Fungal strain of the genus <i>Trichoderma </i>and method for promoting plant growth

Abstract

A fungal strain of the genus Trichoderma with the designation HSA12 and compositions that contain said fungal strain or spores thereof is disclosed. The fungal strain or spores thereof are promoting stabilizing plant growth, increasing the yields of crops, inoculating soil, roots and/or above-ground plant parts with the fungal strain or spores with compositions containing said fungal strain or spores thereof, to increase the efficiency of nutrient intake and to improve the stress tolerance of crops as well as improving the structure and health of the soil or for decontaminating or remediating soil or a body of water and for stabilizing or reestablishing endangered or desired wild plant populations. Also disclosed is a set of primer pairs for amplifying microsatellite loci of the genome of the fungal strain in order to determine molecular markers and to identify the fungal strain. A method for determining the fungal strain is also disclosed.

Claims

1. A method for at least one of promoting plant growth, stabilizing plant growth and increasing yields of crop plants, comprising: inoculating one or more of the group consisting of plant soil, plant roots and above-ground parts of a plant with fungal strain Trichoderma HSA12, which was deposited on Jan. 12, 2018 under the patent deposit number DSM 32722 at the Leibniz institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, or spores thereof.

2. The method according to claim 1, wherein plant growth is suffering under abiotic stress, selected from the group consisting of drought stress, cold stress and oxidative stress.

3. The method according to claim 2, wherein the abiotic stress results from a sowing or planting date for a crop plant that is advanced or delayed in relation to a respective region.

4. The method according to claim 3, wherein the crop plant is an agricultural plant.

5. The method according to claim 4, wherein the agricultural plant is selected from the group consisting of tomato plants, maize plants and soybean plants.

6. A method for improving structure and health of a soil, or for decontaminating or remediating a soil containing toxic organic substances, comprising: inoculating the soil with fungal strain Trichoderma HSA12, which was deposited on Jan. 12, 2018 under the patent deposit number DSM 32722 at the Leibniz institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, and cultivating the Trichoderma HSA12 in the soil.

7. The method of claim 1, wherein inoculating the soil, the plant roots and the above-ground parts of the plant is carried out with a composition comprising the Trichoderma HSA12 and additional microorganisms or biological fertilizers and adjuvants.

8. A method for stabilizing or resettling wild plant populations, comprising: inoculating soil of the wild plant population, and at least one of roots of the wild plant population and above-ground parts of the wild plant population, with fungal strain Trichoderma HSA12, which was deposited on Jan. 12, 2018 under the patent deposit number DSM 32722 at the Leibniz institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH.

9. A method for improving a crop soil or a body of water containing toxic organic substances, comprising: inoculating the soil or the body of water with fungal strain Trichoderma HSA12, which was deposited on Jan. 12, 2018 under the patent deposit number DSM 32722 at the Leibniz institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, and cultivating the Trichoderma HSA12 in the crop soil or the body of water.

10. A method for improving structure and health of a body of water containing toxic organic substances, comprising: inoculating the body of water with fungal strain Trichoderma HSA12, which was deposited on Jan. 12, 2018 under the patent deposit number DSM 32722 at the Leibniz institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, and cultivating the Trichoderma HSA12 in the body of water.

Description

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING

(1) Further details, features and advantages of embodiments of the invention will become evident from the following description of exemplary embodiments with reference to the appended drawings, which show in:

(2) FIG. 1: a bar graph comparing the yields of dwarf tomatoes with and without T. sp. HSA12 treatment,

(3) FIGS. 2A-C: the results of potted experiments with differently treated maize plants in form of bar graphs,

(4) FIG. 2D: the results of potted experiments with differently treated maize plants with reference to a photographic image,

(5) FIG. 3: a photographic image which compares tomato plants after different cultivation conditions at the time of the final harvest,

(6) FIG. 4: a bar graph comparing the respective number of damaged leaves in the cultivation of maize under different conditions,

(7) FIGS. 5A-B: bar graphs showing an induction of an early flowering time and the increase in valuable ingredients in the tomato harvest due to the early flowering when Trichoderma sp. HSA12 was added,

(8) FIGS. 6A-B: the results of potted experiments with tomato plants of the type Harzfeuer in form of bar graphs, and

(9) FIG. 6C: the results of potted experiments with tomato plants of the type Harzfeuer with reference to a photographic image.

