**4. Conclusion**

amounts to 770 bp in rice and 710 bp in tobacco. In *Marchantia polymorpha* it is 188 bp [151]. This region is located between the tRNA genes, just as the non-coding sequence located be‐ tween the *trnL* (UAA) 3' exon and the *trnF* (GAA). Due to its catalytic properties and its sec‐ ondary structure, the *trnL* (UAA) intron, which belongs to type I introns, is less variable and therefore of better utility for evolutionary studies at higher taxonomic levels [113]. More‐ over, depending on the species, they show high frequency of insertions or deletions, which

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

60°C [99] (tobacco)

60°C [99] (tobacco)

60°C [113] (soybean)

56°C [99] (tobacco)

58°C [99] (tobacco)

56°C [99] (tobacco)

50°C [148] (soybean)

50°C [148] (soybean)

56°C [99] (tobacco)

[150] (soybean)

[99] (tobacco)

[99] (tobacco)

[99] (tobacco)

[99] (tobacco)

[99] (tobacco)

[148] (soybean)

[148] (soybean)

[99] (tobacco)

**Region Primer sequence (5 - 3)\*** Annealing temperature **Reference**

r: GCTAACCTTGGTATGGAAGT

r: TTACCACTAAACTATACCCGC

r: TTAAGTCCGTAGCGTCTACC

r: AGATTTGAACTGGTGACACG

r: CAACACTTGCTTTAGTCTCTG

r: TTCAACAGTTTGTGTAGCCA

r: TGAATAACTTACCCATGAATC

r: ATCCGAAGCGATGCGTTG

r: TCAACTCGTATCAACCAATC

**Table 4.** Primers used for amplification of nine non-coding regions of soybean cpDNA.

In most studied species, the *trnL* (UAA) intron ranges in size from 254 - 767 bp. Its smaller fragment – the P6 loop – reaches a length of 10 - 143 bp. It is commonly applied in DNA barcoding. Its main limitation lies in its low homologousness with the species from the Gene Bank, which amounts to 67.3%, while the homologousness of the P6 loop is 19.5%. However, it also has some advantages: conservative primers projected form and trouble-free amplifi‐ cation process. Amplification of the P6 loop can be performed even in a very degraded DNA. The intron is well known and its sequences are used to determine phylogenetic rela‐ tionships between closely related species or to identify a plant species [152]. The first univer‐ sal primers for this region were designed more than 20 years ago [119]. However, it does not

makes them potentially useful as genetic markers.

Relationships

568

*trnH–psbA* f:TGATCCACTTGGCTACATCCGCC

*trnS–trnG* f: GATTAGCAATCCGCCGCTTT

*trnT–trnL* f: GGATTCGAACCGATGACCAT

*trnL–trnF* f: TCGTGAGGGTTCAAGTCC

*atpB–rbcL* f: GAAGTAGTAGGATTGATTCTC

*psbB–psbH* f: AGATGTTTTTGCTGGTATTGA

*rps11–rpl36* f:GTATGGATATATCCATTTCGTG

*rpl16–rps3* f: ACTGAACAGGCGGGTACA

*ndhD–ndhE* f: GAAAATTAAGGAACCCGCAA

\*f, forward primer; r, reverse primer

In phylogenetic and population studies of *Glycine*, genetic information contained not only in cpDNA but also in mtDNA are often analysed. Organelle DNA can be used to find speciesspecific molecular markers. Molecular markers are an important tool to systematize the spe‐ cies because their use allows for detecting the differences in the genes directly. The selection of appropriate sequences, which depends on the taxonomic level at which reconstruction of the origin is carried out is very important. The initial selection concerns non-conservative se‐ quences, which are subject to fast evolution, because the more related the specimen are, the more changeable the region should be. The relatively slow rate of evolution of certain se‐ quences may exclude statistically significant analyses within families or species, while the study of relationship between species, which phylogenetically are very distant, using more slowly evolving sequences can be very useful. Non-coding sequences show a faster rate of evolution than the coding sequences. These regions accumulate a greater number of inser‐ tion/deletion or substitution than the non-coding regions, and therefore may be more suita‐ ble for research at inter-or intra-genus levels.
