring tolerance to aluminium were identified. The study used F(2) progeny derived from a single cross between Yambla (moderately tolerant of Al) and WB229 (tolerant of Al) and developed hydroponic pulse-recovery screening methods to assess tolerance of phenotypes based on root growth. The segregation ratios of tolerant and sensitive genotypes and F(3) progeny testing suggest that a single major gene controlled Al tolerance ( Alt). In order to determine the chromosomal location of the Alt gene, we used the AFLP technique coupled with bulk segregant analysis. We evaluated tolerant and sensitive bulks using 30 combinations of EcoRI/ MseI primers, and 12 of these permitted differentiation of the sensitive and tolerant bulks. More than 1,000 amplified fragments were obtained, and 98 polymorphic bands were scored. AFLP analysis of wheat-barley chromosome addition lines indicated that the Alt gene was located on barley chromosome 4H. Four chromosome 4H-specific microsatellite markers (Bmac310, Bmag353, HVM68 and HVMCABG) were tightly linked to Alt. The large allelic variation detected with microsatellite marker Bmag353 allowed us to implement this marker for routine marker-assisted selection for Al tolerance, and 396 plants could be screened on a single gel.     

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye

 To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08 ₄₁₅ and OPR01 ₆₀₀ RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08 ₄₁₅ and SCR01 ₆₀₀ ) and linked to Alt1 gene (SCR01 ₆₀₀ -2.1 cM-Alt1-33.5 cM-SCA08 ₄₁₅ ). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat. Received: 9 June 1997 / Accepted: 19 September 1997     

Inheritance and fine mapping of fertility restoration for cytoplasmic male sterility in Gossypium hirsutum L

Genetics of CMS fertility restoration was presented through the analysis of classic genetics and molecular markers. Based on F(2) segregation of the crosses between CMS and the restoring lines, the testcrosses and F(1) x F(1) populations, together with RAPD and SSR mapping, one dominant gene was identified to control the CMS fertility restoration in cotton. The strategy of genotype representation analysis (GRA) was put forward to screen the markers linked with the Rf(1) locus. Using 1,025 random decamer primers and 282 pairs of SSR primers, two RAPD and three SSR markers were identified to be closely linked to the Rf(1) gene. Among the five markers, three were co-dominantly inherited. Additionally, based on the analysis of monosomic and telesomic lines with one SSR maker, the Rf(1) locus could be located on the long arm of chromosome 4. The molecular markers available here are helpful in the development of the elite restoring lines in cotton by marker-assisted selection.     

Genetic control of aluminium tolerance in rye (Secale cereale L.)

 Aluminium (Al) tolerance in roots of two cultivars (“Ailés” and “JNK”) and two inbred lines (“Riodeva” and “Pool”) of rye was studied using intact roots immersed in a nutrient solution at a controlled pH and temperature. Both the cultivars and the inbred lines analysed showed high Al tolerance, this character being under multigenic control. The inbred line “Riodeva” was sensitive (non-telerant) at a concentration of 150 μM, whereas the “Ailes” cultivar showed the highest level of Al tolerance at this concentration. The segregation of aluminium-tolerance genes and several isozyme loci in different F₁s, F₂s and backcrosses between plants of “Ailés” and “Riodeva” were also studied. The segregation ratios obtained for aluminium tolerance in the F₂s analysed were 3 : 1 and 15 : 1 (tolerant : non-tolerant) while in backcrosses they were 1 : 1 and 3 : 1. These results indicated that Al tolerance is controlled by, at least, two major dominant and independent loci in rye (Alt1 and Alt3). Linkage analyses carried out between Al-tolerance genes and several isozyme loci revealed that the Alt1 locus was linked to the aconitase-1 (Aco1), nicotinamide adenine dinucleotide dehydrogenase-2 (Ndh2), esterase-6 (Est6) and esterase-8 (Est8) loci, located on chromosome arm 6RL. The order obtained was Alt1-Aco1-Ndh2-Est6-Est8. The Alt3 locus was not linked to the Lap1, Aco1 and Ndh2 loci, located on chromosome arms, 6RS, 6RL and 6RL respectively. Therefore, the Alt3 locus is probably on a different chromosome. Received: 18 March 1997 / Accepted: 21 March 1997     

Rye SCAR markers for male fertility restoration in the P cytoplasm are also applicable to marker-assisted selection in the C cytoplasm

The study aimed at testing the usefulness of recently developed SCAR markers on rye (Secale cereale L.) chromosome 4R in hybrid breeding based on the C source of male sterility-inducing cytoplasm. Of 10 markers studied, 4 revealed polymorphisms between 2 inbred lines (544cms-C and Ot0-20) crossed to develop F2 and BC1 mapping populations. Analyses performed on 94 F2 and 93 BC1 plants allowed to extend a formerly constructed genetic map of chromosome arm 4RL. Three SCAR markers (SCP14M55, SCP15M55 and SCP16M58) were mapped in the vicinity of gene Rfc1, which restores male fertility in the C cytoplasm. The 3 tested SCAR markers proved to be effective in marker-assisted selection (MAS) for male fertility/sterility.     