(10) Table 1 shows, based on a comparison with a known bioeffector product, the induction of root growth and thereby improved stability and drought stress tolerance of the host plants, especially in maize, through inoculation with the fungal strain HSA12 of the genus Trichoderma, hereinafter referred to as Trichoderma species HSA12 or T. sp. HSA12, wherein the increase is 130 and 190%, respectively.

(11) TABLE-US-00001 TABLE 1 Phosphorus content of Shoot-Biomass Root Length the Shoot NO.sub.3 + phosphate in 100% 100% 100% solution (111 g) (4900 cm) (24 mg/Plant) NH.sub.4 RP 59 126 73 NH.sub.4RP Trianum ™ 75 b 117 84 b NH.sub.4RP T.sp.HSA12 86 ab 190 b 78 RP = Raw Phosphate a = not significantly greater than with supplied phosphate; b = significantly different from comparison value of NH.sub.4 RP

(12) The induced growth in maize depends on the form of the nitrogen and phosphorus supply. The tests were carried out on low-phosphorus loam soil with 20 mg phosphorus per kg of soil available to the plant. The amount of phosphorus was determined from calcium acetate-lactate extract, abbreviated as CAL extract, as a generally known method for the extraction of phosphorus available to plants. The abbreviation RP stands for rock phosphate. T. sp. HSA12 induces stronger growth than the bioeffector product Trianum™. Unlike Trianum™, the T. sp. HSA12 has no effect on the proportion of phosphorus in the shoot, but induces a strong increase in root growth (190%), which, as evident from the comparison of the respective root lengths, Is significantly stronger than with treatment with Trianum™. This shows that T. sp. HSA12 promotes the development of maize by increasing root growth.

(13) Table 2 shows the respective content of nitrogen N, phosphorus P, potassium K and manganese Mn in the shoot of maize, whereby it becomes evident from Table 2 how this content is influenced by to the inoculation with T. sp. HSA12 and—related to it—by the type of nitrogen and phosphorus supply. The stronger growth-promoting effect of T. sp. HSA12 on the plant compared to the Trianum™ product can be explained by improved access to several nutrients, namely nitrogen N, potassium K and manganese Mn, brought about by increased root growth. Maize has improved nutrient and trace element uptake in particular for nitrogen N and manganese Mn.

(14) TABLE-US-00002 TABLE 2 N (mg P (mg K (mg Mn (mg per Plant) per Plant) per Plant) per Plant) NO.sub.3 + 100% (289) 100% (24) 100% (34) 100% (0.26) phosphate in NH.sub.4 RP  93 a 73 70  94 a NR.sub.4RP 109 a 84 b 85 b 111 a Trianum ™ NH.sub.4RP 122 ab 78 87 ab 128 ab T.sp.HSA12 RP = Raw Phosphate a = not significantly greater than with supplied phosphate; b = significantly different from comparison value of NH.sub.4 RP

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(15) FIG. 1 shows, with reference to a bar graph, the influence of T. sp. HSA12 on the yield of dwarf tomatoes (Tomato cultivar DwarfTom) compared to the untreated controls in dwarf tomatoes. The yield information is given as the fresh weight of the tomato fruits. The tests were carried out in loess black earth soil without phosphorus fertilization and a pH value of 7.2.

(16) The value N is the sample size per test element. If N=5, this corresponds to a sample size of 5 plants per treatment. The value assigned to the lower-case letter p in the diagram is an indication of the significance level. A value p=0.004, which is significantly smaller than 0.05, indicates a very strong significance.

(17) The abbreviation BE stands for a “BioEffector” for microbial growth promotion. In contrast, BEO is the abbreviation for an untreated control. BE1 refers to Trianum®P, a commercial product from the company Koppert Ltd. from the Netherlands, which contains a Trichoderma fungal strain labelled T22. BE2 refers to Proradix, a commercial bacterial product (Pseudomonas) from the company Sourcon-Padena GmbH, Tübingen. BE3 refers to the strain FZB42 of the bacterial genus Bacillus amyloliquefaciens, which is also manufactured and sold under the name RhizoVital™ by the company AbiTEP Berlin. CombifectA consists of a combination of T. sp. HSA12 in combination with 5 bacilli BactoConc.

(18) OMG8, RapB5 and OMG16 are abbreviations for other Trichoderma strains from the University Anhalt as T. sp. HSA12. BactoConc is a bacterial product made from five different Bacillus strains, BactoProf is a bacterial product made from different bacteria and additives. Both are products from the company Bactiva GmbH, Straelen. Zinc and manganese (Mn/Zn) are used as trace elements in some samples. Furthermore, mixtures of the above listed components are used. The specification PO applies to all samples, meaning that no additional phosphate fertilization takes place.

(19) When incubated with T. sp. HSA12, a fresh weight of >1700 g was determined. With BE2, it was 1650 mg, with BactoConc 1650 g, with CombifectA>1500 g. Increased tomato yields are obtained compared to the untreated controls.

(20) FIGS. 2A to D show the results of a growth test in the form of potted experiments with maize. In these experiments, as the bar graphs in FIGS. 2A to C and the photographic image FIG. 2D show, the influence of Trichoderma sp. HSA12 on the height in cm and the fresh weight in grams of the above-ground parts of the maize plant compared with the untreated controls become evident. The value for N indicates the sample size, which is 5 and 23 plants, respectively, while the value for p indicates the level of significance.

(21) The results confirm the significant growth-promoting influence of Trichoderma sp. HSA12.

(22) FIG. 3 shows a photographic image which compares the tomato plants after different cultivation conditions at the time of the final harvest. Unfertilized plants and plants fertilized in compost soil are each cultivated with and without Trichoderma sp. HSA12 inoculation. The dry weight of the unfertilized tomato plant without T. sp. HSA12 inoculation was 32.6 g, the dry weight of the unfertilized tomato plant with T. sp. HSA12 inoculation was 49 g. The tomato plant grown without T. sp. HSA12 inoculation on composted soil had a dry weight of 46.2 g, the tomato plant on composted soil with T. sp. HSA12 inoculation had a dry weight of 53 g. In comparison, a plant that was fertilized with dissolved phosphate (triple-superphosphate) had a dry weight of 95.2 g. The latter serves as a positive control for what can only be achieved with complex and environmentally harmful chemical fertilization.

(23) Table 3 shows the change in the defense reactions of maize compared to potted experiments with maize in soil containing nitrate (NO.sub.3), with the data being given in percent. The results show that the addition of ammonium (NH.sub.4.sup.+) reduces leaf damage and activates protective compounds such as proline, silicon (Si) and superoxide dismutase (SOD). The addition of T. sp. HSA12 produces additional defense compounds, such as ascorbate peroxidase (APX), antioxidants, root SOD and root proline, which is associated with an additional reduction in leaf damage. In comparison, zinc and manganese, which are not approved for organic farming, silicon or “AlgaVyt”, an algae product that is very rich in minerals, or Superfifty®, a seaweed extract for use as a growth stimulator in plants, or aqueous extracts from compost, so-called compost tea, are added to the soil or leaves in further controls. The increased activity of Mn-dependent superoxide dismutase (SOD) in stems and leaves results in an increased tolerance to oxidative stress from reactive oxygen species in maize.