Isolation and characterization of a new MATE gene located in the same chromosome arm of the aluminium tolerance (Alt1) rye locus

• Aluminium (Al) toxicity is the major constraint for crop productivity in acid soils. Wild rye species (Secale spp.) exhibit high Al tolerance, being a good source of genes related to this trait. The Alt1 locus located on the 6RS chromosome arm is one of the four main loci controlling Al tolerance in rye and is known to harbour major genes but, so far, none have been found.
• Through synteny among the short arm of the rye chromosome 6R and the main grass species, we found a candidate MATE gene for the Atl1 locus, later named ScMATE3, which was isolated and characterized in different Secale species.
• The sequence comparisons revealed both intraspecific and interspecific variability, with high sequence conservation in the Secale genus. SNP with replacement substitution that changed the structure of the protein and can be involved in the Al tolerance trait were found in ScMATE3 gene. The predicted subcellular localization of ScMATE3 is the vacuolar membrane which, together with the phylogenetic relationships performed with other MATE genes of the Poaceae related to Al detoxification, suggest involvement of ScMATE3 in an internal tolerance mechanism. Moreover, expression studies of this gene in rye corroborate its contribution in some Al resistance mechanisms.
• The ScMATE3 gene is located on the 6RS chromosome arm between the same markers in which the Alt1 locus is involved in Al resistance mechanisms in rye, thus being a good candidate gene for this function.

     

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F₂ progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.Communicated by J. Dvorak     

Genetic structure and diversity of European flint maize populations determined with SSR analyses of individuals and bulks

Characterization and manipulation of aluminum (Al) tolerance genes offers a solution to Al toxicity problems in crop cultivation on acid soil, which composes approximately 40% of all arable land. By exploiting the rice (Oryza sativa L.)/rye (Secale cereale L.) syntenic relationship, the potential for map-based cloning of genes controlling Al tolerance in rye (the most Al-tolerant cereal) was explored. An attempt to clone an Al tolerance gene (Alt3) from rye was initiated by using DNA markers flanking the rye Alt3 gene, from many cereals. Two rice-derived, PCR-based markers flanking the Alt3 gene, B1 and B4, were used to screen 1,123 plants of a rye F₂ population segregating for Alt3. Fifteen recombinant plants were identified. Four additional RFLP markers developed from rice genes/putative genes, spanning 10 kb of a 160-kb rice BAC, were mapped to the Alt3 region. Two rice markers flanked the Alt3 locus at a distance of 0.05 cM, while two others co-segregated with it. The rice/rye micro-colinearity worked very well to delineate and map the Alt3 gene region in rye. A rye fragment suspected to be part of the Alt3 candidate gene was identified, but at this level, the rye/rice microsynteny relationship broke down. Because of sequence differences between rice and rye and the complexity of the rye sequence, we have been unable to clone a full-length candidate gene in rye. Further attempts to clone a full-length rye Alt3 candidate gene will necessitate the creation of a rye large-insert library.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. x Secale cereale L.) x T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat--rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)2, 2R(2D)3, 3R(3B), 6R(6A)2. The chromosome composition of lines IR(1A), 2R(W)1, 5R(W), 5R(5A), and 6R(W)1, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)1, 5R(5D), 5R(5A), and 6R(6A)1, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The "combined" long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising "secondary gene pools" for the purpose of plant breeding.     

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.     

应用荧光原位杂交和RFLP标记检测多小穗小麦新种质10-A中的黑麦染色质

利用ＡＰＡＧＥ、荧光原位杂交技术和ＲＦＬＰ标记,对导入黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）多小穗等性状创制的小麦新种质１０＿Ａ进行了分子标记检测。ＡＰＡＧＥ分析发现,１０＿Ａ与其他１ＲＳ／１ＢＬ易位系一样,含有１ＲＳ的醇溶蛋白标记位点Ｇｌｄ１Ｂ３。以黑麦基因组总ＤＮＡ作探针,用中国春（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍｃｖ．ＣｈｉｎｅｓｅＳｐｒｉｎｇ）基因组ＤＮＡ作封阻,与１０＿Ａ根尖细胞有丝分裂染色体进行荧光原位杂交。结果表明,黑麦的１ＲＳ易位到１０＿Ａ中。用２５个ＲＦＬＰ探针进行Ｓｏｕｔｈｅｒｎ分析,进一步发现１０＿Ａ的１ＢＳ特异限制性片段发生丢失,代之以黑麦１ＲＳ的特异限制性片段,而位于其他染色体上的特异限制性片段未发生缺失。据此认为,多小穗小麦新种质１０＿Ａ属于１ＲＳ／１ＢＬ易位系。同时还讨论了１０＿Ａ在小麦遗传改良中的利用情况。     

Genetic and physical characterization of chromosome 4DL in wheat.

The long arm of chromosome 4D in wheat (Triticum aestivum L.) has been shown in previous studies to harbor genes of agronomic importance. A major dominant gene conferring Aluminum (Al) tolerance (Alt2 in 'Chinese Spring' and AltBH in 'BH 1146'), and the Knal locus controlling the K+/Na+ discrimination in saline environments have been mapped to this chromosome arm. However, accurate information on the genetic and physical location of markers related to any of these genes is not available and would be useful for map-based cloning and marker-assisted plant breeding. In the present study, using a population of 91 recombinant inbred lines segregating for Al tolerance, we provide a more extensive genetic linkage map of the chromosome arm 4DL based on RFLP, SSR, and AFLP markers, delimiting the AltBH gene to a 5.9-cM interval between markers Xgdm125 and Xpsr914. In addition, utilizing a set of wheat deletion lines for chromosome arm 4DL, the AltBH gene was physically mapped to the distal region of the chromosome, between deletion breakpoints 0.70 and 0.86, where the kilobase/centimorgan ratio is assumed to be low, making the map-based cloning of the gene a more realistic goal. The polymorphism rates in chromosome arm 4DL for the different types of markers used were extremely low, as confirmed by the physical mapping of AFLPs. Finally, analysis of 1 Mb of contiguous sequence of Arabidopsis chromosome 5 flanking the gene homologous to the BCD1230 clone (a cosegregating marker in our population coding for a ribulose-5-phosphate-3-epimerase gene), revealed a previously identified region of stress-related and disease-resistance genes. This could explain the collinearity observed in comparative mapping studies among different species and the low level of polymorphism detected in the chromosome arm 4DL in hexaploid wheat.