(24) TABLE-US-00003 TABLE 3 Defense Reaction [% Change compared to Leaf Treatment NO.sub.3— Only Soil Treatment AlgaVyt Compost Control] NH.sub.4+ HSA12 Zn/Mn Si Zn/Mn Superfifty ® tea Root length k.A. k.A. k.A. +44 k.A. k.A. k.A. Oxidative Stress Leaf −17 −42 −41 −33 −44 −34 −45 damage SOD (Shoot) +41 +26 +15 +51 +44 +60 +51 SOD (Root) k.A. +89 +95 +110 k.A. k.A. k.A. APX (Shoot) k.A. +47 +62 +54 +54 +59 +53 Antioxidants +19 +86 +79 +75 +76 +76 +76 Phenols k.A. +157 +119 +109 +96 +84 +96 Protective dissolved substances Proline (Shoot) +275 +227 +333 +233 +120 +87 +35 Proline (Roots) k.A. +60 +96 +78 +180 +120 +80 Nutrient level Zinc +33 +16 +105 +34 +56 +56 +56 Si +43 +43 +43 +50 +40 +53 +37 k.A. = no details

(25) FIG. 4 shows the formation of leaf necrosis during cold stress in maize at a root temperature of 12° C. The number of damaged leaves when cultivating maize under various conditions is compared in a bar graph. The plants were fertilized with either nitrate (NO.sub.3.sup.−) or ammonium (NH.sub.4.sup.+). In addition, there was no further addition of trace elements or cultures in the untreated, cooled control noBE. Another control was carried out under uncooled conditions, i.e. at a root temperature of 18° C. The other comparative cultivations for cultivation according to T. sp. HSA12 Inoculation were carried out either with the addition of the trace elements zinc and manganese (Zn, Mn) or Abi02, a Bacillus preparation from the company ABITEP GmbH, or BFOD-Penicillium (fungus), a preparation from the company Bayer Crop Science.

(26) After the start of the cold period of 14 days with temperatures of 12° C., oxidative leaf damage, namely necrosis, chlorosis and anthocyanin formation, develops rapidly. The leaf damage decreases in the following order:

(27) Abi 02+ZnMn>ZnMn>BFOD+ZnMn>T. sp. HSA12>uncooled control.

(28) The root space temperature of the cooled plants is 12° C. In general, plants treated with ammonium incur less damage than plants treated with nitrate. Only the uncooled plants show less leaf damage than the plants treated with T. sp. HSA12. In other words: The uncooled control naturally has the fewest necroses, but the cultivation after T. sp. HSA12 inoculation, which protects maize very well against damage caused by the cold, shows the second-best results. The protection is better with ammonium fertilization than with nitrate fertilization, as the significantly smaller columns indicate.

(29) Cold stress tolerance in maize increases after the inoculation with T. sp. HSA12, which leads to increased levels of polyphenol/flavonoid/proline as anti-stress metabolites in stems and leaves and reduced leaf necrosis.

(30) FIGS. 5A and 5B show in form of bar graphs an induction of an early flowering time and the increase in valuable ingredients in the harvest of tomatoes of the variety “MOBIL” from Hungary due to the early flowering when Trichoderma sp. HSA12 is added. The letters above the columns indicate the significance, meaning that wherever the same letters appear, there are no significant differences and the level of significance is at least p<0.05.

(31) FIG. 5A shows the concentrations of citric acid and maleic acid, and FIG. 5B shows the concentrations of glucose and fructose under four different conditions in each case, once in August and once in September. A control, a sample with the addition of Trichoderma sp. HSA12, abbreviated as BE, a sample with two bacterial additives, abbreviated as BR1 and BR2, and a sample with a combined addition of Trichoderma sp. HSA12 and one of the bacterial additives, BR2, are compared in the bar graphs.

(32) The Induction of an earlier flowering time leads to an increase in valuable ingredients in crop plants, in the illustrated example in tomatoes. While the citric acid content of the controls in August was 220 mg per 100 g of fruit, the plant that was treated with Trichoderma sp. HSA12 had at this time already a significantly higher citric acid content with 250 mg per 100 g of fruit. In September, this difference was with 380 mg citric acid per 100 g fruit for the plants treated with Trichoderma sp. HSA12 was even greater compared to the control which had only 280 mg citric acid per 100 g fruit.

(33) The maleic acid content in the fruits after treatment with Trichoderma sp. HSA was about the same in August and was slightly lower in September than in the control.

(34) In August, as a result of the earlier flowering time, the glucose content in fruits from the plants treated with Trichoderma sp. HSA12 was with 850 mg per 100 g of fruit significantly higher than in the corresponding control, with the control having a glucose content of 480 mg per 100 g of fruit. In September, the glucose content in fruits from the plants treated with Trichoderma sp. HSA12 was with 560 mg per 100 g of fruit somewhat lower than in the corresponding control, where the value was 620 mg per 100 g of fruit.

(35) Both in August and also in September, the values for fructose content in fruits from the plants treated with Trichoderma sp. HSA12 was significantly higher than in the control. The fructose content of the plants treated with Trichoderma sp. HSA12 was 400 mg per 100 g of fruit in August and 420 mg per 100 g of fruit in September, whereas the control values were 260 and 220 mg, respectively, per 100 g of fruit.

(36) FIGS. 6A and 6B show in the form of bar charts the results of potted experiments with tomato plants of the type Harzfeuer. Plant roots were hereby each inoculated individually with T. sp. HSA12 and with the Bacillus amyloliquedaciens strain FZB42, respectively, and the results were compared, on the one hand, with a control and, on the other hand, with roots that were inoculated with a mixture of T. sp. HSA12 and FZB42. The strain FZB42 was purchased as the product RhizoVital™ from the company AbiTEP Berlin.

(37) FIG. 6A shows a comparison of the plant heights in cm, while in FIG. 6B the root weights are compared with one another as a measurable variable for root growth.

(38) The plant height, measured in cm, increases in the order control<FZB42<T. sp. HSA12<<<combination of T. sp. HSA12 and FZB42. The difference between the plant height of the plant treated with a combination of T. sp. HSA12 and FZB42 and the next greater plant height, namely the plant height of the plant treated only with T. sp. HSA12, is significantly greater than the respective differences between the individual comparison samples.

(39) The root weight (dry weight) in g increases in the order of control<T. sp. HSA12<FZB42<<<combination of T. sp. HSA12 and FZB42. The difference between the root weight of the plant treated with a combination of T. sp. HSA12 and FZB 42 and the next higher root weight, namely that of the plant treated only with FZB, is significantly greater than the respective differences between the individual comparison samples. FIG. 6C shows photographic images of the roots of the differently treated plants and of the control.

(40) In both cases, both in terms of plant height and in terms of root weight, no purely additive enhancement of the effectiveness of the two cultures contained in the mixture was observed; instead, the corresponding increases in plant height and root weight are clear signs of synergism.

(41) Table 4a shows the sequences of the primer pairs for the determination of the genetic fingerprint of the fungal strain of the genus Trichoderma with the designation HSA12. Due to the availability of the complete genome sequence, T. sp. HSA12 is unambiguously characterized and unequivocally identifiable at any time, even when admixed to other products. A strain is characterized by its genome sequence. If the genome sequence is determined from two separate cultures and is identical, then this is by definition also the same strain. This would also apply if the strain were isolated from nature at a second time. But if the genome sequences exhibit even slight differences, then this is a different strain, another individual of the species. Since microorganisms and also micro-fungi have very few morphological characteristics, a gene segment of the ribosomal DNA (rDNA) is sequenced for associating micro-fungi to species or genera, and the obtained DNA sequence is compared with reference sequences stored in databases, which is known as DNA barcoding, from Schoch et al., Barcoding Consortium (2012), Nuclear ribosomal internal transcribed spacer as a universal DNA barcode marker for Fungi, PNAS 109 (16): 6241-6246. This analysis showed an association of HSA12 to the species Trichoderma harzanium with a probability of 98.8%. Another and more precise method, the comparison of the entire genome sequences (phylogenomics) of HSA12 with a T. Harzanum reference strain (Voucher Strain), only showed a match of 92% of all base pairs. However, a match of at least 97% is required in the case of fungi in order to be able to clearly associate the DNA sequences with a species. Therefore, HSA12 is definitely not Trichoderma harzianum, but possibly a previously unknown Trichoderma species or a known Trichoderma species whose genome sequence is not yet known. Therefore, the fungal strain is referred to as fungal strain HSA of the genus Trichoderma or as Trichoderma species HSA12, abbreviated as T. sp. HSA12.

(42) Since at best species, but only genera can be determined with certainty using DNA barcoding, and since phylogenomics is very complex for the routine identification of individuals, and since only a few entire fungal genomes have been sequenced, the “genetic fingerprint” is used in routine diagnostics to identify individuals, see also Geistlinger et al., “SSR Markers for Trichoderma viruses: Their Evaluation and Application to identify and Quantify Root-Endophytic Strains”, Diversity 2015, 7, 360-384. So-called hypervariable genome sections, so-called simple ones, are hereby used, so-called simple sequence repeats (SSR), also called microsatellites or SSR markers. Consequently, hypervariable genome segments from the T. sp. HSA12 genome are analyzed and a genetic fingerprint is generated. This genetic fingerprint serves, on the one hand, to again recognize the individual T. sp. HSA12 and, on the other hand, as an exclusion criterion for differentiating from other individuals from the same species or genus.

(43) The genetic fingerprint is created using the polymerase chain reaction (PCR). To this end, the corresponding hypervariable genome segments, also called SSR markers, are amplified at each of the fifteen HSA12 loci. A pair of so-called primers, referred to below as a primer pair, defines the starting point of DNA synthesis on each of the two single strands of DNA, thereby delimiting the region to be replicated on both sides. The specified section is then replicated with the help of DNA polymerase, thereby amplifying the DNA sequence sections. The set of fifteen primer pairs used, referred to below as the primer set, is specific for the corresponding genome segments of the fungal strain Trichoderma sp. HSA12. Table 4a lists the thirty primer sequences with the corresponding SEQ.-numbers 1 to 30, of which two consecutive sequences in the sequence numbering (SEQ-NO.) form a primer pair.

(44) The fragment length of the respective genome segments was determined at a total of fifteen HSA12 loci (L1 to L15) with single sequence repeats (SSRs), corresponding to the sequences with the SEQ-ID-NO. 31 to 45, by polymerase chain reaction (PCR) and compared with other Trichoderma products. This combination of fragment lengths according to Table 4b, given in base pairs (bp), was not obtained in any other isolate. Table 4b also lists the altogether fifteen repeating sequences with SEQ-ID-NO. 31 to 45 and the respective number of sequence repetitions (repeat number) in the genome sections which are amplified by the altogether fifteen primer pairs formed from the primer sequences with the SEQ-ID-NO. 1 to 30.

(45) TABLE-US-00004 TABLE 4a SEQ- Loci Primer designations ID-No. Primer sequences 5′-3′ L1 HSA12S51GAA11f 1 5′-CGGATGTGAGACGCAATATG-3′ HSA12S51GAA11r 2 F-CAACAGCGAAGTGTTGATGG-3′ L2 HSA12S52TCC12f 3 F-TCAACTICGCCCTCATTTTC-3′ HSA12S52TCC12r 4 5′-CGATCTCGAAGCTGACACAG-3′ L3 HSA12S53CAT11f 5 5′-GTCTGGCTACATTGGCCTTC-3′ HSA12S53CAT11r 6 5′-AGACGGAGGGGGAGATTATG-3′ L4 HSA12S54CTT15f 7 5′-TCCTCCTCAATCACCTTTGC-3′ HSA12554CTT15r 8 5′-TTTCCCGAAGAAATCACAGG-3′ L5 HSA12S55AGT13f 9 5′-GCCACAGAGAGAAGCCAGTC-3′ HSA12S55AGT13r 10 5′-GCGTCATGTCCCCATCTATC-3′ L.sup.6 HSA12S56GAAGTGAAG7f 11 5′-TTTCTTCGTGTTTCCCCATC-3′ HSA12S56GAAGTGAAG7r 12 5′-GACAAAGAAGCCGAGGACAG-3′ L.sup.7 HSA12S56GTTTGT8f 13 5′-ATCAATAGACGGGGCATACG-3′ HSA12S56GTTTGT8r 14 5′-CGAAAAGAGAGCCAAAAACG-3′ L8 HSA12S58CT14f 15 5′-GGAGAACGAAGCTTGACCTG-3′ HSA12S58CT14r 16 5′-TATACCCCGCCTCAACAGTC-3′ L9 HSA12S59TA12f 17 5′-TGGIGGIGIGTACGAAATGG-3′ HSA12S59TA12r 18 5′-GGCATCGTAGCGAAGTAAGC-3′ L10 HSA12S6OTCAGG5f 19 5′-TCCAAACCCTGACTGAGGTC-3′ HSA12S6OTCAGG5r 20 5′-AGATGCAGATCGTCGTGTTG-3′ L11 HSA12S6OCAG1Of 21 5′-CTGCCTCTCCAGAACACTCC-3′ HSA12S6OCAG1Or 22 5′-CATTATAAGGGGCCACAACG-3′ L12 HSA12S61AGG6f 23 5′-TACAGCACGAAGACGCTCTC-3′ HSA12S61AGG6r 24 5′-AACAGCGACCAAGCATAACC-3′ L13 HSA12S63TGC7f 25 5′-CTGTCGAGATTGCTGCTGAG-3′ HSA12S63TGC7r 26 5′-ATGTACTTTTCCGCGTCCAG-3′ L14 HSA12S66AGTGCC11f 27 5′-TTCAACAGCGTCAACCTCAG-3′ HSA12S66AGTGCC11r 28 5′-CCGGATTTATTTTGGTGGTG-3′ L15 HSA12S66AT17f 29 5′-CATTTGGGGTGGGTATTCTG-3′ HSA12S66AT17r 30 5′-ATTGTCACCGATGGAGGAAG-3′

(46) TABLE-US-00005 TABLE 4b Fragment SEQ-ID- SEQ-ID- Length NO. Tm [°0] Locus NO. Repeat Number [bp] 1 60.10 L1 31 (GAA).sub.11 488 2 60.30 3 60.19 L2 32 (TCC).sub.12 426 4 59.73 5 59.70 L3 33 (CAT).sub.11 365 6 60.29 7 60.20 L4 34 (CTT).sub.15 492 8 60.04 9 60.14 L5 35 (AGT).sub.13 204 10 60.31 11 59.91 L6 36 (GAAGTGAAG).sub.7 236 12 59.99 13 59.81 L7 37 (TTTGT).sub.8 287 14 69.99 15 59.99 L8 38 (GT).sub.14 289 16 59.96 17 60.28 L9 39 (TA).sub.12 468 18 60.01 19 60.09 L10 40 (TCAGG).sub.5 481 20 59.86 21 59.99 L11 41 (CAG).sub.10 460 22 60.20 23 59.34 L12 42 (AGG).sub.6 473 24 60.14 25 59.88 L13 43 (TGC).sub.7 401 26 60.13 27 60.02 L14 44 (AGTGCC).sub.11 306 28 60.05 29 60.04 L15 45 (AT).sub.17  33 30 59.93